WO2013024121A2

WO2013024121A2 - Increase of sucrose transporter activity in the seeds of plants

Info

Publication number: WO2013024121A2
Application number: PCT/EP2012/065958
Authority: WO
Inventors: Norbert Sauer; Benjamin Peter Ulrich POMMERRENIG
Original assignee: Basf Plant Science Company Gmbh; Friedrich-Alexander-Universität Erlangen-Nürnberg
Priority date: 2011-08-18
Filing date: 2012-08-15
Publication date: 2013-02-21
Also published as: AR087597A1; WO2013024121A3; UY34272A

Abstract

The present invention relates to the use of nucleic acid sequences encoding a sucrose transporter polypeptide in transgenic plants. In particular, the invention is directed to methods for increasing fatty acid-related compounds and for increasing oil level and altering the fatty acid composition in the seeds of plants.

Description

Increase of Sucrose Transporter Activity in the Seeds of Plants

Described herein are inventions in the field of genetic engineering of plants, including isolated nucleic acid molecules encoding a sucrose transporter protein to improve agronomic, horticultural and quality traits. These inventions relate generally to nucleic acid sequences encoding proteins that are related to the presence of seed storage compounds in plants. More specifically, the present invention relates to a sucrose transporter 5 nucleic acid sequence SEQ ID NO: 1 and the use of this sequence in transgenic plants. In particular, the invention is directed to methods for manipulating the content of sugar-related compounds, for increasing the oil level and/or altering the fatty acid composition in plants and seeds.

The study and genetic manipulation of plants has a long history that began even before the famed studies of Gregor Mendel. In perfecting this science, scientists have accomplished modification of particular traits in plants ranging from potato tubers having increased starch content to oilseed plants such as canola and sunflower having increased fatty acid content or altered fatty acid composition. With the increased consumption and use of plant oils, the modification of seed oil content and seed oil levels has become increasingly widespread (e.g. Topfer et al. 1995, Science 268:681 -686). Manipulation of biosynthetic pathways in transgenic plants provides a number of opportunities for molecular biologists and plant bio- chemists to affect plant metabolism giving rise to the production of specific higher-value products. The seed oil production or composition has been altered in numerous traditional oilseed plants such as soybean (U.S. Patent No. 5,955,650), canola (U.S. Patent No.

5,955,650), sunflower (U.S. Patent No. 6,084,164), and rapeseed (Topfer et al. 1995, Science 268:681 -686), and non-traditional oil seed plants such as tobacco (Cahoon et al.

1992, Proc. Natl. Acad. Sci. USA 89:1 1 184-1 1188).

Plant seed oils comprise both neutral and polar lipids (see Table 1). The neutral lipids consist primarily of triacylglycerol, which is the main storage lipid that accumulates in oil bodies in seeds. The polar lipids are mainly found in the various membranes of the seed cells, e.g. microsomal membranes, the cell membrane and the mitochondrial and plastidial membranes. The neutral and polar lipids contain several common fatty acids (see Table 2) and a range of less common fatty acids. The fatty acid composition of membrane lipids is highly regulated and only a select number of fatty acids are found in membrane lipids. On the other hand, a large number of unusual fatty acids can be incorporated into the neutral stor- age lipids in seeds of many plant species (Van de Loo F.J. et al. 1993, Unusual Fatty Acids in Lipid Metabolism in Plants pp. 91 -126, editor TS Moore Jr. CRC Press; Millar et al. 2000, Trends Plant Sci. 5:95-101 ).

Lipids are synthesized from fatty acids and their synthesis may be divided into two parts: the prokaryotic pathway and the eukaryotic pathway (Browse et al. 1986, Biochemical J. 235:25-31 ; Ohlrogge & Browse 1995, Plant Cell 7:957-970). The prokaryotic pathway is located in plastids that are the primary site of fatty acid biosynthesis. Fatty acid synthesis begins with the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (AC- Case). Malonyl-CoA is converted to malonyl-acyl carrier protein (ACP) by the malonyl- CoA:ACP transacylase. The enzyme beta-keto-acyl-ACP-synthase III (KAS III) catalyzes a condensation reaction, in which the acyl group from acetyl-CoA is transferred to malonyl- ACP to form 3-ketobutyryl-ACP. In a subsequent series of condensation, reduction and dehydration reactions the nascent fatty acid chain on the ACP cofactor is elongated by the step-by-step addition (condensation) of two carbon atoms donated by malonyl-ACP until a 16- or 18-carbon saturated fatty acid chain is formed. The plastidial delta-9 acyl-ACP de- saturase introduces the first unsaturated double bond into the fatty acid. Thioesterases cleave the fatty acids from the ACP cofactor and free fatty acids are exported to the cytoplasm where they participate as fatty acyl-CoA esters in the eukaryotic pathway. In this pathway the fatty acids are esterified by glycerol-3-phosphate acyltransferase and lyso- phosphatidic acid acyl-transferase to the sn-1 and sn-2 positions of glycerol-3-phosphate, respectively, to yield phosphatidic acid (PA). The PA is the precursor for other polar and neutral lipids, the latter being formed in the Kennedy pathway (Voelker 1996, Genetic Engineering ed.: Setlow 18:11 1 -1 13; Shanklin & Cahoon 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:61 1 -641 ; Frentzen 1998, Lipids 100:161 -166; Millar et al. 2000, Trends Plant Sci. 5:95-101 ).

Storage lipids in seeds are synthesized from carbohydrate-derived precursors. Plants have a complete glycolytic pathway in the cytosol (Plaxton 1996, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:185-214) and it has been shown that a complete pathway also exists in the plastids of rapeseeds (Kang & Rawsthorne 1994, Plant J. 6:795-805). Sucrose is the pri- mary source of carbon and energy, transported from the leaves into the developing seeds. During the storage phase of seeds, sucrose is converted in the cytosol to provide the metabolic precursors glucose-6-phosphate and pyruvate. These are transported into the plastids and converted into acetyl-CoA that serves as the primary precursor for the synthesis of fatty acids. Acetyl-CoA in the plastids is the central precursor for lipid biosynthesis. Acetyl-CoA can be formed in the plastids by different reactions and the exact contribution of each reaction is still being debated (Ohlrogge & Browse 1995, Plant Cell 7:957-970). It is however accepted that a large part of the acetyl-CoA is derived from glucose-6-phospate, phos- phoenolpyruvate and pyruvate that are imported from the cytoplasm into the plastids. Sucrose is produced in the source organs (leaves, or anywhere that photosynthesis occurs) and is transported to the developing seeds that are also termed sink organs. In the developing seeds, sucrose is the precursor for all the storage compounds, i.e. starch or lipids. Therefore, it is clear that carbohydrate metabolism, in which sucrose plays a central role is very important to the accumulation of seed storage compounds. Although the lipid and fatty acid content and/or composition of seed oil can be modified by the traditional methods of plant breeding, the advent of recombinant DNA technology has allowed for easier manipulation of the seed oil content of a plant, and in some cases, has allowed for the alteration of seed oils in ways that could not be accomplished by breeding alone (see, e.g., Topfer et al., 1995, Science 268:681 -686). For example, introduction of a A¹²-hydroxylase nucleic acid sequence into transgenic tobacco resulted in the introduction of a novel fatty acid, ricinoleic acid, into the tobacco seed oil (Van de Loo et al. 1995, Proc. Natl. Acad. Sci USA 92:6743-6747). Tobacco plants have also been engineered to produce low levels of petroselinic acid by the introduction and expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992, Proc. Natl. Acad. Sci USA 89:1 1 184-1 1188). The modification of seed oil content in plants has significant medical, nutritional and economic ramifications. With regard to the medical ramifications, the long chain fatty acids (C18 and longer) found in many seed oils have been linked to reductions in hypercholesterolemia and other clinical disorders related to coronary heart disease (Brenner 1976, Adv. Exp. Med. Biol. 83:85-101 ). Therefore, consumption of a plant having increased levels of these types of fatty acids may reduce the risk of heart disease. Enhanced levels of seed oil content also increase large-scale production of seed oils and thereby reduce the cost of these oils.

In order to increase or alter the levels of compounds such as seed oils in plants, nucleic acid sequences and proteins regulating lipid and fatty acid metabolism must be identified. As mentioned earlier, several desaturase nucleic acids such as the A⁶-desaturase nucleic acid, A¹²-desaturase nucleic acid and acyl-ACP desaturase nucleic acid have been cloned and demonstrated to encode enzymes required for fatty acid synthesis in various plant species. Oleosin nucleic acid sequences from such different species as canola, soybean, car- rot, pine and Arabidopsis thaliana have also been cloned and determined to encode proteins associated with the phospholipid monolayer membrane of oil bodies in those plants.

It has also been determined that two phytohormones, gibberellic acid (GA) and absisic acid (ABA), are involved in overall regulatory processes in seed development (e.g. Ritchie & Gil- roy, 1998, Plant Physiol. 1 16:765-776; Arenas-Huertero et al., 2000, Genes Dev. 14:2085- 2096). Both the GA and ABA pathways are affected by okadaic acid, a protein phosphatase inhibitor (Kuo et al. 1996, Plant Cell. 8:259-269). The regulation of protein phosphorylation by kinases and phosphatases is accepted as a universal mechanism of cellular control (Cohen, 1992, Trends Biochem. Sci. 17:408-413. Likewise, the plant hormones eth- ylene (e.g. Zhou et al., 1998, Proc. Natl. Acad. Sci. USA 95:10294-10299; Beaudoin et al., 2000, Plant Cell 2000:1 103-1 115) and auxin (e.g. Colon-Carmona et al., 2000, Plant Physiol. 124:1728-1738) are involved in controlling plant development as well.

Although several compounds are known that generally affect plant and seed development, there is a clear need to specifically identify factors that are more specific for the developmental regulation of storage compound accumulation and to identify genes which have the capacity to confer altered or increased oil production to its host plant and to other plant species.

Thus, the technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below. These nucleic acid sequences can be used to alter or increase the level of oil in plants, including transgenic plants, such as canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor, peanut, cranmbe and Jatropha, which are oilseed plants containing high amounts of lipid compounds.

The Arabidopsis sucrose transporter 5 (SUC5 protein - SEQ ID NO: 2) represents a su- crose/H⁺ symporter. Its gene was previously shown to be expressed in the endosperm. In Example 1 it is shown that the SUC5 protein is also essential for the delivery of biotin to the endosperm and the embryo in developing seeds of biotin biosynthesis-defective Arabidopsis mutants (biol and bio2). Embryo development, seed germination, seedling development, triacylglycerol (TAG) accumulation, and fatty acid composition were compared in single mutant (suc5, biol or bio2), double mutant (suc5/bio1 and suc5/bio2) and wild type seeds. Whereas suc5 mutants were like wild type plants, biol and bio2 mutants showed multiple developmental defects and had reduced TAG contents and altered fatty acid compositions in their dry seeds. These phenotypes were severely enhanced in suc5/bio1 and suc5/bio2 double mutants. Externally supplied biotin suppressed the phenotypes of biol and bio2 single and suc5/bio1 and suc5/bio2 double mutants, but higher biotin concentration were needed for double than for single mutants. Results of genetic and metabolic anal- yses demonstrate that the SUC5 protein acts as biotin transporter in plants. In Example 15 and 16 it was shown that the over-expression of the SUC 5 gene in plants alone or in combination with the GPT1 (SEQ ID NO: 83) nucleic acid sequence and the NTT1 (SEQ ID NO: 84) nucleic acid sequence results in an increased fatty acid content in plant seeds. Biotin (vitamin B₇ or vitamin H) is a prosthetic group in a small number of enzymes catalysing essential carboxylation, decarboxylation and transcarboxylation reactions (Knowles, 1989; Nikolau et al., 2003; Smith et al., 2007). Bacteria, plants, some fungi and few animals are capable of synthesizing the biotin needed for these reactions. For the identification of possible plant biotin transporters, a yeast mutant deleted in the

VHT1 gene (Avht1) was complemented with an Arabidopsis cDNA library and screened for growth on low extracellular biotin concentrations (Ludwig et al., 2000). Surprisingly, this screening identified a sequence with high similarity to sucrose transporter cDNAs from different plant species [e.g. AtSUCI and AtSUC2 from Arabidopsis or SoSUTI from spinach (Spinacea oleracea; Sauer, 2007)]. Functional analyses of the encoded protein demonstrated that it was, in fact, a Arabidopsis sucrose transporter (named SUC5; At1 g71890) with transport characteristics similar to those of previously published sucrose transporters (Ludwig et ai, 2000). Analyses with radiolabeled biotin, however, doubtlessly confirmed that SUC5 could also transport biotin (Ludwig et ai, 2000), a molecule with no structural similarity to sucrose. SUC5-driven biotin uptake was sensitive to cyanide-m- chlorophenylhydrazone (CCCP), an uncoupler of transmembrane proton gradients, and to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS), a compound widely used to inhibit the activity of plant sucrose transporters (M'Batchi and Delrot, 1984).

As for the Arabidopsis SUC5 protein, uptake of both substrates was sensitive to PCMBS and CCCP, and PmSUC2 complemented the growth defect of Avht1 yeast cells (Ludwig et ai, 2000). Evidence for a role of plant sucrose transporters in the catalysis of transmembrane biotin transport in planta has so far not been provided.

Arabidopsis plants with defects in biotin biosynthesis were first identified in analyses of em- bryo-lethal mutants. One mutant, bio1. 1 (At5g57590), was shown to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid (Schneider et ai, 1989; Muralla et ai, 2008), the other, bio2. 1 (At2g43360), in the conversion of dethiobiotin to biotin (Baldet and Ruffet, 1996; Patton et ai, 1996; Weaver et ai, 1996; Patton et ai, 1998). The developmental arrest observed in homozygous (bio1. 1/bio1. 1 or bio2. 1/bio2. 1) mutant embryos in the siliques of heterozygous (BI01/bio1. 1 or BI02/bio2. 1) mother plants was rescued by watering the soil-grown heterozygotes with 0.5 mM biotin. This demonstrated that the supplied biotin was taken up by the roots, translocated to the developing seeds, imported into the female gametophyte, and eventually into the endosperm and the developing embryo. Genetic and biochemical studies of suc5 transporter mutants in the background of Arabidopsis lines defective in the biosynthesis of biotin, one of the putatively transported substrates, were carried out. Two allelic mutants disrupted in the SUC5 gene (suc5.4 and suc5.5) were isolated. These lines were crossed with the biol. 1 or bio2. 1 mutants. The development of embryos, seedlings and seeds in the different single and double mutants were compared and the triacylglycerol (TAG) content and the fatty acid composition in dry seeds of the respective plants quantified. While both suc5 single mutants were like wt plants, the absence of SUC5 strongly enhanced all developmental and biochemical phenotypes observed in bio1. 1 and bio2. 1 mutant plants. Most importantly, all of these phenotypes were fully reversed by externally supplied biotin. However, the concentrations needed to rescue suc5/bio1. 1 or suc5/bio2. 1 double mutants were significantly higher than those needed to rescue bio1. 1 and bio2. 1 single mutants. In summary, the data demonstrate that SUC5 transfers biotin from the maternal tissue into the endosperm and embryos of developing Arabidopsis seeds and that this activity is essential under conditions of biotin limitation. Identification of two allelic mutant lines defective in SUC5 The suc5.1 to suc5.3 mutants published by Baud et al. (2005) had been generated in the Wassilewskija background. As the bio1.1 and bio2.1 mutants were in ecotype Columbia (Col-0), two novel suc5 mutants (SAIL_367_D07 = suc5.4; SALK_092412 = suc5.5; http://signal.salk.edu/cgi-bin/tdnaexpress) were characterized in the same ecotype. These mutants carry insertions either in the 2^nd intron (suc5.4: after nucleotide 250 of the 2^nd in- tron) or 3^rd exon (suc5.5: between nucleotides 98 and 105 of the 3^rd exon; 6 nucleotides deleted) with suc5.5 having a tandem insertion with the two right borders facing each other (Figure 1 a). For the suc5.4 allele only the orientation of the left border was determined. Comparative RT-PCRs with RNA isolated from flower tissue (Figure 1 b) demonstrated that both lines failed to produce intact SUC5 mRNA. This characterized both lines as k.o.- mutants.

Analyses of p SUC5/sGFP and pSUC5/tmGFP9 plants

Two different pSL/C5/reporter lines under the control of a 2030-bp SUC5 promoter were generated. These lines expressed the open reading frames (ORFs) of the soluble and freely mobile green fluorescent protein (sGFP) or of a non-mobile version of GFP (tmGFP9) that is membrane-attached by N-terminal transmembrane helices (Stadler et al., 2005a). After BASTA-selection of T1 seedlings, we obtained numerous transformed T1 plants for both constructs. Analyses of these plants by confocal microscopy confirmed the published (Baud et al., 2005) SUC5 expression in the endosperm (Figure 2a, b) and the accumulation of GFP at the chalazal end of the endosperm (Figure 2b). Also in agreement with the data of Baud et al. (2005), no expression of GFP was observed in globular-stage (Figure 2c) or heart-stage embryos (Figure 2d). Unexpectedly, pSUC5 activity with both constructs during the later stages of embryo development were found (Figures 2e to 2i). This SUC5 expression in the embryo has not been observed in earlier analyses performed with standard fluorescence microscopy and a shorter, 1500-bp promoter fragment (Baud et al., 2005).

Optical sections through pSUC5/tmGFP9 embryos confined the pSUC5 activity to the em- bryo epidermis (Figure 2g, h), specifically to the epidermis of the outer surface of the cotyledons, i.e. to their anatomical underside. In previous analyses (Stadler et al., 2005a) it had been shown that sGFP synthesized in the embryo epidermis from Arabidopsis GLABRA2 promoter (pGL2)/sGFP constructs can move symplastically into all other cells of the developing embryo due to the presence of large plasmodesmata. This explains the homogenous fluorescence seen in the early-torpedo-stage embryo expressing sGFP from pSUC5 (Figure 2e). Embryos from wt seeds showed no fluorescence at any developmental stage (Figure 2i).

Comparative analyses of embryo and seed development

The originally published bio1.1 and bio2.1 mutations (Schneider et al., 1989; Patton et al., 1998) were induced by chemical mutagenesis. Homozygous suc5.4 or suc5.5 plants were crossed with these bio1.1 or bio2.1 mutants, and double-homozygosity was determined by PCR (for the T-DNA insertions in suc5.4 and suc5.5) and by confirming the growth defect on biotin-free medium (for bio1.1 and bio2.1) in the following generations. On soil, these double-homozygotes developed normal rosettes, flowered and produced fertile seeds, when watered with 1 mM biotin as described for the biol.1 and bio2.1 single mutants (Schneider et al., 1989; Patton et al., 1998). Without supplemented biotin, the homozygous bio1.1 and bio2.1 plants and all double-homozygotes germinated poorly, developed tiny plants, turned pale and eventually died. However, when the seeds were first germinated on biotin-containing (1 mM) agar medium for about 10 d and then transferred to soil, normal-looking plants developed that flowered even without further biotin supplement.

To compare the development of seeds and embryos in wt plants, in suc5.4, suc5.5, bio1. 1 and bio2.1 single mutants, and in the different bio/suc5 double mutants, seeds of all lines were germinated on agar medium with 1 mM biotin. After 12 d, all seedlings had the identical size and were transferred to soil. These seedlings were then watered either without additional biotin, with 0.1 mM supplemental biotin, or with 1 mM supplemental biotin. All plants flowered at the same time and produced normal looking siliques. When comparing developing seeds from siliques harvested at the same developmental stage, phenotypic differences were observed (Figure 3a). Whereas developing wt seeds looked absolutely normal under all growth conditions, and whereas seeds from all suc5 single mutants where indistinguishable from the wt seeds, bio2.1 seeds showed a biotin- dependent phenotype. When the plants were grown without a biotin supplement (0 mM bio- tin in Figure 3a), the bio2.1 seeds were yellowish and pale. With increasing concentrations (0.1 mM or 1 mM biotin) of supplemented biotin, this phenotype disappeared gradually. Interestingly, and in agreement with a suspected biotin transporter function of SUC5, a significantly stronger phenotype was observed in developing seeds of bio2.1/suc5.5 double mutants that were not supplemented with biotin (0 mM biotin in Figure 3a). Seeds from these plants were white and smaller than seeds of bio2. 1 single mutants, which indicated a stronger biotin-deficiency. As in the single mutants, increasing concentrations of supplemented biotin gradually reduced this phenotype.

Isolated embryos from the developing-seed batches were analysed - see Figure 3a. As for the seeds, no differences were observed between wt and suc5 embryos under the different growth conditions. bio1.1 and bio2.1 embryos, however, showed a strongly retarded development in non-supplemented plants (Figure 3b). This defect was only partly rescued in plants supplemented with 0.1 mM biotin. Embryos from these plants had the same size as wt embryos; however, they were unable to synthesize chlorophyll and had a faint yellowish colour. Embryos from bio2.1 plants supplemented with 1 mM biotin were able to synthesize chlorophyll and looked essentially like wt embryos. Embryos from bio2.1/suc5.5 double-mutant seeds revealed a significantly stronger biotin- dependent phenotype than bio2.1 single-mutant embryos (Figure 3b). Without a biotin supplement, no embryos could be detected, and even the embryos from plants supple- mented with 0.1 mM biotin showed a strong developmental phenotype. Only in seeds from plants that were supplemented with 1 mM biotin wt-like embryos were formed.

Finally the morphology of dry seeds were compared from homozygous single and double mutants, and from wt plants (Figure 4). As expected, wt and suc5.5 mutant seeds looked normal under all growth conditions (supplemented with 0, 0.1 or 1 mM biotin, respectively). In contrast, the seeds of bio2.1 and of bio2.1/suc5.5 plants had a slightly (bio2.1) or strongly wrinkled and empty appearance (bio2.1/suc5.5), when the plants had not been supplied with biotin. A supplement of 0.1 mM biotin complemented these phenotypes to various extents. Whereas the bio2.1 seeds looked almost normal, 100% of the bio2.1/suc5.5 seeds still had a wrinkled appearance. Watering of the parent plants with 1 mM biotin completely (bio2.1) or almost completely (bio2.1/suc5.5) reverted this phenotype.

Similar results were obtained for developing seeds and embryos and for dry seeds from bio1. 1 and bio1.1/suc5.4 plants.

Phenotypic comparison of seedlings

Homozygous single-mutant seedlings (bio1.1, bio2. 1, suc5.4 and suc5.5) developed normally on high-biotin medium and were indistinguishable from wt plants (Figure 5: MS + biotin). Similar analyses on biotin-free medium (Figure 5: MS), however, revealed clear pheno- typic differences and confirmed the previously described defects for biol.1 and bio2.1 seedlings (Schneider et ai, 1989; Patton et ai, 1998). As germinable homozygous bio1.1 and bio2. 1 seeds were obtained exclusively from biotin-watered plants, the seedlings started to form normally sized, green cotyledons. Already the first pair of rosette leaves, however, developed poorly, was tiny and yellowish (Figure 5), and eventually these plants would die. In contrast, suc5.4 and suc5.5 (parent plants not watered with biotin) showed no phenotypic difference to wt seedlings on biotin-free medium. Reproducibly, also the wt, suc5.4 and suc5.5 seedlings developed slightly better on medium supplemented with 1 mM biotin.

When the same analyses were performed with seeds of bio1.1/suc5.4, bio1.1/suc5.5, bio2. 1/suc5.4 and bio2.1/suc5.5 double-homozygotes (all parent plants watered with biotin; Figure 5, right panel), we observed a high percentage of seedlings with severely aberrant cotyledon phenotypes on biotin-free medium (Figure 5, right panel: MS). The cotyledons of almost all seedlings were slightly (few plants) or significantly (most plants) smaller than those of wt seedlings, and in a high percentage of seedlings these cotyledons were partial- ly or completely white. Again, this demonstrated that the lack of functional SUC5 protein strongly enhanced the effect of the bio1. 1 or bio2. 1 mutations. Interestingly, even these strong phenotypes could be rescued with 1 mM biotin in the growth medium (Figure 5, right panel: MS + biotin). Compared to rescued bio1.1 or bio2.1 seedlings, however, the development of these rescued double-mutant seedlings was delayed. When these double- mutant seedlings were transferred to soil and watered with biotin, they formed normal ro- settes, flowered, and produced double-homozygous seeds. Upon germination, these seeds exhibited the same seedling phenotypes as their parent plants.

A more detailed and quantitative analysis of these double-mutant phenotypes is shown in Figure 6. The cotyledons of many seedlings failed to expand and were too small to lift the seed coat (Figure 6a). However, even when the expanding cotyledons lifted the seed coat, they stayed tiny and failed to turn green (Figure 6c). Other seedlings had misshaped, only partially green cotyledons (Figure 6b), and the few seedlings with normally shaped cotyledons were smaller than wt seedlings (Figure 6d). A significant number of seeds did not germinate at all or only after prolonged incubation. Interestingly, after prolonged incubation on biotin-containing medium (sometimes for several weeks), even seemingly cotyledon- less seedlings (Figure 6a) produced a first pair of rosette leaves. After this, even these seedlings could be transferred to soil, and formed normal-looking rosettes.

A quantification of the observed phenotypes (Figure 6e) demonstrated that the percentage of misshaped seedlings or of non-germinating and late-germinating seeds was negligible in the different homozygous single mutants, but prominent in the double mutants.

Analyses of triacylglycerol (TAG) content and fatty acid composition in seeds

Doubtlessly, the morphological phenotypes of bio1.1 and bio2.1 single mutants (Figures 3 to 6) result from a lack in biotin. Whereas in the very same analyses no phenotypic differences were detected between wt and suc5.4 or suc5.5 single mutants, the observed phenotypes were significantly stronger with bio2.1/suc5.5 (Figures 3 to 6) or other combinations of bio/suc5 double mutants (Figures 5 and 6). This demonstrated that defects in the SUC5 gene are not critical when biotin biosynthetic activity is normal, but become obvious under conditions of biotin limitation.

As biotin-dependent enzymes are involved in central steps of fatty acid biosynthesis (Ohl- rogge and Jaworski, 1997), biotin limitation should not only affect seed and seedling morphology, but also reduce the capacity to synthesize TAG. If SUC5 acts as biotin trans- porter, an additional suc5 mutation should further reduce these TAG levels. In fact, when the total TAG content in seeds of wt plants, single and double mutants were analysed, we observed an 80% reduction of the TAG content in seeds from bio2.1 single mutants that were not supplemented with biotin (0 mM biotin), and this reduction was even more pronounced (almost 95%) in seeds from non-supplemented bio2.1/suc5.5 double mutants (Figure 7a). A reduction in the TAG content was also seen in seeds from bio2.1 single and bio2. 1/suc5.5 double mutants supplemented with 0.1 mM biotin, and again the reduction was stronger in bio2.1/suc5.5 seeds (75% reduction versus 50% reduction in bio2.1 seeds, respectively). Seeds from plants supplemented with 1 mM biotin, had wt-like (bio2.1) or almost wt-like (bio2.1/suc5.5) TAG levels demonstrating that the observed biochemical phenotypes can be reverted solely with high biotin concentrations. The TAG content of wt and suc5.5 single mutant seed was unaffected under all conditions analysed (Figure 7a). Analyses of the fatty acid composition in the different TAG samples revealed that the reduced TAG contents in seeds of bio2.1 and bio2.1/suc5.5 plants (0 mM or 0.1 mM biotin) were paralleled by altered fatty acid compositions. Seeds from single and double mutants grown on 0-mM or 0.1 -mM biotin had 3 to 8-fold higher contents of monounsaturated n7 fatty acids (16:1 n7, 18:1 n7 and 20:1 n7) and 1.5 to 4-fold lower contents of monounsaturated n9 fatty acids (18:1 n9 and 20:1 n9; Figure 7b, c). Moreover, these seeds showed a reduced content of 18:0 and of 18:2n6 fatty acids. All of these alterations were more pronounced (up to 2-fold) in bio2.1/suc5.5 than in bio2. 1 seeds (Figure 7b, c). The fatty acid composition in seeds from bio2.1 and bio2.1/suc5.5 plants supplemented with 1 mM biotin were comparable to those in suc5.5 or wt seeds. The seeds of suc5.5 or wt plants showed the same fatty acid composition under all conditions (Figure 7c).

Again these results demonstrated that the observed alterations resulted exclusively from a reduced availability of biotin, and that a suc5 mutation enhanced the biotin-requirement in seeds of mutants defective in biotin synthesis.

The examples presented address the question whether or not the Arabidopsis SUC5 protein does act as biotin transporter in planta. The results show that SUC5 is expressed in the epidermis of torpedo-stage or older embryos (Figure 2e to 2i) demonstrating that SUC5 is not only involved in the transport of its substrate(s) from the maternal tissue into the endosperm but also in the transport from the endosperm into the embryo. The results also demonstrate that SUC5 is important for the transport of biotin across these boundaries and provide the first direct evidence that biotin transport by SUC proteins is physiologically relevant in planta. In Example 1 comparative analyses of two newly characterized suc5 mutants (suc5.4 and suc5.5), of two previously characterized biotin biosynthetic mutants (bio1.1 and bio2.1; Schneider et al., 1989; Patton et al., 1998), and of double mutants (bio1/suc5 or bio2/suc5) resulting from crosses of transport-defective and biosynthesis-defective lines are presented. Whereas the suc5 sin- gle mutants showed no phenotypic alterations under the conditions analysed, bio1.1 and bio2. 1 single mutants revealed severe phenotypes, such as impaired germination and seedling development (Figures 5 and 6), altered seed morphology (Figures 3 and 4), reduced TAG content (Figure 7a), and altered fatty acid composition in dry seeds (Figure 7c).

All of these developmental, morphological and biochemical defects were significantly en- hanced in bio/suc5 double-mutants, and as in the bio1.1 and bio2.1 single mutants, these more severe defects of bio/suc5 double mutants could be rescued solely by externally supplied biotin. Under all conditions, the biotin concentrations needed to reverse the defects in double mutants were higher than those needed for single mutants. Sucrose was not needed to rescue the stronger defect of double mutants. Together, this can only be explained by a biotin transport activity of SUC5.

SUC5 is responsible for biotin transport in planta

It is unclear, if biotin uptake by the roots is carrier- mediated, but within the sporophytic symplast, the distribution may occur mainly by diffusion. The transfer from the sporophyte to the endosperm and the developing embryo, however, involves strictly apoplastic steps and uptake by the endosperm and the embryo epidermis is likely to be carrier-mediated. Most importantly, the biotin requirement within the embryo exceeds that of other organs, e.g. of leaves and roots, as in addition to the production of membranes or cuticle waxes, large amounts of fatty acids are synthesized for storage lipid (TAG) production.

As published by Baud et al. (2005), the comparisons of wt plants and suc5.5 mutants revealed no differences in embryo and seedling development, no significant decrease in TAG accumulation and no alteration in the fatty acid compositions (Figure 7). However, as suggested by the wrinkled appearance of dry seeds from biotin -starved bio2.1 plants (Figure 4), reduced TAG contents in these seeds were found, and this reduction was paralleled by an increase in the relative content of n7 fatty acids and a decrease in n9 fatty acids (Figure 7b, c). Moreover, in seeds from the corresponding biotin-starved bio2.1/suc5.5 plants, these changes were significantly stronger. Most importantly, in bio2.1 and in bio2.1/suc5.5 plants these phenotypic alterations were gradually reverted to wt levels, when the plants were supplemented with 0.1 mM or 1 mM biotin (Figure 7c). These biotin supplements were always more effective in bio2.1 than in bio2.1/suc5.5 plants (Figure 7c) demonstrating that higher biotin concentrations are needed in the absence of the SUC5 transporter.

Again, these data can be explained only by a lack of biotin transport activity in suc5 mutants. Obviously, this lack has little effect when biotin biosynthesis in seeds is normal, but becomes increasingly important under conditions of biotin limitation. An essential role of SUC5 in the supply of sucrose is rather unlikely, as in suc5 seeds neither the sugar content nor the total fatty acid or TAG content were affected (Figure 7c and Baud et al., 2005). The previously described transient decrease in the fatty acid content in suc5 mutants at 8 days after flowering at the onset of fatty acid biosynthesis (Baud et al., 2005) may result from a transient lack of biotin that is rapidly overcome by the biotin synthesis in developing wt seeds.

Sufficiently high biotin levels are essential for fatty acid and TAG biosynthesis Acetyl-CoA carboxylase (ACCase), a biotin enzyme that catalyses the first and rate-limiting step in fatty acid biosynthesis (Nikolau et al., 2003), is active both in the endosperm and the embryo. Under conditions of biotin limitation (e.g. in biol or bio2 mutants), when biotin supply from the maternal tissue becomes increasingly important, sufficiently high biotin concen- trations in the endosperm are more easily warranted than in the embryo, as the endosperm is in direct contact with the maternal tissue. Only the surplus of biotin that is not used by biotin-binding proteins of the endosperm will be available to the embryo.

In fact, the importance of ACCase biotinylation for optimal fatty acid biosynthesis has been reported (Thelen and Ohlrogge, 2002). In these analyses, overexpression of BIOTIN CARBOXYL CARRIER PROTEIN ISOFORM 2 (BCCP2) under the seed-specific napin promoter resulted in a 10-fold increase in BCCP2 protein levels. However, 60% of the BCCP2 protein was not biotinylated (= apo-BCCP2) and integration of apo-BCCP2 with another ACCase subunit resulted in a 65% reduction in ACCase activity. Earlier analyses suggested that the amount of free biotin is not sufficient to allow complete biotinylation of the additional BCCP2 protein (Chapman-Smith et al., 1994; Thelen et al., 2001 ).

Example 1 underlines the importance of sufficiently high biotin concentrations for optimal TAG formation in Arabidopsis embryos. In fact, the biotin-depleted seeds of bio2.1/suc5.5 (Figure 4) look empty, have a wrinkled appearance, and resemble seeds of the low-seed- oil mutant wrinkledl (writ, Focks and Benning, 1998).

In line with these results, data shown in Example 1 suggest that fatty acid biosynthesis and ACCase activity are strongly affected by changes in the availability of biotin. Moreover, the data suggest that the concentration of biotin is adjusted to the specific needs of an organ under different developmental conditions. Besides biotin biosynthesis, biotin supply from adjacent tissues is an alternative mechanism to adjust cellular biotin concentrations. The results presented in Example 1 demonstrate that SUC5 is responsible for the supply of biotin to the endosperm and the embryo under conditions of biotin limitation.

The present invention relates to a polynucleotide comprising a nucleic acid sequences selected from the group consisting of:

(a) a nucleic acid sequence as shown in SEQ ID NO: 1 ;

(b) a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2;

(c) a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence of (a) or (b), wherein said nucleic acid sequence encodes a polypeptide or biologically active portion thereof having sucrose transporter 5 activity ; and

(d) a nucleic acid sequence being a fragment of any one of (a) to (c), wherein said frag- ment encodes a polypeptide or biologically active portion thereof having sucrose transporter 5 activity. The term "polynucleotide" as used in accordance with the present invention relates to a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide being a biotin and sucrose transporter, i.e. a polypeptide capable of transporting biotin and sucrose through a membrane. The sucrose transporter 5 polypeptides encoded by the polynucleo- tides of the present invention shall be capable of increasing the amount of seed storage compounds, preferably, fatty acids or lipids, when present in plant seeds. The polypeptides encoded by the polynucleotide of the present invention are also referred to as SUC 5 protein herein below. Suitable assays for measuring the activities mentioned before are described in the accompanying Examples.

Preferably, the polynucleotide of the present invention upon expression in the seed of a transgenic plant is capable of significantly increasing the amount by weight of at least one seed storage compound. More preferably, such an increase as referred to in accordance with the present invention is an increase of the amount by weight of at least 1 , 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 % as compared to a control. Whether an increase is significant can be determined by statistical tests well known in the art including, e.g., Student^'s t-test. The percent increase rates of a seed storage compound are, preferably, determined compared to an empty vector control. An empty vector control is a transgenic plant, which has been transformed with the same vector or construct as a transgenic plant according to the present invention except for such a vector or construct is lacking the polynucleotide of the present invention. Alternatively, an untreated plant (i.e. a plant which has not been genetically manipulated) or a wildtype regenerate from the in vitro culture may be used as a control.

A polynucleotide encoding a polypeptide having a biological activity as specified above has been obtained in accordance with the present invention from Arabidopsis thaliana. The corresponding polynucleotides, preferably, comprises the nucleic acid sequence shown in SEQ ID NO: 1 encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. It is to be understood that a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 may be also encoded due to the degenerated genetic code by other polynucleotides as well.

Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Said variants may represent orthologs, paralogs or other homologs of the polynucleotide of the present invention.

The polynucleotide variants, preferably, also comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid se- quences shown in SEQ ID NO: 1 by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having a biological activity as specified above. Variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecu- lar Biology, John Wiley & Sons, N. Y. (1989), 6.3.1 -6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 χ sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 χ SSC, 0.1 % SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under "standard hybridization conditions" the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 χ SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for

DNA:DNA hybrids are, preferably, 0.1 χ SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA:RNA hybrids are, preferably, 0.1 χ SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled work- er knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991 , "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants are obtainable by PCR- based techniques such as mixed oligonucleotide primer- based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present invention. Conserved domains of the polypeptide of the present invention may be identified by a sequence comparison of the nucleic acid sequences of the polynucleotides or the ami- no acid sequences of the polypeptides of the present invention. Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from bacteria, fungi, plants or animals may be used. Further, variants include polynucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequences shown in SEQ ID NO: 1 retaining a biological activity as specified above. Moreover, also encompassed are polynucleotides which comprise nucleic acid sequences encoding amino acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences shown in SEQ ID NO: 2 wherein the polypeptide comprising the amino acid sequence retains a biological activity as specified above. The percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit (Needle- man and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981 ))), which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 (1991 )], are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, us- ing the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences can be also determined using the Vector NTI 7.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814). A gap- opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap-opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide sequence is equivalent to an uracil nucleotide. A polynucleotide comprising a fragment of any of the aforementioned nucleic acid sequences is also encompassed as a polynucleotide of the present invention. The fragment shall encode a polypeptide which still has a biological activity as specified above. Accordingly, the polypeptide may comprise or consist of the domains of the polypeptide of the present invention conferring the said biological activity. A fragment as meant herein, preferably, comprises at least 20, at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of any one of the aforementioned nucleic acid sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any one of the aforementioned amino acid sequences.

The variant polynucleotides or fragments referred to above, preferably, encode polypeptides retaining at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the sucrose transporter 5 activity exhibited by the polypeptide shown in SEQ ID NO: 2. The activity may be tested as described in the accompanying Examples. The polynucleotides of the present invention either essentially consist of the aforementioned nucleic acid sequences or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well. Preferably, the polynucleotide of the present invention may comprise in addition to an open reading frame further un- translated sequence at the 3' and at the 5' terminus of the coding gene region: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5' terminus of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' terminus of the coding gene region. Furthermore, the polynucleotides of the present invention may encode fusion proteins wherein one partner of the fusion protein is a polypeptide being encoded by a nucleic acid sequence recited above. Such fusion proteins may comprise as additional part other enzymes of the fatty acid or lipid biosynthesis pathways, polypeptides for monitoring expression (e.g., green, yellow, blue or red fluorescent proteins, alkaline phosphatase and the like) or so called "tags" which may serve as a detectable marker or as an auxiliary measure for purification purposes. Tags for the different purposes are well known in the art and comprise FLAG-tags, 6-histidine-tags, MYC-tags and the like.

Variant polynucleotides as referred to in accordance with the present invention may be ob- tained by various natural as well as artificial sources. For example, polynucleotides may be obtained by in vitro and in vivo mutagenesis approaches using the above mentioned mentioned specific polynucleotides as a basis. Moreover, polynucleotids being homologs or orthologs may be obtained from various animal, plant, bacteria or fungus species. Paralogs may be identified from Arabidopsis thaliana.

The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context such as a gene locus) or in genetically modified or exogenously (i.e. artificially) manipulated form. An isolated polynucleotide can, for example, comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. The polynucleotide, preferably, is double or single stranded DNA including cDNA or RNA. The term encompasses single- as well as double-stranded polynucleotides. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides.

The polynucleotide encoding a polypeptide having a biological activity as specified encompassed by the present invention is also, preferably, a polynucleotide having a nucleic acid sequence which has been adapted to the specific codon- usage of the organism, e.g., the plant species, in which the polynucleotide shall be expressed (i.e. the target organism). This is, in general, achieved by changing the codons of a nucleic acid sequence obtained from a first organism (i.e. the donor organism) encoding a given amino acid sequence into the codons normally used by the target organism whereby the amino acid sequence is retained. It is in principle acknowleged that the genetic code is redundant (i.e. degenerated). Specifical- ly, 61 codons are used to encode only 20 amino acids. Thus, a majority of the 20 amino acids will be encoded by more than one codon. The codons for the amino acids are well known in the art and are universal to all organisms. However, among the different codons which may be used to encode a given amino acid, each organism may preferably use certain codons. The presence of rarely used codons in a nucleic acid sequence will result a depletion of the respective tRNA pools and, thereby, lower the translation efficiency. Thus, it may be advantageous to provide a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as referred to above wherein said nucleic acid sequence is optimized for expression in the target organism with respect to the codon usage. In order to optimize the codon usage for a target organism, a plurality of known genes from the said organism may be investigated for the most commonly used codons encoding the amino acids. In a subsequent step, the codons of a nuclei acid sequence from the donor organism will be optimized by replacing the codons in the donor sequence by the codons most commonly used by the target organism for encoding the same amino acids. It is to be understood that if the same codon is used preferably by both organisms, no replacement will be necessary. For various target organisms, tables with the preferred codon usages are already known in the art; see e.g., http://www.kazusa.or.jp/Kodon/E.html. Moreover, computer programs exist for the optimization, e.g., the Leto software, version 1.0 (Entelechon GmbH, Germany) or the GeneOptimizer (Geneart AG, Germany). For the optimization of a nucleic acid sequence, several criteria may be taken into account. For example, for a given amino acid, always the most commonly used codon may be selected for each codon to be exchanged. Alternatively, the codons used by the target organism may replace those in a donor sequence according to their naturally frequency. Accordingly, at some positions even less commonly used codons of the target organism will appear in the optimized nucleic acid sequence. The distribution of the different replacement codons of the target organism to the donor nucleic acid sequence may be randomly. Preferred target organisms in accordance with the present invention are soybean or canola (Brassica) species. Preferably, the polynucleotide of the present invention has an optimized nucleic acid for codon usage in the envisaged target organism wherein at least 20%, at least 40%, at least 60%, at least 80% or all of the relevant codons are adapted.

It has been found in the studies underlying the present invention that the polypeptides being encoded by the polynucleotides of the present invention is a sucrose transporter 5 polypeptide involved in the regulation of seed storage compounds. Moreover, the polypeptides encoded by the polynucleotides of the present invention are, advantageously, capable of in- creasing the amount of seed storage compounds in plants significantly. Thus, the polynucleotides of the present invention are, in principle, useful for the enrichment and synthesis of seed storage compounds such as fatty acids or lipids. Moreover, they may be used to generate transgenic plants or seeds thereof having a modified, preferably increased, amount of seed storage compounds. Such transgenic plants or seeds may be used for the manufacture of seed oil or other lipid and/or fatty acid containing compositions.

Further, the present invention relates to vector comprising the polynucleotide of the present invention. Preferably, the vector is an expression vector.

The term "vector", preferably, encompasses phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous recombination or heterologous insertion as described in detail below. The vector encompassing the polynucleotides of the pre- sent invention, preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. If introduced into a host cell, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion, see below. Vectors can be introduced into prokaryotic or eukary- otic cells via conventional transformation or transfection techniques. An "expression vector" according to the present invention is characterized in that it comprises an expression control sequence such as promoter and/or enhancer sequence operatively linked to the polynucleotide of the present invention. Preferred vectors, expression vectors and transfor- mation or transfection techniques are specified elsewhere in this specification in detail.

Furthermore, the present invention encompasses a host cell comprising the polynucleotide or vector of the present invention. Host cells are primary cells or cell lines derived from multicellular organisms such as plants or animals. Furthermore, host cells encompass prokaryotic or eukaryotic single cell organisms (also referred to as microorganisms), e.g. bacteria or fungi including yeast or bacteria. Primary cells or cell lines to be used as host cells in accordance with the present invention may be derived from the multicellular organisms, preferably from plants. Specifically pre- ferred host cells, microorganisms or multicellular organism from which host cells may be obtained are disclosed below.

The polynucleotides or vectors of the present invention may be incorporated into a host cell or a cell of a transgenic non-human organism by heterologous insertion or homologous re- combination. "Heterologous" as used in the context of the present invention refers to a polynucleotide which is inserted (e.g., by ligation) or is manipulated to become inserted to a nucleic acid sequence context which does not naturally encompass the said polynucleotide, e.g., an artificial nucleic acid sequence in a genome of an organism. Thus, a heterologous polynucleotide is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous polynucle- otides encode proteins that are normally not produced by the cell expressing the said heterologous polynucleotide. An expression control sequence as used in a targeting construct or expression vector is considered to be "heterologous" in relation to another sequence (e.g., encoding a marker sequence or an agronomically relevant trait) if said two sequences are either not combined or operatively linked in a different way in their natural environment. Preferably, said sequences are not operatively linked in their natural environment (i.e. originate from different genes). Most preferably, said regulatory sequence is covalently joined (i.e. ligated) and adjacent to a nucleic acid to which it is not adjacent in its natural environment. "Homologous" as used in accordance with the present invention relates to the insertion of a polynucleotide in the sequence context in which the said polynucleotide naturally occurs. Usually, a heterologous polynucleotide is also incorporated into a cell by homologous recombination. To this end, the heterologous polynucleotide is flanked by nucleic acid sequences being homologous to a target sequence in the genome of a host cell or a non- human organism. Homologous recombination now occurs between the homologous sequences. However, as a result of the homologous recombination of the flanking sequences, the heterologous polynucleotide will be inserted, too. How to prepare suitable target constructs for homologous recombination and how to carry out the said homologous recombination is well known in the art.

Also provided in accordance with the present invention is a method for the manufacture of a polypeptide having sucrose transporter 5 activity comprising:

(a) expressing the polynucleotide of the present invention in a host cell; and

(b) obtaining the polypeptide encoded by said polynucleotide from the host cell.

The polypeptide may be obtained, for example, by all conventional purification techniques including affinity chromatography, size exclusion chromatography, high pressure liquid chromatography (HPLC) and precipitation techniques including antibody precipitation. It is to be understood that the method may - although preferred -not necessarily yield an essentially pure preparation of the polypeptide. It is to be understood that depending on the host cell which is used for the aforementioned method, the polypeptides produced thereby may become posttranslationally modified or processed otherwise.

The present invention, moreover, pertains to a polypeptide encoded by the polynucleotide of the present invention or which is obtainable by the aforementioned method of the present invention. The term "polypeptide" as used herein encompasses essentially purified polypeptides or polypeptide preparations comprising other proteins in addition. Further, the term also relates to the fusion proteins or polypeptide fragments being at least partially encoded by the polynucleotide of the present invention referred to above. Moreover, it includes chemically modified polypeptides. Such modifications may be artificial modifications or naturally occurring modifications such as phosphorylation, glycosylation, myristylation and the like. The terms "polypeptide", "peptide" or "protein" are used interchangeable throughout this specification. The polypeptide of the present invention shall exhibit the biological activities referred to above, i.e. it should be a sucrose transporter 5 and, more preferably, it shall be capable of increasing the amount of seed storage compounds, preferably, fatty acids or lipids, when present in plant seeds as referred to above. Most preferably, if present in plant seeds, the polypeptide shall be capable of significantly increasing the seed storage of lipids.

Encompassed by the present invention is, furthermore, an antibody which specifically rec- ognizes the polypeptide of the invention.

Antibodies against the polypeptides of the invention can be prepared by well known methods using a purified polypeptide according to the invention or a suitable fragment derived therefrom as an antigen. A fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Such fragments may be obtained either from the polypeptide of the invention by proteolytic digestion or may be a synthetic peptide. Preferably, the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human or humanized antibody or primatized, chimerized or fragment thereof. Also comprised as antibodies by the present invention are: a bispecific antibody, a synthetic antibody, an antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. The antibody of the present invention shall specifically bind (i.e. does significantly not cross react with other polypeptides or peptides) to the polypeptide of the invention. Specific binding can be tested by various well known techniques. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques originally described in Kohler and Milstein, Nature 256 (1975) 495, and Galfre, Meth. Enzymol. 73 (1981 ) 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. The antibodies can be used, for example, for the im- munoprecipitation, immunolocalization or purification (e.g., by affinity chromatography) of the polypeptides of the invention as well as for the monitoring of the presence of said variant polypeptides, for example, in recombinant organisms, and for the identification of compounds interacting with the proteins according to the invention. The present invention also relates to a transgenic non-human organism comprising the polynucleotide, the vector or the host cell of the present invention. Preferably, said non-human transgenic organism is a plant. The term "non-human transgenic organism", preferably, relates to a plant, an animal or a multicellular microorganism. The polynucleotide or vector may be present in the cytoplasm of the organism or may be incorporated into the genome either heterologous or by homologous recombination. Host cells, in particular those obtained from plants or animals, may be introduced into a developing embryo in order to obtain mosaic or chimeric organisms, i.e. non-human transgenic organisms comprising the host cells of the present invention. Preferably, the non-human transgenic organism expresses the polynucleotide of the present invention in order to produce the polypeptide in an amount resulting in a detectable sucrose transporter 5 activity due to the presence of the said polypeptide. Suitable transgenic organisms are, preferably, all those organisms which are capable of synthesizing fatty acids or lipids. Preferred organisms and methods for transgenesis are disclosed in detail below. A transgenic organism or tissue may comprise one or more transgenic cells. Preferably, the organism or tissue is substantially consisting of transgenic cells (i.e., more than 80%, preferably 90%, more preferably 95%, most preferably 99% of the cells in said organism or tissue are transgenic). The term "transgene" as used herein refers to any nucleic acid se- quence, which is introduced into the genome of a cell or which has been manipulated by experimental manipulations including techniques such as chimera- or genoplasty. Preferably, said sequence is resulting in a genome which is significantly different from the overall genome of an organism (e.g., said sequence, if endogenous to said organism, is introduced into a location different from its natural location, or its copy number is increased or de- creased). A transgene may comprise an endogenous polynucleotide (i.e. a polynucleotide having a nucleic acid sequence obtained from the same organism or host cell) or may be obtained from a different organism or hast cell, wherein said different organism is, preferably an organism of another species and the said different host cell is, preferably, a different microorganism, a host cell of a different origin or derived from a an organism of a different species.

Particularly preferred as a plant to be used in accordance with the present invention are oil producing plant species. Most preferably, the said plant is selected from the group consisting of canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, ta- getes, cotton, oil palm, coconut palm, flax, castor and peanut,

The present invention relates to a method for the manufacture of a lipid and/or a fatty acid comprising the steps of:

(a) cultivating the host cell or the transgenic non-human organism of the present invention under conditions allowing synthesis of the said lipid or fatty acid; and (b) obtaining the said lipid and/or fatty acid from the host cell or the transgenic non- human organism.

The term "lipid" and "fatty acid" as used herein refer, preferably, to those recited in Table 1 (for lipids) and Table 2 (for fatty acids), below. However, the terms, in principle, also encompass other lipids or fatty acids which can be obtained by the lipid metabolism in a host cell or an organism referred to in accordance with the present invention.

In a preferred embodiment of the aforementioned method of the present invention, the said lipid and/or fatty acids constitute seed oil.

Moreover, the present invention pertains to a method for the manufacture of a plant having a modified amount of a seed storage compound, preferably a lipid or a fatty acid, comprising the steps of:

(a) introducing the polynucleotide or the vector of the present invention into a plant cell; and

(b) generating a transgenic plant from the said plant cell, wherein the polypeptide encoded by the polynucleotide modifies the amount of the said seed storage compound in the transgenic plant.

The term "seed storage compound" as used herein, preferably, refers to compounds being a sugar or, more preferably, a lipid or a fatty acid. Preferably, the amount of said seed storage compound is significantly increased compared to a control, preferably an empty vector control as specified above. The increase is, more preferably, an increase in the amount by weight of at least 1 , 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 % as compared to a control.

It is to be understood that the polynucleotides or the vector referred to in accordance with the above method of the present invention may be introduced into the plant cell by any of the aforementioned insertion or recombination techniques.

The aforementioned method of the present invention may be also used to manufacture a plant having altered total oil content in its seeds or a plant having altered total seed oil content and/or altered levels of seed storage compounds in its seeds. Such plants are suitable sources for seed oil and may be used for the large scale manufacture thereof.

Further preferred embodiments of the compounds, methods and uses according to the present invention are described in the following. Moreover, the terms used above will be explained in more detail. The present invention provides novel isolated nucleic acid and amino acid sequences, i.e., the polynucleotides and polypeptides of the present invention, associated with the metabolism of seed storage compounds in plants. Preferably provided is a polynucleotide comprising a nucleic acid from Arabidopsis thaliana encoding the sucrose transporter s polypeptide of the present invention, i.e. a Lipid Metabolism Protein (SUC 5 protein), or a portion thereof. These sequences may be used to modify or increase lipids and fatty acids, cofactors and enzymes in microorganisms and plants. Arabidopsis plants are known to produce considerable amounts of fatty acids like linoleic and linolenic acid (see, e.g., Table 2) and for their close similarity in many aspects (gene homology etc.) to the oil crop plant Brassica. Therefore, nucleic acid molecules originating from a plant like Brassica napus or related organisms including Arabidopsis (i.e. the polynucleotides of the present invention) are especially suited to modify the lipid and fatty acid metabolism in a host such as the host cells or transgenic non-human organisms of the present invention, especially in microorganisms and plants. Furthermore, nucleic acids from the plant Arabidopsis thaliana or related organisms can be used to identify those DNA sequences and enzymes in other species, which are useful to modify the biosynthesis of precursor molecules of fatty acids in the respective organisms.

The present invention further provides an isolated nucleic acid comprising a fragment of at least 15 nucleotides of a polynucleotide of the present invention, preferably, a polynucleotide comprising a nucleic acid from a plant encoding the polypeptides of the present invention.

The present invention, thus, also encompasses an oligonucleotide which specifically binds to the polynucleotides of the present invention. Binding as meant in this context refers to hybridization by Watson-Crick base pairing discussed elsewhere in the specification in detail. An oligonucleotide as used herein has a length of at most 100, at most 50, at most 40, at most 30 or at most 20 nucleotides in length which are complementary to the nucleic acid sequence of the polynucleotides of the present invention. The sequence of the oligonucleotide is, preferably, selected so that a perfect match by Watson-Crick base pairing will be obtained. The oligonucleotides of the present invention may be suitable as primers for PCR- based amplification techniques.

Also provided by the present invention are polypeptides encoded by the nucleic acids, and heterologous polypeptides comprising polypeptides encoded by the nucleic acids, and antibodies to those polypeptides. Additionally, the present invention relates to and provides the use of the polynucleotides of the present invention in the production of transgenic plants having a modified level or com- position of a seed storage compound. In regard to an altered composition, the present invention can be used to, for example, increase the percentage of oleic acid relative to other plant oils. A method of producing a transgenic plant with a modified level or composition of a seed storage compound includes the steps of transforming a plant cell with an expression vector comprising a polynucleotide of the present invention, and generating a plant with a modified level or composition of the seed storage compound from the plant cell. In a preferred embodiment, the plant is an oil producing species selected from the group consisting of canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut, for example.

According to the present invention, the compositions and methods described herein can be used to alter the composition of a SUC 5 protein in a transgenic plant and to increase or decrease the level of a SUC 5 protein in a transgenic plant comprising increasing or decreasing the expression of a SUC protein nucleic acid in the plant. Increased or decreased expression of the SUC 5 protein nucleic acid can be achieved through transgenic overex- pression, co-suppression approaches, antisense approaches, and in vivo mutagenesis of the SUC 5 protein nucleic acid or micro-RNA based techniques. The present invention can also be used to increase or decrease the level of a lipid in a seed oil, or to increase or decrease the level of a fatty acid in seed oil.

More specifically, the present invention includes and provides a method for altering (increasing or decreasing or changing the specific profile) of the total oil content in a seeds comprising: Transforming a plant with a nucleic acid construct that comprises as operably linked components, a promoter and nucleic acid sequences capable of modulating the level of the polynucleotides or polypeptides of the present invention, and growing the plant. Furthermore, the present invention includes and provides a method for altering (increasing or decreasing) the level of eicosenoic acid in a seed comprising: transforming a plant with a nucleic acid construct that comprises as operably linked components, a promoter, a struc- tual nucleic acid sequence capable of altering (increasing or decreasing) the level of ei- cosenoic acid, and growing the plant

Also included herein is a seed produced by a transgenic plant transformed by a polynucleotide of the present invention, wherein the seed contains the said polynucleotide and wherein the plant is true breeding for a modified level of a seed storage compound. The present invention additionally includes seed oil produced by the aforementioned seed.

Further provided by the present invention are vectors comprising a polynucleotide of the present invention, host cells containing the vectors, and descendent plant materials produced by transforming a plant cell with the nucleic acids and/or vectors. According to the present invention, the compounds, compositions, and methods described herein can be used to increase or decrease the relative percentages of a lipid in a seed oil, increase or decrease the level of a lipid in a seed oil, or to increase or decrease the level of a fatty acid in a seed oil, or to increase or decrease the level of a starch or other carbohy- drate in a seed or plant. The manipulations described herein can also be used to improve seed germination and growth of the young seedlings and plants and to enhance plant yield of seed storage compounds.

It is further provided a method of producing a higher or lower than normal or typical level of storage compound in a transgenic plant expressing a polynucleotide of the present invention from Arabisopsis thalaiana in the transgenic plant, wherein the transgenic plant is Ara- bidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Zea mays, Triticum aestivum, Helianthus anuus or Beta vulgaris or a species different from Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Zea mays, Triticum aestivum, Helianthus anuus or Beta vulgaris. Also included herein are compositions and methods of the modification of the efficiency of production of a seed storage compound. As used herein, where the phrase Arabidopsis thaliana, Brassica napus, Glycine max, Oryza sativa, Zea mays, Triticum aestivum, Helianthus anuus or Beta vulgaris is used, this also means Arabidopsis thaliana and/or Brassica napus and/or Glycine max and/or Oryza sativa and/or Triticum aestivum and/or Zea mays and/or Helianthus anuus and/or Beta vulgaris.

Accordingly, it is an object of the present invention to provide polynucleotides encoding a SUC 5 protein as well as the corresponding polypeptide from Arabidopsis thaliana as well as active fragments, analogs, and orthologs thereof. Those active fragments, analogs, and orthologs can also be from different plant species as one skilled in the art will appreciate that other plant species will also contain those or related nucleic acids.

It is another object of the present invention to provide transgenic plants having modified levels of seed storage compounds, and in particular, modified levels of a lipid, a fatty acid, or a sugar.

The polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have also uses that include modulating plant growth, and potentially plant yield, preferably increasing plant growth under adverse conditions (drought, cold, light, UV). In addition, antagonists of the present invention may have uses that include modulating plant growth and/or yield, through preferably increasing plant growth and yield. In yet another embodiment, over-expression polypeptides of the present invention using a constitutive promoter may be useful for increasing plant yield under stress conditions (drought, light, cold, UV) by modulating light utilization efficiency. Moreover, polynucleotides and pol- ypeptides of the present invention will improve seed germination and seed dormancy and, hence, will improve plant growth and/or yield of seed storage compounds. The polynucleotides of the present invention may further comprise an operably linked promoter or partial promoter region. The promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter. The constitutive promoter can be, for example, the superpromoter (Ni et al., Plant J. 7:661 -676, 1995; US5955646) or the PtxA promoter (WO 05/085450, Song H. et al. ). The tissue-specific promoter can be active in vegetative tissue or reproductive tissue. The tissue-specific promoter active in reproductive tissue can be a seed-specific promoter. The tissue-specific promoter active in vegetative tissue can be a root-specific, shoot-specific, meristem-specific, or leaf-specific promoter. The polynucleotides of the present invention can still further comprise a 5' non-translated sequence, 3' non-translated sequence, introns, or the combination thereof.

The present invention also provides a method for altering (increasing or decreasing) the number and/or size of one or more plant organs of a plant expressing a polynucleotide of the present invention, preferably, from Arabidopsis thaliana encoding a polypeptide of the present invention. More specifically, seed size and/or seed number and/or weight might be manipulated. Moreover, root length can be increased. Longer roots can alleviate not only the effects of water depletion from soil but also improve plant anchorage/standability, thus reducing lodging. Also, longer roots have the ability to cover a larger volume of soil and improve nutrient uptake. All of these advantages of altered root architecture have the po- tential to increase crop yield. Additionally, the number and size of leaves might be increased by the nucleic acid sequences provided in this application. This will have the advantage of improving photosynthetic light utilization efficiency by increasing photosynthetic light-capture capacity and photosynthetic efficiency. It is a further object of the present invention to provide methods for producing such aforementioned transgenic plants.

It is another object of the present invention to provide seeds and seed oils from such aforementioned transgenic plants.

Before the present compounds, compositions, methods and preferred embodiments thereof are disclosed and described in more detail, it is to be understood that this invention is not limited to specific polynucleotides, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell up to a plu- rality of cells can be utilized. The present invention is based, in part, on the isolation and characterization of nucleic acid molecules a sucrose 5 transporter from plants including Arabidopsis, canola (Brassica napus or Brassica oleracea) and other related crop species like maize, barley, linseed, sugar beet, or sunflower.

In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, provides an isolated nucleic acid from a plant (Arabidopsis thaliana) encoding a SUC 5 protein, or a portion thereof. One aspect of the invention pertains to an isolated nucleic acid molecule that encodes a SUC 5 polypeptide or a biologically active portion thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of a SUC 5 protein-encoding nucleic acid (e.g., SUC 5 protein DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of a gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded

DNA. An "isolated" nucleic acid molecule is one which is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is substantially free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism, from which the nucleic acid is derived. For example, in various embodiments, the isolated SUC 5 protein nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., a Brassica napus cell). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a nucleotide sequence of the polynucleotide of the present invention, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a Arabidopsis thaliana or Brassica napus SUC 5 protein cDNA can be isolated from an a Arabidopsis thaliana or Brassica napus library using all or portion of one of the sequences of the polynucleotide of the present invention as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence of SEQ ID NO: 1). For example, mRNA can be isolated from plant cells (e.g., by the guanidinium- thiocyanate extraction procedure of Chirgwin et al. 1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO: 1. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a SUC 5 protein nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid of the invention comprises one of the nucleotide sequences shown of the polynucleotide of the present invention. The sequence of SEQ ID NO: 1 corresponds to the Arabidopsis thaliana SUC 5 protein cDNA of the invention. These cDNAs comprise sequences encoding SUC 5 proteins (i.e., the "coding region", indicated in SEQ ID NO: 1 ), as well as 5' untranslated sequences and 3' untranslated sequences. Alternatively, the nucleic acid molecules can comprise only the coding region of any of the sequences in SEQ ID NO: 1 or can contain whole genomic fragments isolated from genomic DNA.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule, which is a complement of one of the nucleotide sequences shown in SEQ ID NO: 1 , or a portion thereof. A nucleic acid molecule which is complemen- tary to one of the nucleotide sequences shown in SEQ ID NO: 1 is one which is sufficiently complementary to one of the nucleotide sequences shown in SEQ ID NO: 1 such that it can hybridize to one of the nucleotide sequences shown in SEQ ID NO: 1 , thereby forming a stable duplex. In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleotide sequence shown in SEQ ID NO: 1 , or a portion thereof. Specific algorithms for the determination of the degree of identity are found elsewhere in this specification. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention com- prises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences shown in SEQ ID NO: 1 , or a portion thereof. These hybridization conditions include washing with a solution having a salt concentration of about 0.02 molar at pH 7 at about 60°C. Specific hybridization conditions are to be found elsewhere in this specification. Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the sequences in SEQ ID NO: 1 , for ex- ample a fragment, which can be used as a probe or primer or a fragment encoding a biologically active portion of a SUC 5 protein. The nucleotide sequences determined from the cloning of the SUC 5 protein gene from Arabidopsis thaliana allows for the generation of probes and primers designed for use in identifying and/or cloning SUC 5 protein homo- logues in other cell types and organisms, as well as SUC 5 protein homologues from other plants or related species. Therefore this invention also provides compounds comprising the nucleic acids disclosed herein, or fragments thereof. These compounds include the nucleic acids attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. The probe/primer typically comprises substantially purified oligo- nucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in SEQ ID NO: 1 , an anti-sense sequence of one of the sequences set forth in SEQ ID NO: 1 , or naturally occurring mutants thereof. Primers based on a nucleotide se- quence of SEQ ID NO: 1 can be used in PCR reactions to clone SUC 5 protein homologues. Probes based on the SUC 5 protein nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express a SUC 5 protein, such as by measuring a level of a SUC 5 protein-encoding nucleic acid in a sample of cells, e.g., detecting SUC 5 protein mRNA levels or determining whether a genomic SUC 5 protein gene has been mutated or deleted. In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid encoded by a sequence of SEQ ID NO: 2 such that the protein or portion thereof maintains the same or a similar function as the wild-type protein. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue, which has a simi- lar side chain as an amino acid residue in one of the ORFs of a sequence of SEQ ID NO: 2) amino acid residues to an amino acid sequence such that the protein or portion thereof is able to participate in the metabolism of compounds necessary for the production of seed storage compounds in plants, construction of cellular membranes in microorganisms or plants, or in the transport of molecules across these membranes. How to determine the de- gree of identical or equivalent amino acids between two sequences is set forth elsewhere in this specification in detail. Transport proteins, such as the sucrose transporter 5, play a role in the biosynthesis of seed storage compounds. Examples of such activities are described herein. Examples of SUC 5 protein-encoding nucleic acid sequences are set forth in SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 and 79.

As altered or increased sugar and/or fatty acid production is a general trait wished to be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, canola, manihot, pepper, sunflower, sugar beet and tagetes, solana- ceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut) and perennial grasses and forage crops, these crop plants are also preferred target plants for genetic engineering as one further embodiment of the present invention.

Portions of proteins encoded by the SUC 5 protein nucleic acid molecules of the invention are preferably biologically active portions of one of the SUC 5 proteins. As used herein, the term "biologically active portion of a SUC 5 protein" is intended to include a portion, e.g., a domain/ motif, of a SUC 5 protein that has an activity as set forth above. To determine whether a SUC 5 protein or a biologically active portion thereof can participate in the metabolism of compounds necessary for the production of seed storage compounds and cellu- lar membranes, an assay of enzymatic activity may be performed. Such assay methods are well known to those skilled in the art, and as described in Example 14 of the Exemplification.

Biologically active portions of a SUC 5 protein include peptides comprising amino acid sequences derived from the amino acid sequence of a SUC 5 protein (e.g., an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 or the amino acid sequence of a protein homologous to a SUC 5 protein, which include fewer amino acids than a full length SUC 5 protein or the full length protein which is homologous to a SUC 5 protein) and exhibit at least one activity of a SUC 5 protein. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of a SUC 5 protein and in accordance with the present invention, preferably, the sucrose transporter 5 activity. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of a SUC 5 pro- tein include one or more selected domains/motifs or portions thereof having biological activity. Additional nucleic acid fragments encoding biologically active portions of a SUC 5 protein can be prepared by isolating a portion of one of the sequences, expressing the encoded portion of the SUC 5 protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the SUC 5 protein or peptide. The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO: 1 (and portions thereof) due to degeneracy of the genetic code and thus encode the same SUC 5 protein as that encoded by the nucleotide sequences shown in SEQ ID NO: 1. In a further embodiment, the nucleic acid molecule of the invention encodes a full length protein which is substantially homologous to an amino acid sequence of a polypeptide encoded by an open reading frame shown in SEQ ID NO: 1. In one embodiment, the full-length nucleic acid or protein or fragment of the nucleic acid or protein is from Arabidopsis thaliana. In addition to the SUC 5 protein nucleotide sequences shown in SEQ ID NO:1 , it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of SUC 5 proteins may exist within a population (e.g., the Arabidopsis thaliana population). Such genetic polymorphism in the SUC 5 protein gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a SUC 5 protein, preferably, an Arabidopsis thaliana SUC 5 protein. Such natural variations can typically result in 1 -40% variance in the nucleotide sequence of the SUC 5 protein gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in SUC 5 protein that are the result of natural variation and that do not alter the functional activity of SUC 5 proteins are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural variants and non- Arabidopsis thaliana orthologs of the SUC 5 protein cDNA of the invention can be isolated based on their homology SUC 5 protein nucleic acid disclosed herein using the cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. As used herein, the term

"orthologs" refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode proteins having the same or similar functions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989: 6.3.1 -6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural Arabidopsis thaliana SUC 5 protein. In addition to naturally-occurring variants of the SUC 5 protein sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO: 1 , thereby leading to changes in the amino acid sequence of the encoded SUC 5 protein, without altering the functional ability of the SUC 5 protein. For example, nucleotide substitutions leading to amino acid substitutions at "nonessential" amino acid residues can be made in a sequence of SEQ ID NO: 2. A "non- essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the SUC 5 proteins (SEQ ID NO: 2) without altering the activity of said SUC 5 protein, whereas an "essential" amino acid residue is required for SUC 5 protein activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having SUC 5 protein activity) may not be essential for activity and thus are likely to be amenable to alteration without altering SUC 5 protein activity.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding SUC 5 proteins that contain changes in amino acid residues that are not essential for SUC 5 protein activity. Such SUC 5 proteins differ in amino acid sequence from a sequence yet retain at least one of the SUC 5 protein activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and has one or more activities set forth above. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79, more preferably at least about 60-70% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the sequences encod- ed by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79.

To determine the percent homology of two amino acid sequences (e.g., one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the sequence selected from the polypeptide encoded by a nucleic acid of SEQ ID NO: 1 ), then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = numbers of identical positions/total numbers of positions x 100). An isolated nucleic acid molecule encoding a SUC 5 protein homologous to a protein sequence encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 by stand- ard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted nonessential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in a SUC 5 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a SUC 5 protein coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a SUC 5 protein activity de- scribed herein to identify mutants that retain SUC 5 protein activity. Following mutagenesis of one of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Examples 1 1 -13 of the Exemplification).

SUC 5 proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described herein), and the SUC 5 protein is expressed in the host cell. The SUC 5 protein can then be isolat- ed from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a SUC 5 protein or peptide thereof can be synthesized chemically using standard peptide synthesis techniques. Moreover, native SUC 5 protein can be isolated from cells, for example using an anti-SUC 5 protein antibody, which can be produced by standard techniques utilizing a SUC 5 protein or fragment there- of of this invention.

The invention also provides SUC 5 protein chimeric or fusion proteins. As used herein, a SUC 5 protein "chimeric protein" or "fusion protein" comprises a SUC 5 protein polypeptide operatively linked to a non-SUC 5 protein polypeptide. An "SUC 5 protein polypeptide" re- fers to a polypeptide having an amino acid sequence corresponding to a SUC 5 protein, whereas a "non-SUC 5 protein polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the SUC 5 protein, e.g., a protein which is different from the SUC 5 protein, and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the SUC 5 protein polypeptide and the non-SUC 5 protein polypeptide are fused to each other so that both sequences fulfil the proposed function attributed to the sequence used. The non-SUC 5 protein polypeptide can be fused to the N-terminus or C-terminus of the SUC 5 protein polypeptide. For example, in one embodiment, the fusion protein is a GST-SUC 5 protein (glutathione S-transferase) fusion protein in which the SUC 5 protein sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant SUC 5 protein s. In another embodiment, the fusion protein is a SUC 5 protein containing a heterologous signal sequence at its N- terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a SUC 5 protein can be increased through use of a heterologous signal sequence.

Preferably, a SUC 5 protein chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An SUC 5 protein-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SUC 5 protein.

In addition to the nucleic acid molecules encoding SUC 5 proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can be hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire SUC 5 protein coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a SUC 5 protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding SUC 5 protein. The term "noncoding region" refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding SUC 5 protein disclosed herein (e.g., the sequences set forth in SEQ ID NO:1 ), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of SUC 5 protein mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of SUC 5 protein mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SUC 5 protein mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense or sense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Ex- amples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylamino-methyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydro-uracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine, 1 -methyl-guanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl-cytosine, N-6-adenine, 7- methylguanine, 5-methyl-aminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyl-uracil, 5-methoxyuracil, 2-methylthio-N-6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyace- tic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diamino-purine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

In another variation of the antisense technology, a double-strand interfering RNA construct can be used to cause a down-regulation of the SUC 5 protein mRNA level and SUC 5 pro- tein activity in transgenic plants. This requires transforming the plants with a chimeric construct containing a portion of the SUC 5 protein sequence in the sense orientation fused to the antisense sequence of the same portion of the SUC 5 protein sequence. A DNA linker region of variable length can be used to separate the sense and antisense fragments of SUC 5 protein sequences in the construct.

The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a SUC 5 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complement to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic including plant promoters are preferred. In yet another embodiment, the antisense nucleic acid molecule of the invention is anomehc nucleic acid molecule. An anomehc nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual units, the strands run parallel to each other (Gaultier et al. 1987, Nucleic Acids Res. 15:6625-6641 ). The antisense nucleic acid molecule can also comprise a 2'-o-methyl-ribonucleotide (Inoue et al. 1987, Nucleic Acids Res. 15:6131 -6148) or a chimeric RNA-DNA analogue (Inoue et al. 1987, FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribo- zymes are catalytic RNA molecules with ribonuclease activity, which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff & Gerlach 1988, Nature 334:585-591 )) can be used to catalytically cleave SUC 5 protein mRNA transcripts to thereby inhibit translation of SUC 5 protein mRNA. A ribozyme having specificity for a SUC 5 protein -encoding nucleic acid can be designed based upon the nucleotide sequence of a SUC 5 protein cDNA disclosed herein or on the basis of a heterologous sequence to be isolated according to methods taught in this invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a SUC 5 protein-encoding mRNA (see, e.g., Cech et al., U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No. 5,1 16,742). Alternatively, SUC 5 protein mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel, D. & Szostak J.W. 1993, Science 261 :1411 -1418). Alternatively, SUC 5 protein gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a SUC 5 protein nucleotide sequence (e.g., a SUC 5 protein promoter and/or enhancers) to form triple helical structures that prevent transcription of a SUC 5 protein gene in target cells (See generally, Helene C. 1991 , Anticancer Drug Des. 6:569-84; Helene C. et al. 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. 1992, Bioassays 14:807-15).

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a SUC 5 protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicat- ed along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used inter-changeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence^) in a manner which allows for expression of the nucleotide sequence and both sequences are fused to each other so that each fulfils its proposed function (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton, Florida, eds.: Glick & Thompson, Chapter 7, 89-108 including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., SUC 5 proteins, mutant forms of SUC 5 proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of SUC 5 proteins in prokaryotic or eukaryotic cells. For example, SUC 5 protein genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos M.A. et al. 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J.J. et al. 1991 , Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, Bennet & Lasure, eds., p. 396-428:Academic Press: an Diego; and van den Hondel & Punt 1991 , Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1 -28, Cambridge University Press: Cambridge), algae (Fal- ciatore et al. 1999, Marine Biotechnology 1 :239-251 ), ciliates of the types: Holotrichia, Perit- richia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma,

Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especially of the genus Stylonychia lemnae with vectors following a transformation method as described in WO 98/01572 and multicellular plant cells (see Schmidt & Willmitzer 1988, High efficiency Agrobacterium tumefaciens-med\ated transformation of Arabidopsis thaliana leaf and cotyledon plants, Plant Cell Rep.:583-586); Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Florida, chapter 6/7, S.71 -1 19 (1993); White, Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds.: Kung and Wu, Academic Press 1993, 128-43; Potrykus 1991 , Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225 (and references cited therein) or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in En- zymology 185, Academic Press, San Diego, CA 1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve one or more of the following purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson 1988, Gene 67:31 -40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the SUC 5 protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant SUC 5 protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. 1988, Gene 69:301 -315) and pET 1 1d (Studier et al. 1990, Gene Expression Technolo- gy: Methods in Enzymology 185, Academic Press, San Diego, California 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11 d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA pol- ymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or

HMS174 (DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein

(Gottesman S. 1990, Gene Expression Technology: Methods in Enzymology 185:119-128, Academic Press, San Diego, California). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression (Wada et al. 1992, Nucleic Acids Res. 20:211 1 -2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the SUC 5 protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSed (Baldari et al. 1987, Embo J. 6:229-234), pMFa (Kurjan & Herskowitz 1982, Cell 30:933-943), pJRY88 (Schultz et al. 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel & Punt 1991 , "Gene transfer systems and vector development for filamentous fungi," in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1 -28, Cambridge University Press: Cambridge.

Alternatively, the SUC 5 proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow & Summers 1989, Virology 170:31 -39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed 1987, Nature 329:840) and pMT2PC (Kaufman et al. 1987, EMBO J. 6:187- 195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sam- brook, Fritsh and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. In another embodiment, the SUC 5 proteins of the invention may be expressed in unicellular plant cells (such as algae, see Falciatore et al. (1999, Marine Biotechnology 1 :239- 251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in:

Becker, Kemper, Schell and Masterson (1992 "New plant binary vectors with selectable markers located proximal to the left border," Plant Mol. Biol. 20:1 195-1 197) and Bevan (1984 "Binary Agrobacterium vectors for plant transformation," Nucleic Acids Res. 12:871 1 - 8721 ; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds.: Kung und R. Wu, Academic Press, 1993, S. 15-38).

A plant expression cassette preferably contains regulatory sequences capable to drive gene expression in plant cells, and which are operably linked so that each sequence can fulfil its function such as termination of transcription, including polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al. 1984, EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptional levels a plant expres- sion cassette preferably contains other operably linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al. 1987, Nucleic Acids Res. 15:8693-871 1 ). Plant gene expression has to be operably linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Preferred are promoters driving constitutive expression (Benfey et al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CAMV (Franck et al. 1980, Cell 21 :285-294), the 19S CaMV (see also US 5,352,605 and WO 84/02913) or plant promoters like those from Rubisco small subunit described in US 4,962,028. Even more preferred are seed-specific promoters driving expression of SUC 5 protein proteins during all or selected stages of seed development. Seed-specific plant promoters are known to those of ordinary skill in the art and are identified and characterized using seed-specific mRNA libraries and expression profiling techniques. Seed-specific promoters include the napin-gene promoter from rapeseed (US 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al. 1991 , Mol. Gen. Genetics 225:459-67), the oleosin-promoter from Arabidopsis (WO 98/45461 ), the phaseolin- promoter from Phaseolus vulgaris (US 5,504,200), the Bce4-promoter from Brassica (W091 13980) or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant J. 2:233- 239) as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice etc. Suitable promoters to note are the Ipt2 or Ipt1 -gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (pro- moters from the barley hordein-gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the Sorghum kasirin-gene, and the rye secalin gene). Plant gene expression can also be facilitated via an inducible promoter (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is desired in a time specific manner. Examples for such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404), and an ethanol inducible promoter (WO 93/21334).

Promoters responding to biotic or abiotic stress conditions are also suitable promoters such as the pathogen inducible PRP1-gene promoter (Ward et al., 1993, Plant. Mol. Biol. 22:361 - 366), the heat inducible hsp80-promoter from tomato (US 5,187,267), cold inducible alpha- amylase promoter from potato (WO 96/12814) or the wound-inducible pinll-promoter (EP 375091).

Other preferred sequences for use in plant gene expression cassettes are targeting- sequences necessary to direct the gene-product in its appropriate cell compartment (for review see Kermode 1996, Crit. Rev. Plant Sci. 15:285-423 and references cited therein) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chro- moplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells. Also especially suited are promoters that confer plastid-specific gene expression, as plastids are the compartment where precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters such as the viral RNA-polymerase promoter are described in WO 95/16783 and WO 97/06250 and the clpP-promoter from Arabidopsis described in WO 99/46394.

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to SUC 5 protein mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the anti- sense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (1986, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 ) and Mol et al. (1990, FEBS Lett. 268:427-430).

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokar- yotic or eukaryotic cell. For example, a SUC 5 protein can be expressed in bacterial cells, insect cells, fungal cells, mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, or plant cells. Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection," "conjugation" and "transduction" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and other laboratory manuals, such as Methods in Molecular Biology 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, New Jersey.

For stable transfection of mammalian and plant cells, it is known that, depending upon the used expression vector and transfection technique, only a small fraction of cells may inte- grate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, kanamycin, and methotrexate or in plants that confer resistance towards an herbicide such as glyphosate or glufosinate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a SUC 5 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of a SUC 5 protein gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the SUC 5 protein gene. Preferably, this SUC 5 protein gene is an Arabidopsis thaliana or Brassica napus SUC 5 protein gene, but it can be a homologue from a related plant or even from a mammalian, yeast, or insect source. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous SUC 5 protein gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a knock-out vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous SUC 5 protein gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous SUC 5 protein). To create a point mutation via homologous recombination, DNA- RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al. 1999, Nucleic Acids Res. 27:1323-1330 and Kmiec 1999, American Scientist 87:240-247). Ho- mologous recombination procedures in Arabidopsis thaliana or other crops are also well known in the art and are contemplated for use herein.

In a homologous recombination vector, the altered portion of the SUC 5 protein gene is flanked at its 5' and 3' ends by additional nucleic acid of the SUC 5 protein gene to allow for homologous recombination to occur between the exogenous SUC 5 protein gene carried by the vector and an endogenous SUC 5 protein gene in a microorganism or plant. The additional flanking SUC 5 protein nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas & Capecchi 1987, Cell 51 :503, for a description of homologous recombination vectors). The vector is introduced into a microorganism or plant cell (e.g., via polyethylenegly- col mediated DNA). Cells in which the introduced SUC 5 protein gene has homologously recombined with the endogenous SUC 5 protein gene are selected using art-known techniques.

In another embodiment, recombinant microorganisms can be produced which contain selected systems, which allow for regulated expression of the introduced gene. For example, inclusion of a SUC 5 protein gene on a vector placing it under control of the lac operon permits expression of the SUC 5 protein gene only in the presence of IPTG. Such regulatory systems are well known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture can be used to produce (i.e., express) a SUC 5 protein. Accordingly, the invention further provides methods for producing SUC 5 proteins using the host cells of the invention. In one embod- iment, the method comprises culturing a host cell of the invention (into which a recombinant expression vector encoding a SUC 5 protein has been introduced, or which contains a wild- type or altered SUC 5 protein gene in its genome) in a suitable medium until SUC 5 protein is produced. In another embodiment, the method further comprises isolating SUC 5 proteins from the medium or the host cell. Another aspect of the invention pertains to isolated SUC 5 proteins SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 ,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80 and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of SUC 5 protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of SUC 5 protein having less than about 30% (by dry weight) of non-SUC 5 protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-SUC 5 protein, still more preferably less than about 10% of non-SUC 5 protein, and most preferably less than about 5% non-SUC 5 protein. When the SUC 5 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of SUC 5 protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes prepara- tions of SUC 5 protein having less than about 30% (by dry weight) of chemical precursors or non-SUC 5 protein chemicals, more preferably less than about 20% chemical precursors or non-SUC 5 protein chemicals, still more preferably less than about 10% chemical precursors or non-SUC 5 protein chemicals, and most preferably less than about 5% chemical precursors or non-SUC 5 protein chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the SUC 5 protein is derived. Typically, such proteins are produced by recombinant expression of, for example, an Arabidopsis thaliana or Brassica napus SUC 5 protein in other plants than Arabidopsis thaliana or Brassica napus or microorganisms, algae or fungi.

An isolated SUC 5 protein or a portion thereof of the invention can participate in the metabolism of compounds necessary for the production of seed storage compounds in Brassica napus or of cellular membranes, or has one or more of the activities set forth above. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 such that the protein or portion thereof maintains its sucrose transporter 5 activity. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, a SUC 5 protein of the invention has an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1. In yet another preferred embodiment, the SUC 5 protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1. In still another preferred embodiment, the SUC 5 protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50- 60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90- 95%, and even more preferably at least about 96%, 97%, 98%, 99% or more homologous to one of the amino acid sequences encoded by a nucleic acid of SEQ ID NO: 1. The preferred SUC 5 proteins of the present invention also preferably possess at least one of the SUC 5 protein activities described herein. For example, a preferred SUC 5 protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1 , and which has one or more of the activities set forth above.

In other embodiments, the SUC 5 protein is substantially homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1 and retains the functional activity of the protein of one of the sequences encoded by a nucleic acid of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 or 79 yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail above. Accordingly, in another embodiment, the SUC 5 protein is a protein which comprises an amino acid sequence which is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence and which has at least one of the SUC 5 protein activities SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 ,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80 described herein. In another embodiment, the invention pertains to a full Arabidopsis thali- ana protein which is substantially homologous to an entire amino acid sequence encoded by a nucleic acid of SEQ ID NO: 1.

Dominant negative mutations or trans-dominant suppression can be used to reduce the activity of a SUC 5 protein in transgenic seeds in order to change the levels of seed storage compounds. To achieve this, a mutation that abolishes the activity of the SUC 5 protein is created and the inactive non-functional SUC 5 protein gene is overexpressed in the transgenic plant. The inactive trans-dominant SUC 5 protein competes with the active endogenous SUC 5 protein for substrate or interactions with other proteins and dilutes out the activity of the active SUC 5 protein. In this way the biological activity of the SUC 5 protein is reduced without actually modifying the expression of the endogenous SUC 5 protein gene. This strategy was used by Pontier et al to modulate the activity of plant transcription factors (Pontier D, Miao ZH, Lam E, Plant J 2001 Sep. 27(6): 529-38, Trans-dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responses).

Homologues of the SUC 5 protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the SUC 5 protein. As used herein, the term "homologue" refers to a variant form of the SUC 5 protein that acts as an agonist or antagonist of the activity of the SUC 5 protein. An agonist of the SUC 5 protein can retain substantially the same, or a subset, of the biological activities of the SUC 5 protein. An antagonist of the SUC 5 protein can inhibit one or more of the activities of the naturally occurring form of the SUC 5 protein, by, for example, competitively binding to a downstream or upstream member of the cell membrane component metabolic cascade which includes the SUC 5 protein, or by binding to a SUC 5 protein which mediates transport of compounds across such membranes, thereby preventing translocation from taking place. In an alternative embodiment, homologues of the SUC 5 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the SUC 5 protein for SUC 5 protein agonist or antagonist activity. In one embodiment, a variegated library of SUC 5 protein variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of SUC 5 protein variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SUC 5 protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of SUC 5 protein sequences therein. There are a variety of methods that can be used to produce libraries of potential SUC 5 protein homo- logues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential SUC 5 protein sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang 1983, Tetrahedron 39:3; Itakura et al. 1984, Annu. Rev. Biochem. 53:323; Itakura et al. 1984, Science 198:1056; Ike et al. 1983, Nucleic Acids Res. 1 1 :477).

In addition, libraries of fragments of the SUC 5 protein coding sequences can be used to generate a variegated population of SUC 5 protein fragments for screening and subsequent selection of homologues of a SUC 5 protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a SUC 5 protein coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nu- clease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the SUC 5 protein. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of SUC 5 protein homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify SUC 5 protein homologues (Arkin & Yourvan 1992, Proc. Natl. Acad. Sci. USA 89:781 1 - 7815; Delgrave et al. 1993, Protein Engineering 6:327-331 ).

In another embodiment, cell based assays can be exploited to analyze a variegated SUC 5 protein library, using methods well known in the art.

The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of Arabidopsis thaliana and related organisms; mapping of genomes of organisms related to Arabidopsis thaliana; identification and localization of Arabidopsis thaliana sequences of interest; evolutionary studies; determination of SUC 5 protein regions required for function; modulation of a SUC 5 protein activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; and modulation of seed storage compound accumulation.

Higher plants like Arabidopsis thaliana or Brassica napus share a high degree of homology on the DNA sequence and polypeptide level, allowing the use of heterologous screening of DNA molecules with probes evolving from other plants or organisms, thus enabling the derivation of a consensus sequence suitable for heterologous screening or functional annota- tion and prediction of gene functions in third species. The ability to identify such functions can therefore have significant relevance, e.g., prediction of substrate specificity of enzymes. Further, these nucleic acid molecules may serve as reference points for the mapping of Arabidopsis genomes, or of genomes of related organisms. The SUC 5 protein nucleic acid molecules of the invention have a variety of uses. First, the nucleic acid and protein molecules of the invention may serve as markers for specific re- gions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of Arabidopsis thaliana or Brassica napus proteins. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related plants.

The SUC 5 protein nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.

Manipulation of the SUC 5 protein nucleic acid molecules of the invention may result in the production of SUC 5 proteins having functional differences from the wild-type SUC 5 pro- teins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a SUC 5 protein of the invention may directly affect the accumulation and/or composition of seed storage compounds. In the case of plants expressing SUC 5 proteins, increased transport can lead to altered accumulation of compounds and/or solute partitioning within the plant tissue and organs which ultimately could be used to affect the accumulation of one or more seed storage compounds during seed development. An example is provided by Mitsukawa et al. (1997, Proc. Natl. Acad. Sci. USA 94:7098-7102), where overexpression of an Arabidopsis high- affinity phosphate transporter gene in tobacco cultured cells enhanced cell growth under phosphate-limited conditions. Phosphate availability also affects significantly the production of sugars and metabolic intermediates (Hurry et al. 2000, Plant J. 24:383-396) and the lipid composition in leaves and roots (Hartel et al. 2000, Proc. Natl. Acad. Sci. USA 97:10649- 10654). Likewise, the activity of the plant ACCase has been demonstrated to be regulated by phosphorylation (Savage & Ohlrogge 1999, Plant J. 18:521 -527) and alterations in the activity of the kinases and phosphatases (SUC 5 proteins) that act on the ACCase could lead to increased or decreased levels of seed lipid accumulation.

Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and Examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims included herein.

Furthermore the invention is disclosed as follows:

A. A method of producing a transgenic plant having an increased level of fatty acids in the seed comprising, transforming a plant cell with an expression vector comprising a nucle- ic acid sequence encoding a sucrose transporter 5 polypeptide sequence, generating from the plant cell the transgenic plant, analyzing the production of fatty acids in the seed of the transgenic plant, and selecting a transgenic plant having an increased level of fatty acids as compared to a corresponding untransformed wild type variant of the plant, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence as defined in SEQ ID NO:1 ;

b) a polynucleotide sequence encoding a polypeptide as defined in SEQ ID NO:2; c) a polynucleotide sequence encoding a polypeptide having at least 70 % sequence identity with the polypeptide as defined in SEQ ID NO: 2;

a polynucleotide sequence having at least 70% sequence identity with the polynucleotide sequence of a), b) or c); and

e) a polynucleotide sequence that hybridizes under stringent conditions to the polynucleotide sequence of a), b), c) or d).

wherein the polynucleotide sequence is operatively linked to a seed-specific promoter. B. The method of producing a transgenic plant according to A, wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence comprises a polynucleotide having at least 90% sequence identity with the polynucleotide sequence of a), b) or c). C. The method of producing a transgenic plant according to A and B, wherein the seed- specific promoter is the USP promoter or the SUC5 promoter.

D. A method of increasing the level of the total fatty acids in the seed of a plant comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence, generating from the cell a transgenic plant, and selecting a transgenic plant having an increased level of fatty acids as compared to a corresponding untransformed wild type variety of the plant, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide sequence as defined in SEQ ID NO:1 ;

d) a polynucleotide sequence having at least 70% sequence identity with the polynucleotide sequence of a), b) or c); and

e) a polynucleotide sequence that hybridizes under stringent conditions to the pol- ynucleotide sequence of a), b), c) or d),

wherein the polynucleotide sequence is operatively linked to a seed-specific promoter.

E. A method of increasing the level of the total fatty acids in the seed of a plant according to D, wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide se- quence comprises a polynucleotide sequence having at least 90% sequence identity with the polynucleotide sequence of a), b) or c).

F. A method of increasing the level of the total fatty acids in the seed of a plant according to D and E, wherein the seed-specific promoter is the USP promoter or the SUC5 promoter.

G. A transgenic plant with increased total fatty acids content in the seed of the plant as compared to a wild type variety of the plant comprising a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence as defined in SEQ ID NO:1 ;

e) a polynucleotide sequence that hybridizes under stringent conditions to the polynucleotide sequence of a), b), c) or d),

H. A transgenic plant with an increased total fatty acids content in the seed of the plant as compared to a wild type variety of the plant comprising a polypeptide encoding the polypeptide as described by SEQ ID NO: 2 or a polypeptide having at least 70% sequence identity with the polypeptide as defined by SEQ ID NO: 2.

I. Transgenic seed produced by a transgenic plant according to G or H. J. A method of producing a transgenic plant having an increased level of 20:1 (1 1 ) fatty acid in the seed comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5, a plastidic translocator GPT1 and a plastidic translocator NTT1 polypeptide sequence, generating from the plant cell a transgenic plant, analyzing the production of fatty acid in the seed of the transgenic plant, and selecting a transgenic plant having an increased level of 20:1 fatty acid as compared to a corresponding untransformed wild type variant of the plant, wherein the expression vector comprises polynucleotide sequences selected from the group consisting of:

a) polynucleotide sequences as defined in SEQ ID NO:1 , 83 and 84;

b) polynucleotide sequences having at least 70% sequence identity with the polynucleotide sequences of a); and

c) polynucleotide sequences that hybridize under stringent conditions to the polynucleotides sequences of a) or b),

wherein the polynucleotide sequences are operatively linked to a seed-specific promoter.

K. The method of producing a transgenic plant according to J, wherein the nucleic acid sequences encoding a sucrose transporter 5, a plastidic translocator GPT1 and a plastidic translocator NTT1 polypeptide sequence comprise polynucleotides having at least 90% sequence identity with the polynucleotide sequences of a), b) or c).

L. The method of producing a transgenic plant according to J and K, wherein the seed- specific promoter is the USP promoter.

M. The method of producing a transgenic plant according to claim J and K, wherein the seed- specific promoter is the SUC5 promoter.

N. A method of increasing the level of 20:1 (11) fatty acid in the seed of a plant comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence, a plastidic translocator GPT1 and a plastidic translocator NTT1 , generating from the cell a transgenic plant, and selecting a transgenic plant having an increased level of 20:1 fatty acid as compared to a corresponding untransformed wild type variety of the plant, wherein the nucleic acid comprises polynucleotide sequences selected from the group consisting of:

a) polynucleotide sequences as defined in SEQ ID NO:1 , SEQ ID NO: 83 and

SEQ ID NO: 84;

c) polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequences of a) or b), wherein the polynucleotide sequences are operatively linked to a seed-specific promoter.

O. A method of increasing the level of 20: 1 (11 ) fatty acid in the seed of a plant according to N, wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide, a plastidic translocator GPT1 and a plastidic translocator NTT1 sequence comprises polynucleotide sequences having at least 90% sequence identity with the polynucleotide sequences of a), b) or e).

P. A method of increasing the level of 20: 1 (11 ) fatty acid in the seed of a plant according to claim N and O, wherein the seed-specific promoter is the USP promoter.

Q. A method of increasing the level of 20:1 (11 ) fatty acid in the seed of a plant according to claim N and O, wherein the seed-specific promoter is the SUC5 promoter. R. A transgenic plant with increased 20:1 (11 ) fatty acid content in the seed of the plant as compared to a wild type variety of the plant comprising polynucleotide sequences selected from the group consisting of:

a) polynucleotide sequences as defined in SEQ ID NO:1 , SEQ ID NO: 83 and SEQ ID NO: 84;

c) polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequences of a) or b),

wherein the polynucleotide sequences are operatively linked to a seed-specific pro- moter.

S. Transgenic seed produced by a transgenic plant according to R.

FIGURE LEGENDS

Figure 1. Characterization of the suc5.4 and suc5.5 mutant alleles in Col-0.

(a) Schematic depiction of the SUC5 gene with its three exons (thick lines) and of the T- DNA insertions in the suc5.4 and suc5.5 mutants. Start (ATG) and stop of translation (asterisk) are indicated. Arrows show positions and directions of primers used in (b).

(b) RT-PCRs with actin-specific primers (left) and with the primers 1 to 3 shown in (a) on total RNA isolated from flowers of wt, suc5.4 and suc5.5 plants. Primers binding to the region upstream from the T-DNA insertion sites amplified bands in the wt and in both mutant lines. Bands indicating the presence of an intact SUC5 mRNA were not amplified in the mutants, but were obtained in the wt.

Figure 2. Analysis of pSUC5/sGFP and pSUC5/tmGFP9 plants. Confocal images of developing seeds [(a) to (e)] and isolated embryos [(f) to (i)] from pSUC5/sGFP plants [(a), (d) and (e)] or pSUC5/tmGFP9 [(b), (c), and (f) to (i)] plants are presented.

(a) Young seed with syncytial endosperm and no detectable embryo showing GFP fluorescence in the endosperm nuclei (maximum projection).

(b) Young seed with syncytial endosperm showing strong GFP fluorescence in the chalazal region (maximum projection; ch).

(c) Developing seed showing GFP fluorescence (optical section) in the ER of the endosperm but not in the globular embryo (arrow).

(d) Slightly older seed with GFP fluorescence in the endosperm nuclei (maximal projection) but not in the heart-stage embryo (arrow).

(e) Developing seed with GFP fluorescence in the early-torpedo-stage embryo and in the endosperm.

(f) Isolated walking stick-stage embryo (maximum projection) showing highest GFP fluorescence at the physiological underside of the forming cotyledons (white arrows) and, no GFP fluorescence on the upper side (black arrow), and little fluorescence in the hypocotyl.

(g) Higher magnification of an embryo (optical section) at the same developmental stage as shown in (f).

(h) Higher magnification (optical section) of a section through the underside of a forming cotyledon [same embryo as in (g)] showing GFP fluorescence only in the epidermis.

(i) Comparison of the fluorescence in an isolated wt embryo [slightly older (mid-torpedo stage) than the embryo shown in (e)] and pSUC5/tmGFP9 embryo [similar stage as in (f) and (g)].

Red colour results from autofluorescence of chlorophyll. Scale bars are 10 m in (h), 25 μιη in (c), 50 μιη in (a), 100 μιη in (b), (d), (e) and (g), and 200 μιη in (f) and (i).

Figure 3. Development of seeds and embryos in siliques of wt plants and of different single and double mutants. After an initial growth (12 d) on agar medium with 1 mM biotin, plants were transferred to soil and watered with the indicated supplements of biotin.

(a) Developing seeds isolated from siliques of comparable developmental stages.

(b) Embryos isolated from the developing-seed batches analysed in (a).

Space bars are 20 μιη in (a) and (b).

Figure 4. Phenotypes of dry seeds from wt plants, single and double mutants supplemented with different biotin concentrations.

Dry seeds from the indicated plant lines. Seeds of wt and suc5.5 plants are shaped nor- mally under all growth conditions. Seeds of bio2. 1 plants are wrinkled and seeds of bio2. 1/suc5.5 double mutants have the appearance of "empty bags", when their parent plants were not supplemented with biotin. A supplement of 0.1 mM biotin complemented this defect partly (bio2.1 seeds are almost normal looking; bio2.1/suc5.5 plants are still wrinkled), and the seeds looked normal, when the parent plants were watered with 1 mM biotin. Bars are 50 μιη.

Figure 5. Comparative analysis of seedlings from wt plants and from homozygous single or double mutants on MS medium or MS medium supplemented with biotin. Seedlings were photographed after 10 days at 21 °C under long-day conditions (16 h light / 8 h dark). Par- ent plants of all seeds with a bio mutation had been watered with 1 mM biotin, parent plants of wt and suc5 mutant seeds only with water. Typical phenotypes are shown for pairs of seedlings grown either on biotin-free MS medium (left) or on MS medium supplemented with biotin (right; final biotin concentration: 1 mM). Names of different double mutant lines are given in brackets. All pictures were taken at the same magnification. Bar is 2 mm.

Figure 6. Phenotypes of double-homozygous bio1/suc5 and bio2/suc5 seedlings. Seedlings grown on high-biotin medium were photographed after a 12-d incubation of the plates at 21 °C (16 h light/8 h dark).

(a) biol.1/suc5.4 seedling with no visible cotyledons.

(b) bio2.1/suc5.5 seedling with a green cotyledon (upper arrow) and a white, callus-like cotyledon (lower arrow).

(c) bio1.1/suc5.4 seedling with small yellowish cotyledons (arrows).

(d) Seedling of a wt plant with normal cotyledons (arrows) photographed at the same time of development.

(e) The percentage of the different phenotypes (numbers inside the bars) was quantified in selected lines of homozygous single or double mutants. Green bars show the percentage of seedlings forming green, normally shaped cotyledons (typically these cotyledons were smaller than wt seedlings), yellow bars show the percentage of seedlings with severe coty- ledon phenotypes [as in (a), (b) or (c) or similar], brown bars show the percentage of seeds that were not yet germinated at the time of analysis. The total number of analysed plants is shown below the bars.

Scale bars are 500 μιη in (a), (b) and (c), and 1 mm in (d). Figure 7. TAG content of dry seeds and fatty acid composition in these TAGs.

(a) TAG content in seeds from wt plants, and from single and double mutants that were supplemented with the indicated biotin concentrations (** i-test: p-value≤ 0.0001 . p-Values were calculated for 0 mM versus 0.1 mM, 0.1 mM versus 1 mM, and 0 mM versus 1 mM).

(b) Fatty acid composition in the TAGs shown in (a).

(a) and (b) Data (± SD) were obtained from two independent biological experiments and three technical replicates per experiment (n = 6). Data sets labelled with a white asterisk represent three technical replicates from one experiment (n = 3).

(c) Mol% values of n7 and n9 fatty acids extracted from the data shown in (b). In wt and suc5.5 seeds, the content of n9 fatty acids is about 9-fold higher than that of n7 fatty acids, and these values are not affected by added biotin. In seeds of bio2. 1 plants, however, and even more pronounced in seeds of bio2. 1/suc5.5 plants, the mol% of n7 and n9 fatty acids are increased or decreased, respectively, and these changes can be reverted to wt-levels by biotin. Thick bars, thin bars or triangles indicate high, low, increasing or decreasing mol% values, respectively.

Figure 8. Schematic map of the expression vector pDEST-USP:SUC5, which was used for transformation of Arabidopsis thaliana plants with the SUC5 gene (SEQ ID NO: 1 ) alone under the transcriptional control of the USP promoter. Figure 9. Schematic map of the expression vector pDEST- USP:GPT/USP:NTT/USP:SUC5, which was used for transformation of Arabidopsis thaliana plants. Additionally to the SUC5 gene (SEQ ID NO: 1 ) the plasmid contains genes for the plastidic translocators GPT1 (SEQ ID NO: 83) and NTT1 (SEQ ID NO: 84). All three genes are cloned behind the transcriptional control of the USP-promoter. Transformation of Arabidopsis wt plants with this construct resulted in the generation of independent transgenic lines, which were consecutively named BioOI3-1 to BioOI3-20.

Figure 10. Fatty acid composition of TAGs from dry seeds obtained from wt (black bar) and transgenic BioOI3 plants (=plants transformed with vector pDEST- USP:GPT/USP:NTT/USP:SUC5, white bars) in pg/mg dry seeds. Error bars represent measurements of seeds from at least 10 different plants per line. Absolute values for fatty acids from C18 to C22 are shown. A significant weight increase was detected for the fatty acids C20:0, C20:1 (1 1 ), C20:2, C22:0 and C22: 1. The most drastic increase of 16.5% in g/mg dry seeds was observed for eicosenoic acid [C20:1 (1 1 )]. Figure 11 . Total TAG content of dry seeds obtained from wt (black bar) and transgenic BioOI3 plants (grey bars). Content is shown in g/mg dry seeds for 12 independent transgenic BioOI3 lines. Error bars represent measurements of seeds from 10 different plants per line. An average increase of 3.2% TAG was observed for all BioOI3 plants.

Figure 12. Fatty acid composition of TAG from dry seeds obtained from wt and the 3 BioOI3-lines with the strongest increase in total TAG (lines 3-1 , 3-4 and 3-15). Increase in total TAG is mostly due to an increase in fatty acids from C20 to C22. The increase in the level of C20:1 (1 1 ) fatty acid averages 20% in these 3 lines. Error bars represent meas- urements of seeds from 10 different plants per line.

Figure 13. Histogram showing the total TAG content of different plants from wt, BioOI3-1 , 3-4 and 3-15. Individual TAG content from 10 plants of each line is plotted in 20 g TAG/mg seeds-intervals (x-axis) against the number of individual plants with this TAG content. TAG content in wt and in the 3 plotted transgenic lines shows a Gaussian distribution in which the peak of the distribution curve is shifted towards higher TAG levels in the transgenic lines compared with wt.

Figure 14. In the alignment shown in Figure 14 the nine disaccharide transporters from Arabidopsis thaliana share 25.3% identical positions (black/ white) and 77.0% consensus positions (grey/ white).

Figure 15. Total TAG content of dry seeds obtained from wt (white bar) and transgenic BioOil4 plants (black bars) is shown in Figure 15. Total TAG content is shown in g/mg dry seeds for 1 1 independent transgenic BioOil4 lines. Error bars represent measurements of seeds from 10 different plants per line. An average increase of 4.7% TAG was observed throughout all BioOil4 plants.

Figure 16. Figure 16 shows an elevation of total biotin content in the seeds of SUC5- overexpressing plants.

Figure 17. Figure 17 shows an elevation of total biotin content in the seeds of SUC5- overexpressing plants. Figure 18. Figure 18 shows the SUC5 promoter sequence as used in constructs used for construction of above BioOil4 plants. Sequences highlighted in grey or dark grey show primers used for amplification of the promoter sequence from genomic Arabidopsis DNA.

Figure 19. SL/C5-mRNA levels were measured by qPCR in developing seeds harvested from siliques of wt or SL/C5-overexpressing lines (BioOil3, BioOil4) at the indicated days after flowering (DAF). ACTIN2 (ACT2) was used as internal reference gene for the determination of relative expression.

Figure 20. Sucrose and raffinose content in ripe seeds of wt, BioOil3 and BioOM. Bars and errors represent mean values and standard deviations from 3 independent measurements.

Figure 21. Uptake of sucrose and biotin by wt, BioOil3 and BioOM embryos. Bars and errors in (a) and (b) represent mean values and standard deviations from 3 independent measurements.

(A) Uptake of 2 mM [ ⁴C]-sucrose into wt, BioOH3 and BioOM embryos at 8-DAF. The sucrose taken up per embryo after 90 min is given in pmoles.

(B) Uptake of 10 μΜ [ ⁴C]-biotin into wt and BioOM embryos at 8-DAF. The biotin taken up per embryo after 6 h is given in fmoles.

(C) Isolated wt, BioOil3 and BioOM embryos at 8-DAF used for uptake measurements with radiolabeled biotin or sucrose. Bars are 250 μιη.

EXAMPLES

Example 1 - GENERAL EXPERIMENTAL PROCEDURES Strains, growth conditions and plant transformation

Arabidopsis thaiiana Col-0 and mutant plants were grown in the growth chamber on potting soil under a 16 h light/8 h dark regime at 22°C and 60% relative humidity and watered with the indicated biotin concentrations. bio1. 1 and bio2. 1 mutant lines (N6316 = bio1. 1; N6329 = bio2. 1) and suc5 mutant lines (SAIL_367_D07 = suc5.4; SALK_092412 = suc5.5) were obtained from the Nottingham Arabidopsis Stock Centre. Agrobacterium tumefaciens GV3101 (Holsters et al., 1980) was used for Arabidopsis transformation by floral dip (Clough and Bent, 1998). Escherichia coli strain DH5a (Hanahan, 1983) was used for all cloning steps. Characterization of biol, bio2 and suc5 single mutants and generation of homozygous bio/suc5 double mutants

The position of the T-DNA insertion in the suc5.4 mutant was determined by sequencing PCR fragments that were with the primers LB2 (5'- GCTTCCTATTATATCTTCCCAAATTACCAATACA-3') and AtSUC5g540f (5'- CGCAAACGCGTGTTTCTCCT-3'). The double insertion in suc5.5 was determined with the primers LBa1 (5'-TGGTTCACGTAGTGGGCCATCG-3') and AtSUC5g540f (5'-end of insertion) or with LBa1 and AtSUC5g2136r ( 5 ' -TG C ACAACAATACTGTATT AG ATG G -3 ' ; 3'-end of the insertion). SUC5 and ACTIN2 mRNA levels were determined by RT-PCR using the primers AtSUC5g540f (= primer 1 in Figure 1 ), AtSUC5g1 199r (5'- TCCGGCTTTAATACCACTGC-3'; = primer 2 in Figure 1 ) and AtSUC5g2136r (= primer 3 in Figure 1 ), and the 4C77N2-specific primers Act2g+846f (5'- ATTCAGATGCCCAGAAGTCTTGTT-3') and Act2g+1295r (5'-GAA ACA TTTTCTGTG AACGATTCCT-3') . To obtain homozygous bio1. 1 and bio2. 1 plants, the seeds from heterozygous, biotin- watered BI01/bio1. 1 or BI02/bio2. 1 plants were germinated on commercially available, biotin-free medium. In contrast to the heterozygous plants, homozygous bio1. 1 and bio2. 1 mutant seedlings (about 20%) developed normal cotyledons, but were unable to form rosette leaves, and turned pale about 2 weeks after germination. If transferred to medium enriched with 1 mM biotin (= high-biotin) these seedlings re-greened and developed normally. After about 2 weeks on high-biotin plates, plantlets were transferred to soil and watered with 1 mM biotin (Schneider et al., 1989; Patton et al., 1998) to ensure normal development, flowering, and the production of homozygous bio1. 1 or bio2. 1 seeds. When seeds of these homozygous plants were germinated on biotin-free medium, 100% failed to grow. Homozygous bio1. 1 or bio2. 1 plants were crossed with homozygous suc5.4 or suc5.5 plants. The resulting seeds (cross-0 seeds) were germinated on soil, and the presence or absence of the suc5.4 or suc5.5 insertion was determined by PCR. Next, seeds from plants carrying suc5.4 or suc5.5 alleles (cross-1 seeds) were germinated on biotin-free medium. Pale seedlings (indicating homozygosity for bio1. 1 or bio2. 1) were rescued on high-biotin medium, transferred to soil, and watered with biotin. Eventually, cross-2 seeds were germinated either on biotin-free medium (to re-confirm that 100% of the seedlings turned pale) or on high-biotin medium, and homozygosity for suc5.4 or suc5.5 was determined by PCR. Generation of pSUC5/reporter lines

For construction of pSUC5/sGFP, 2030 bp of pSUC5 were amplified using the primers At- SUC5-2030f (5'-AAGCTTAACAATTTATGTAGTTTAGAACG-3') and AtSUC5-1 r (5'- CCATGGTGAAAAGAAAAACGAGCAGACAA-3') that introduced Hindlll and Ncol cloning sites to the 5' and 3'-ends, respectively. The resulting fragment was used to replace pSUC2 in pEP/pUC19, a plasmid containing a pSUC2/GFP cassette (Imlau et ai, 1999). From the resulting vector, pEP-S5-GFP, the pSUC5/sGFP fragment was excised with Hindlll and Sacl and cloned into the respective sites of pAF16 (Stadler et ai, 2005b). The resulting plasmid was used for Arabidopsis transformation. For construction of pSUC5/tmGFP9, a genomic 1 152-bp fragment encoding the 232 N- terminal amino acids of STP9 (Schneidereit et ai, 2003) was excised from plasmid pMH4 (Stadler et ai, 2005b) with Ncol and inserted into the unique Ncol site separating pSUC5 and the GFP ORF in pEP-S5-GFP. From the resulting plasmid, pMH19, the 3916-bp pSUC5/tmGFP9 cassette was excised with Hindll l/Sacl and cloned into the respective sites of pAF16 yielding pMH21 that was used for Arabidopsis transformation.

Confocal microscopy

For detection of GFP fluorescence in pSUC5/GFP plants, images were made with a confocal microscope (Leica TCS SPII; Leica Microsystems, Bensheim, Germany) as described (Stadler et al., 2005b). The excitation wavelength for GFP was 488 nm. Confocal images were processed using Leica Confocal Software 2 (Leica Microsystems).

Analyses of TAG and fatty acids

Fatty acid methyl esters (FAMEs) of pooled Arabidopsis seeds were obtained by methyla- tion with 0.5 M sulphuric acid in methanol containing 2% (v/v) dimethoxypropane at 80°C for 1 h. FAMEs were extracted in 2 ml of n-hexane, dried under N₂ and analysed by gas- chromatography (GC). The GC analysis was performed with an Agilent GC 6890 system coupled with a flame ionization detector equipped with a capillary 122-2332 DB-23 column (30 m x 0.32 mm; 0.5 μιη coating thickness; Agilent). Helium was used as carrier gas (1 ml min-¹). Samples were injected at 220°C. The temperature gradient was 150°C for 1 min, 150 to 200°C at 15°C min-¹, 200 to 250°C at 2°C min-¹, and 250°C for 10 min. Data were processed using the HP ChemStation Rev. A09.03. FAMEs were identified by comparison with appropriate reference substances and quantified according to an internal standard (tripentadecanoin) of known concentration (Hoffmann et ai, 2008). General Cloning Processes

Cloning processes such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli and yeast cells, growth of bacteria and sequence analysis of recombinant DNA were carried out as de- scribed in Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309- 6) or Kaiser, Michaelis and Mitchell (1994, "Methods in Yeast Genetics", Cold Spring Harbor Laboratory Press: ISBN 0-87969-451 -3).

Chemicals

The chemicals used were obtained, if not mentioned otherwise in the text, in p. a. quality from the companies Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were prepared using purified, pyrogen-free water, designated as H20 in the following text, from a Milli-Q water system water purification plant (Millipore, Eschborn). Restriction endonucleases, DNA-modifying enzymes and mo- lecular biology kits were obtained from the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Gottingen), Boehringer (Mannheim), Genomed (Bad

Oeynnhausen), New England Biolabs (Schwalbach/ Taunus), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Strata- gene (Amsterdam, Netherlands). They were used, if not mentioned otherwise, according to the manufacturer's instructions.

Example 2 - Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up of 1 gram fresh weight of plant material.

CTAB buffer: 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCI pH 8.0; 1.4 M NaCI; 20 mM EDTA. N-Laurylsarcosine buffer: 10% (w/v) N- laurylsarcosine; 100 mM Tris HCI pH 8.0; 20 mM EDTA. The plant material was triturated under liquid nitrogen in a mortar to give a fine powder and transferred to 2 ml Eppendorf vessels. The frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml CTAB buffer, 100 μΙ of N-laurylsarcosine buffer, 20 μΙ of β-mercaptoethanol and 10 μΙ of proteinase K solution, 10 mg/ml) and incubated at 60°C for one hour with continuous shaking. The homogenate obtained was distributed into two Eppendorf vessels (2 ml) and extracted twice by shaking with the same volume of chlo- roform/isoamyl alcohol (24:1 ). For phase separation, centrifugation was carried out at 8000g and RT for 15 min in each case. The DNA was then precipitated at -70°C for 30 min using ice-cold isopropanol. The precipitated DNA was sedimented at 4°C and 10,000 g for 30 min and resuspended in 180 μΙ of TE buffer (Sambrook et al. 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). For further purification, the DNA was treated with NaCI (1.2 M final concentration) and precipitated again at -70°C for 30 min using twice the volume of absolute ethanol. After a washing step with 70% ethanol, the DNA was dried and subsequently taken up in 50 μΙ of H₂0 + RNAse (50 mg/ml final concentration). The DNA was dissolved overnight at 4°C and the RNAse digestion was subsequently carried out at 37°C for 1 h. Storage of the DNA took place at 4°C.

Example 3 - Isolation of Total RNA and poly-(A)+ RNA from Plants: Arabidopsis thaliana

For the investigation of transcripts, both total RNA and poly-(A)+ RNA were isolated.

RNA is isolated from siliques of Arabidopsis plants according to the following procedure: RNA preparation from Arabidopsis seeds - "hot" extraction:

1. Buffers, enzymes and solution

2M KCI

Proteinase K

Phenol (for RNA)

Chloroform:lsoamylalcohol

(Phenokcholoroform 1 :1 ; pH adjusted for RNA)

4 M LiCI, DEPC-treated

DEPC-treated water

3M NaOAc, pH 5, DEPC-treated

Isopropanol

70% ethanol (made up with DEPC-treated water)

Resuspension buffer:0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up with DEPC- treated water as this solution can not be DEPC-treated

Extraction Buffer:

0.2M Na Borate

30 mM EDTA

30 mM EGTA

1 % SDS (250μΙ of 10% SDS-solution for 2.5ml buffer)

1 % Deoxycholate (25mg for 2,5ml buffer)

2% PVPP (insoluble - 50mg for 2.5ml buffer)

2% PVP 40K (50mg for 2.5ml buffer)

10 mM DTT

100 mM β-Mercaptoethanol (fresh, handle under fume hood - use 35μΙ of 14.3M solution for 5ml buffer) 2. Extraction. Heat extraction buffer up to 80°C. Grind tissue in liquid nitrogen-cooled mortar, transfer tissue powder to 1.5ml tube. Tissue should be kept frozen until buffer is added so transfer the sample with pre-cooled spatula and keep the tube in liquid nitrogen all time. Add 350μΙ preheated extraction buffer (here for 100mg tissue, buffer volume can be as much as 500μΙ for bigger samples) to tube, vortex and heat tube to 80°C for ~1 min. Keep then on ice. Vortex sample, grind additionally with electric mortar.

3. Digestion. Add Proteinase K (0.15mg/100mg tissue), vortex and keep at 37°C for one hour.

First Purification. Add 27μΙ 2M KCI. Chill on ice for 10 min. Centrifuge at 12.000 rpm for 10 minutes at room temperature. Transfer supernatant to fresh, RNAase-free tube and do one phenol extraction, followed by a chloroform:isoamylalcohol extraction. Add 1 vol. isopropa- nol to supernatant and chill on ice for 10 min. Pellet RNA by centrifugation (7000 rpm for 10 min at RT). Resolve pellet in 1 ml 4M LiCI by 10 to 15min vortexing. Pellet RNA by 5min centrifugation.

Second Purification. Resuspend pellet in 500μΙ Resuspension buffer. Add 500μΙ phenol and vortex. Add 250μΙ chloroform:isoamylalcohol and vortex. Spin for 5 min. and transfer supernatant to fresh tube. Repeat chloform:isoamylalcohol extraction until interface is clear. Transfer supernatant to fresh tube and add 1/10 vol 3M NaOAc, pH 5 and 600μΙ iso- propanol. Keep at -20 for 20 min or longer. Pellet RNA by 10 min centrifugation. Wash pellet once with 70% ethanol. Remove all remaining alcohol before resolving pellet with 15 to 20μΙ DEPC-water. Determine quantity and quality by measuring the absorbance of a 1 :200 dilution at 260 and 280nm. ^g RNA/ml = 10D260

RNA from wild-type of Arabidopsis is isolated as described (Hosein, 2001 , Plant Mol. Biol. Rep., 19, 65a-65e; Ruuska,S.A., Girke,T., Benning.C, & Ohlrogge,J.B., 2002, Plant Cell, 14, 1 191 -1206).

The mRNA is prepared from total RNA, using the Amersham Pharmacia Biotech mRNA purification kit, which utilizes oligo(dT)-cellulose columns.

Isolation of Poly-(A)+ RNA was isolated using Dyna BeadsR (Dynal, Oslo, Norway) follow- ing the instructions of the manufacturer's protocol. After determination of the concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70°C.

Total RNA was extracted from tissues using RNeasy Maxi kit (Qiagen) according to manufacture's protocol and mRNA was processed from total RNA using Oligotex mRNA Purifica- tion System kit (Qiagen), also according to manufacture's protocol. mRNA was sent to

Hyseq Pharmaceuticals Incorporated (Sunnyville, CA) for further processing of mRNA from each tissue type into cDNA libraries and for use in their proprietary processes in which similar inserts in plasmids are clustered based on hybridization patterns.

Example 4 - cDNA Library Construction

For cDNA library construction, first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12°C (2 h), 16°C (1 h) and 22°C (1 h). The reaction was stopped by incubation at 65°C (10 min) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37°C (30 min). Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12°C, overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37°C, 30 min). This mixture was subjected to separation on a low melting agarose gel. DNA molecules larger than 300 base pairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany) and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.

Brassica napus cDNA libraries were generated at Hyseq Pharmaceuticals Incorporated (Sunnyville, CA) No amplification steps were used in the library production to retain expression information. Hyseq's genomic approach involves grouping the genes into clusters and then sequencing representative members from each cluster. cDNA libraries were generat- ed from oligo dT column purified mRNA. Colonies from transformation of the cDNA library into E.coli were randomly picked and the cDNA insert were amplified by PCR and spotted on nylon membranes. A set of ^33_P radiolabeled oligonucleotides were hybridized to the clones and the resulting hybridization pattern determined to which cluster a particular clone belonged. cDNA clones and their DNA sequences were obtained for use in overexpression in transgenic plants and in other molecular biology processes described herein.

Example 5 - Cloning of full-length cDNAs and orthologs of identified SUC 5 protein genes

Clones corresponding to full-length sequences and partial cDNAs from Arabidopsis thaliana had been identified in the in-house proprietary Hyseq databases. The Hyseq clones of Ar- abidopsis thaliana were sequenced at DNA Landmarks using a ABI 377 slab gel sequencer and BigDye Terminator Ready Reaction kits (PE Biosystems, Foster City, CA). Sequence alignments were done to determine whether the Hyseq clones were full-length or partial clones. In cases where the Hyseq clones were determined to be partial cDNAs the following procedure was used to isolate the full-length sequences. Full-length cDNAs were iso- lated by RACE PCR using the SMART RACE cDNA amplification kit from Clontech allowing both 5'- and 3' rapid amplification of cDNA ends (RACE). The RACE PCR primers were designed based on the Hyseq clone sequences. The isolation of full-length cDNAs and the RACE PCR protocol used were based on the manufacturer's conditions. The RACE product fragments were extracted from agarose gels with a QIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacturer's instructions. Recombinant vectors were transformed into TOP10 cells (Invitrogen) using standard conditions (Sambrook et al. 1989). Transformed cells were grown overnight at 37°C on LB agar containing 50 μg/ml kanamycin and spread with 40μΙ of a 40 mg/ml stock solution of X-gal in dimethylformamide for blue-white selection. Single white colonies were selected and used to inoculate 3 ml of liquid LB containing 50 μg/ml kanamycin and grown overnight at 37°C. Plasmid DNA is extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Subsequent analyses of clones, and restriction mapping, was performed according to standard molecular biology techniques (Sambrook et al. 1989).

Full-length cDNAs were isolated and cloned into binary vectors by using the following pro- cedure: Gene specific primers were designed using the full-length sequences obtained from Hyseq clones or subsequent RACE amplification products. Full-length sequences and genes were amplified utilizing Hyseq clones or cDNA libraries as DNA template using touch-down PCR. In some cases, primers were designed to add an "AACA" Kozak-like sequence just upstream of the gene start codon and two bases downstream were, in some cases, changed to GC to facilitate increased gene expression levels (Chandrashekhar et al. 1997, Plant Molecular Biology 35:993-1001 ). PCR reaction cycles were: 94°C, 5 min; 9 cycles of 94°C, 1 min, 6°C, 1 min, 72°C, 4 min and in which the anneal temperature was lowered by 1 °C each cycle; 20 cycles of 94°C, 1 min, 55°C, 1 min, 72°C, 4 min; and the PCR cycle was ended with 72°C, 10 min. Amplified PCR products were gel purified from 1 % agarose gels using GenElute -EtBr spin columns (Sigma) and after standard enzymatic digestion, were ligated into the plant binary vector pBPS-GB1 for transformation of Ara- bidopsis. The binary vector was amplified by overnight growth in E. coli DH5 in LB media and appropriate antibiotic and plasmid was prepared for downstream steps using Qiagen MiniPrep DNA preparation kit. The insert was verified throughout the various cloning steps by determining its size through restriction digest and inserts were sequenced to ensure the expected gene was used in Arabidopsis transformation.

Gene sequences can be used to identify homologous or heterologous genes (orthologs, the same SUC 5 protein gene from another plant) from cDNA or genomic libraries. This can be done by designing PCR primers to conserved sequences identified by multiple sequence alignments. Orthologs are often identified by designing degenerate primers to full-length or partial sequences of genes of interest.

Gene sequences can be used to identify homologues or orthologs from cDNA or genomic libraries. Homologous genes (e. g. full-length cDNA clones) can be isolated via nucleic acid hybridization using for example cDNA libraries: Depending on the abundance of the gene of interest, 100,000 up to 1 ,000,000 recombinant bacteriophages are plated and transferred to nylon membranes. After denaturation with alkali, DNA is immobilized on the membrane by e.g. UV cross linking. Hybridization is carried out at high stringency conditions. Aqueous solution hybridization and washing is performed at an ionic strength of 1 M NaCI and a tem- perature of 68°C. Hybridization probes are generated by e.g. radioactive (32P) nick transcription labelling (High Prime, Roche, Mannheim, Germany). Signals are detected by autoradiography.

Partially homologous or heterologous genes that are related but not identical can be identi- fied in a procedure analogous to the above-described procedure using low stringency hybridization and washing conditions. For aqueous hybridization, the ionic strength is normally kept at 1 M NaCI while the temperature is progressively lowered from 68 to 42°C.

Isolation of gene sequences with homologies (or sequence identity/similarity) only in a dis- tinct domain of (for example 10-20 amino acids) can be carried out by using synthetic radio labeled oligonucleotide probes. Radio labeled oligonucleotides are prepared by phosphorylation of the 5' end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double stranded concatemers are then radiolabeled by for example nick transcription. Hy- bridization is normally performed at low stringency conditions using high oligonucleotide concentrations.

Oligonucleotide hybridization solution:

6 x SSC

0.01 M sodium phosphate

1 mM EDTA (pH 8)

0.5 % SDS

100 g/ml denaturated salmon sperm DNA

0.1 % nonfat dried milk During hybridization, temperature is lowered stepwise to 5-10°C below the estimated oligonucleotide Tm or down to room temperature followed by washing steps and autoradiography. Washing is performed with low stringency such as 3 washing steps using 4x SSC. Further details are described by Sambrook et al. (1989, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press) or Ausubel et al. (1994, "Current Protocols in Molecular Biology," John Wiley & Sons).

Example 6 - Identification of Genes of Interest by Screening Expression Libraries with Antibodies

c-DNA clones can be used to produce recombinant protein for example in E. coil (e. g. Qi- agen QIAexpress pQE system). Recombinant proteins are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant proteins can be used to produce specific antibodies for example by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Gu et al. (1994, BioTechniques 17:257-262). The antibody can then be used to screen expression cDNA libraries to identify homologous or heterologous genes via an immunological screening (Sambrook et al. 1989, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press or Ausubel et al. 1994, "Current Protocols in Molecular Biology," John Wiley & Sons).

Example 7 - Northern-Hybridization

For RNA hybridization, 20 μg of total RNA or 1 μg of poly-(A)+ RNA is separated by gel electrophoresis in 1.25% agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem. 152:304), transferred by capillary attraction using 10 x SSC to positively charged nylon membranes (Hybond N+, Amersham, Braunschweig), immobilized by UV light and pre-hybridized for 3 hours at 68°C using hybridization buffer (10% dextran sulfate w/v, 1 M NaCI, 1 % SDS, 100 μg/ml of herring sperm DNA). The labelling of the DNA probe with the Highprime DNA labelling kit (Roche, Mannheim, Germany) is carried out during the pre-hybridization using alpha-32P dCTP (Amersham, Braunschweig, Germany). Hybridization is carried out after addition of the labelled DNA probe in the same buffer at 68°C overnight. The washing steps are carried out twice for 15 min using 2 x SSC and twice for 30 min using 1 x SSC, 1 % SDS at 68°C. The exposure of the sealed filters is carried out at - 70°C for a period of 1 day to 14 days.

Example 8 - DNA Sequencing and Computational Functional Analysis

cDNA libraries can be used for DNA sequencing according to standard methods, in particu- lar by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany). Random sequencing can be carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DH10B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands). Plasmid DNA can be prepared from overnight grown E. coli cultures grown in Luria-Broth medium containing am- picillin (see Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969- 309-6) on a Qiagene DNA preparation robot (Qiagen, Hilden) according to the manufacturer's protocols). Sequences can be processed and annotated using the software package EST-MAX commercially provided by Bio-Max (Munich, Germany). The program incorpo- rates bioinformatics methods important for functional and structural characterization of protein sequences. For reference see http://pedant.mips.biochem.mpg.de.

The most important algorithms incorporated in EST-MAX are: FASTA: Very sensitive protein sequence database searches with estimates of statistical significance (Pearson W.R. 1990, Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzy- mol. 183:63-98). BLAST: Very sensitive protein sequence database searches with esti- mates of statistical significance (Altschul S.F., Gish W., Miller W., Myers E.W. and Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 215:403-410). PREDATOR: High- accuracy secondary structure prediction from single and multiple sequences. (Frishman & Argos 1997, 75% accuracy in protein secondary structure prediction. Proteins 27:329-335). CLUSTALW: Multiple sequence alignment (Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1994, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22:4673-4680). TMAP: Transmembrane region prediction from multiply aligned sequences (Persson B. & Argos P. 1994, Prediction of transmembrane segments in proteins utilizing multiple sequence alignments, J. Mol. Biol. 237: 182-192).

ALOM2:Transmembrane region prediction from single sequences (Klein P., Kanehisa M., and DeLisi C. 1984, Prediction of protein function from sequence properties: A discriminant analysis of a database. Biochim. Biophys. Acta 787:221 -226. Version 2 by Dr. K. Nakai). PROSEARCH: Detection of PROSITE protein sequence patterns. Kolakowski L.F. Jr., Leunissen J.A.M. and Smith J.E. 1992, ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function. Biotechniques 13:919- 921 ). BLIMPS: Similarity searches against a database of ungapped blocks (Wallace & Henikoff 1992, PATMAT: A searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford).

Example 9 - Plasmids for Plant Transformation

For plant transformation binary vectors such as pBinAR can be used (Hofgen & Willmitzer 1990, Plant Sci. 66:221 -230). Construction of the binary vectors can be performed by ligation of the cDNA in sense or antisense orientation into the T-DNA. 5' to the cDNA a plant promoter activates transcription of the cDNA. A polyadenylation sequence is located 3' to the cDNA. Tissue-specific expression can be achieved by using a tissue specific promoter. For example, seed-specific expression can be achieved by cloning the napin or LeB4 or USP promoter 5' to the cDNA. Also any other seed specific promoter element can be used. For constitutive expression within the whole plant the CaMV 35S promoter can be used. The expressed protein can be targeted to a cellular compartment using a signal peptide, for example for plastids, mitochondria, or endoplasmic reticulum (Kermode 1996, Crit. Rev. Plant Sci. 15:285-423). The signal peptide is cloned 5-prime in frame to the cDNA to achieve subcellular localization of the fusion protein. Further examples for plant binary vectors are the pBPS-GB1 , pSUN2-GW or pBPS-GB047 vectors into which the SUC 5 protein gene candidates are cloned. These binary vectors contain an antibiotic resistance gene driven under the control of the AtAct2-l promoter and a USP seed-specific promoter or the PtxA promoter in front of the candidate gene with the NOSpA terminator or the OCS terminator. Partial or full-length SUC 5 protein cDNA are cloned into the multiple cloning site of the plant binary vector in sense orientation behind the USP seed-specific or PtxA promoters. The recombinant vector containing the gene of in- terest is transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells are selected for on LB agar containing 50 μg/ml kanamycin grown overnight at 37°C. Plasmid DNA is extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analysis of subsequent clones and restriction mapping is performed according to standard molecular biology techniques (Sambrook et al. 1989, Molecular Cloning, A Laboratory Manual. 2^nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY).

Example 10 - Agrobacterium Mediated Plant Transformation

Agrobacterium mediated plant transformation with the SUC 5 protein nucleic acids described herein can be performed using standard transformation and regeneration techniques (Gelvin, Stanton B. & Schilperoort R.A, Plant Molecular Biology Manual, 2nd ed. Kluwer Academic Publ., Dordrecht 1995 in Sect., Ringbuc Zentrale Signatur:BT1 1 -P; Glick, Bernard R. and Thompson, John E. Methods in Plant Molecular Biology and Biotechnology, S. 360, CRC Press, Boca Raton 1993). For example, Agrobacterium mediated transformation can be performed using the GV3 (pMP90) (Koncz & Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain.

Arabidopsis thaliana can be grown and transformed according to standard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1 194-1 199; Bent et al. 1994, Science 265:1856-

1860). Additionally, rapeseed can be transformed with the LMR nucleic acids of the present invention via cotyledon or hypocotyl transformation (Moloney et al. 1989, Plant Cell Report 8:238-242; De Block et al. 1989, Plant Physiol. 91 :694-701 ). Use of antibiotic for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using a selectable plant marker. Additionally, Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al. (1994, Plant Cell Report 13:282-285). The Arabidopsis thaliana sucrose transporter 5 gene was cloned into a binary vector and expressed either under the USP promoter or the PtxA promoter (the promoter of the Pisum sativum PtxA gene), which is a promoter active in virtually all plant tissues. However, in seeds and flowers, there is no expression activity detectable by GUS staining and low expression activity detectable with the more sensitive method of RT-PCR (Song, H-S. et al., WO 05/085450). Only in plant lines comprising multiple copies of a transgenic ptxA- promoter/GUS expression construct some expression could be detected in part of the flowers and the siliques (for more details see Song, H-S. et al., WO 05/085450). Alternatively, the superpromoter, which is a constitutive promoter (Stanton B. Gelvin, US 5,428,147 and US 5,217,903) or seed-specific promoters like USP (unknown seed protein) from Vicia faba (Baeumlein et al. 1991 , Mol. Gen. Genetics 225:459-67), or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant J. 2:233-239) as well as promoters conferring seed- specific expression in monocot plants like maize, barley, wheat, rye, rice etc. were used.

Transformation of soybean can be performed using for example a technique described in EP 0424 047, U.S. Patent No. 5,322,783 (Pioneer Hi-Bred International) or in EP 0397 687, U.S. Patent No. 5,376,543, or U.S. Patent No. 5,169,770 (University Toledo), or by any of a number of other transformation procedures known in the art. Soybean seeds are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) tween for 20 minutes with con- tinuous shaking. Then the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours. The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air-dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.

The method of plant transformation is also applicable to other crops. In particular, seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05 % (v/v) Tween for 20 minutes, at room temperature with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. The seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period, the seeds lose approximately 85% of their water content. The seeds are then stored at room temperature in a sealed Petri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8.

Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and re-suspended in MS (Murashige & Skoog 1962, Physiol. Plant. 15:473-497) medium supplemented with 100 mM acetosyringone. Bacteria cultures are incubated in this pre- induction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 44% moisture content are imbibed for 2 h at room tempera- ture with the pre-induced Agrobacterium suspension culture. (The imbibition of dry embryos with a culture of Agrobacterium is also applicable to maize embryo axes). The embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at room temperature. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supple- merited with 500 mg/l carbenicillin or 300 mg/l cefotaxime to kill the agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25°C, under 440 μιηοΙ nr^s^'l and 12 hours photoperiod. Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favour the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25°C, under 440 μιηοΙ nr^s^'l light intensity and 12 h photoperiod for about 80 days.

Samples of the primary transgenic plants (To) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization wherein DNA is electro- phoresed on a 1 % agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labeled probe by PCR as recommended by the manufacturer.

In general, a rice (or other monocot) sucrose transporter 5 gene under a plant promoter like PtxA could be transformed into corn, or another crop plant, to generate effects of monocot sucrose transporter 5 genes in other monocots, or dicot sucrose transporter 5 genes in other dicots, or monocot genes in dicots, or vice versa. The plasmids containing these coding sequences, 5' of a promoter and 3' of a terminator would be constructed in a manner similar to those described for construction of other plasmids herein.

Example 11 - In vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by incorporation and passage of the plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) that are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp W.D. 1996, DNA repair mechanisms, in: Escherichia col\ and Salmonella, p. 2277- 2294, ASM: Washington.) Such strains are well known to those skilled in the art. The use of such strains is illustrated, for example, in Greener and Callahan 1994, Strategies 7:32- 34. Transfer of mutated DNA molecules into plants is preferably done after selection and testing in microorganisms. Transgenic plants are generated according to various examples within the exemplification of this document.

Example 12 - Assessment of the mRNA Expression and Activity of a Recombinant Gene Product in the Transformed Organism

The activity of a recombinant gene product in the transformed host organism can be measured on the transcriptional or/and on the translational level. A useful method to ascertain the level of transcription of the gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for exam- pie, Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labelled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information at least partially demonstrates the degree of transcription of the transformed gene. Total cellular RNA can be prepared from plant cells, tissues or organs by several methods, all well-known in the art, such as that described in Bormann et al. (1992, Mol. Microbiol. 6:317-326).

To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed (see, for example, Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label, which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell. The activity of SUC 5 proteins that bind to DNA can be measured by several well- established methods, such as DNA band-shift assays (also called gel retardation assays). The effect of such SUC 5 protein on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar H. et al. 1995, EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both prokaryotic and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.

The determination of activity of lipid metabolism membrane-transport proteins can be performed according to techniques such as those described in Gennis R.B. (1989 Pores, Channels and Transporters, in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137, 199-234 and 270-322).

Example 13 - In vitro Analysis of the Function of Arabidopsis thaliana Sucrose Transporter 5 Genes in Transgenic Plants

The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one skilled in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M. & Webb, E.C. 1979, Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P.D., ed. (1983) The Enzymes, 3rd ed. Academic Press: New York; Bisswanger, H., (1994) Enzymkinetik, 2nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, H.U., Bergmeyer, J., Grai!>\, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes. VCH: Weinheim, p. 352-363.

Example 14 - Purification of the Desired Product from Transformed Organisms

An SUC 5 protein can be recovered from plant material by various methods well known in the art. Organs of plants can be separated mechanically from other tissue or organs prior to isolation of the seed storage compound from the plant organ. Following homogenization of the tissue, cellular debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from cells grown in culture, then the cells are removed from the cul- ture by low-speed centrifugation and the supernatant fraction is retained for further purification.

The supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin, while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One skilled in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.

There is a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for ex- ample, in Bailey J.E. & Ollis D.F. 1986, Biochemical Engineering Fundamentals, McGraw- Hill: New York).

The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, analytical chromatography such as high performance liquid chromatography, NIRS, enzymatic assay, or microbiologically.

Such analysis methods are reviewed in: Patek et al. (1994, Appl. Environ. Microbiol.

60:133-140), Malakhova et al. (1996, Biotekhnologiya 1 1 :27-32) and Schmidt et al. (1998,

Bioprocess Engineer 19:67-70), Ulmann's Encyclopedia of Industrial Chemistry (1996, Vol. A27, VCH: Weinheim, p. 89-90, p. 521 -540, p. 540-547, p. 559-566, 575-581 and p. 581 -

587) and Michal G. (1999, Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17).

The effect of the genetic modification in plants on a desired seed storage compound (such as a sugar, lipid or fatty acid) can be assessed by growing the modified plant under suitable conditions and analyzing the seeds or any other plant organ for increased production of the desired product (i.e., a lipid or a fatty acid). Such analysis techniques are well known to one skilled in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatog- raphy such as high performance liquid chromatography (see, for example, Ullman 1985, Encyclopedia of Industrial Chemistry, vol. A2, pp. 89-90 and 443-613, VCH: Weinheim; Fallon, A. et al. 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993 Product recovery and purification, Biotechnology, vol. 3, Chapter III, pp. 469-714, VCH: Weinheim; Belter, P.A. et al., 1988 Bioseparations: downstream processing for biotechnology, John Wiley & Sons; Kennedy J.F. & Cabral J. M.S. 1992, Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz J.A. & Henry J.D. 1988, Biochemical separations in: Ulmann's Encyclopedia of Industrial Chemistry, Separation and purification techniques in biotechnology, vol. B3, Chapter 1 1 , pp. 1 -27, VCH: Weinheim; and Dechow F.J. 1989).

Besides the above-mentioned methods, plant lipids are extracted from plant material as described by Cahoon et al. (1999, Proc. Natl. Acad. Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal. Biochemistry 442:141-145). Qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W., Advances in Lipid Methodology. Ayr/Scotland :Oily Press. - (Oily Press Lipid Library; Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland:Oily Press, 1989 Repr. 1992. - IX,307 S. - (Oily Press Lipid Library; and "Progress in Lipid Research, Oxford :Pergamon Press, 1 (1952) - 16 (1977) Progress in the Chemistry of Fats and Other Lipids CODEN. Unequivocal proof of the presence of fatty acid products (Table 1 and 2) can be obtained by the analysis of transgenic plants following standard analytical procedures: GC, GC-MS or TLC as variously described by Christie and references therein (1997 in: Advances on Lipid Methodology 4th ed.: Christie, Oily Press, Dundee, pp. 119-169; 1998). Detailed methods are described for leaves by Lemieux et al. (1990, Theor. Appl. Genet. 80:234-240) and for seeds by Focks & Benning (1998, Plant Physiol. 1 18:91 -101 ).

Table 1 : Plant Lipid Classes

Neutral Lipids Triacylglycerol (TAG)

Diacylglycerol (DAG)

Monoacylglycerol (MAG)

Polar Lipids Monogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG)

Phosphatidylglycerol (PG)

Phosphatidylcholine (PC)

Phosphatidylethanolamine (PE)

Phosphatidylinositol (PI)

Phosphatidylsehne (PS)

Sulfoquinovosyldiacylglycerol

Common Plant Fatty Acids

16:0 Palmitic acid

16:1 Palmitoleic acid

16:3 Palmitolenic acid

18:0 Stearic acid

18:1 Oleic acid

18:2 Linoleic acid

18:3 Linolenic acid

Y-18:3 Gamma-linolenic acid*

20:0 Arachidic acid

20:1 Eicosenoic acid

22:6 Docosahexanoic acid (DHA) *

20:2 Eicosadienoic acid

20:4 Arachidonic acid (AA) *

20:5 Eicosapentaenoic acid (EPA) *

22:1 Erucic acid

The marked up (*) fatty acids do not normally occur in plant seed oils, but their production in transgenic plant seed oil is of importance in plant biotechnology.

Positional analysis of the fatty acid composition at the sn-1 , sn-2 or sn-3 positions of the glycerol backbone is determined by lipase digestion (see, e.g., Siebertz & Heinz 1977, Z. Naturforsch. 32c: 193-205, and Christie 1987, Lipid Analysis 2^nd Edition, Pergamon Press, Exeter, ISBN 0-08-023791 -6).

Total seed oil levels can be measured by any appropriate method. Quantification of seed oil contents is often performed with conventional methods, such as near infrared analysis (NIR) or nuclear magnetic resonance imaging (NMR). NIR spectroscopy has become a standard method for screening seed samples whenever the samples of interest have been amenable to this technique. Samples studied include canola, soybean, maize, wheat, rice, and others. NIR analysis of single seeds can be used (see e.g. Velasco et al., "Estimation of seed weight, oil content and fatty acid composition in intact single seeds of rapeseed" (Brassica napus L.) by near-infrared reflectance spectroscopy, "Euphytica," Vol. 106, 1999, pp. 79-85). NMR has also been used to analyze oil content in seeds (see e.g. Robertson & Morrison, "Analysis of oil content of sunflower seed by wide-line NMR," Journal of the American Oil Chemists Society, 1979, Vol. 56, 1979, pp. 961 -964, which is herein incorporated by reference in its entirety).

A typical way to gather information regarding the influence of increased or decreased protein activities on lipid and sugar biosynthetic pathways is for example via analyzing the carbon fluxes by labeling studies with leaves or seeds using ^c-acetate or 14_C -pyruvate

(see, e.g. Focks & Benning 1998, Plant Physiol. 1 18:91 -101 ; Eccleston & Ohlrogge 1998, Plant Cell 10:613-621 ). The distribution of carbon-14 into lipids and aqueous soluble components can be determined by liquid scintillation counting after the respective separation

(for example on TLC plates) including standards like ^c-sucrose and ^c-malate (Eccleston & Ohlrogge 1998, Plant Cell 10:613-621 ).

Material to be analyzed can be disintegrated via sonification, glass milling, liquid nitrogen and grinding or via other applicable methods. The material has to be centrifuged after disintegration. The sediment is re-suspended in distilled water, heated for 10 minutes at 100°C, cooled on ice and centrifuged again followed by extraction in 0.5 M sulfuric acid in methanol containing 2% dimethoxypropane for 1 hour at 90°C leading to hydrolyzed oil and lipid compounds resulting in transmethylated lipids. These fatty acid methyl esters are extracted in petrolether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at a temperature gradient between 170°C and 240°C for 20 minutes and 5 min. at 240°C. The identity of resulting fatty acid methylesters is defined by the use of standards available form commercial sources (i.e., Sigma).

In case of fatty acids where standards are not available, molecule identity is shown via deri- vatization and subsequent GC-MS analysis. For example, the localization of triple bond fatty acids is shown via GC-MS after derivatization via 4,4-Dimethoxy-oxazolin-Derivaten (Christie, Oily Press, Dundee, 1998).

A common standard method for analyzing sugars, especially starch, is published by Stitt M., Lilley R.Mc.C, Gerhardt R. and Heldt M.W. (1989, "Determination of metabolite levels in specific cells and subcellular compartments of plant leaves," Methods Enzymol. 174:518- 552; for other methods see also Hartel et al. 1998, Plant Physiol. Biochem. 36:407-417 and Focks & Benning 1998, Plant Physiol. 1 18:91 -101 ).

For the extraction of soluble sugars and starch, 50 seeds are homogenized in 500 μΙ of 80% (v/v) ethanol in a 1.5-ml polypropylene test tube and incubated at 70°C for 90 min. Follow- ing centrifugation at 16,000 g for 5 min, the supernatant is transferred to a new test tube. The pellet is extracted twice with 500 μΙ of 80% ethanol. The solvent of the combined su- pernatants is evaporated at room temperature under a vacuum. The residue is dissolved in 50 μΙ of water, representing the soluble carbohydrate fraction. The pellet left from the etha- nol extraction, which contains the insoluble carbohydrates including starch, is homogenized in 200 μΙ of 0.2 N KOH, and the suspension is incubated at 95°C for 1 h to dissolve the starch. Following the addition of 35 μΙ of 1 N acetic acid and centrifugation for 5 min at 16,000 g, the supernatant is used for starch quantification.

To quantify soluble sugars, 10 μΙ of the sugar extract is added to 990 μΙ of reaction buffer containing 100 mM imidazole, pH 6.9, 5 mM MgCl2, 2 mM NADP, 1 mM ATP, and 2 units 2 ml^"1 of Glucose-6-P-dehydrogenase. For enzymatic determination of glucose, fructose and sucrose, 4.5 units of hexokinase, 1 unit of phosphoglucoisomerase, and 2 μΙ of a saturated fructosidase solution are added in succession. The production of NADPH is photometrically monitored at a wavelength of 340 nm. Similarly, starch is assayed in 30 μΙ of the insoluble carbohydrate fraction with a kit from Boehringer Mannheim.

Enzymatic assays of hexokinase and fructokinase are performed spectrophotometrically according to Renz et al. (1993, Planta 190:156-165), of phosphogluco-isomerase, ATP- dependent 6-phosphofructokinase, pyrophosphate-dependent 6-phospho-fructokinase, Fructose-1 ,6-bisphosphate aldolase, those phosphate isomerase, glyceral-3-P dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase are performed according to Burrell et al. (1994, Planta 194:95-101 ) and of UDP-Glucose- pyrophosphorylase according to Zrenner et al. (1995, Plant J. 7:97-107).

Intermediates of the carbohydrate metabolism, like Glucose-1 -phosphate, Glucose-6- phosphate, Fructose-6-phosphate, Phosphoenolpyruvate, Pyruvate, and ATP are measured as described in Hartel et al. (1998, Plant Physiol. Biochem. 36:407-417) and metabolites are measured as described in Jelitto et al. (1992, Planta 188:238-244).

In addition to the measurement of the final seed storage compound (i.e., lipid or starch) it is also possible to analyze other components of the metabolic pathways utilized for the production of a desired seed storage compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound (Fiehn et al. 2000, Nature Biotech. 18:1447-1 161 ). For example, yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for alterations in sugar, oil, lipid or fatty acid contents.

Similarly, plant expression vectors comprising the nucleic acids disclosed herein, or frag- ments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soybean, rapeseed, rice, maize, wheat, Medicago truncatula, etc., using standard protocols. The resulting transgenic cells and/or plants derived there from can then be assayed for alterations in sugar, oil, lipid or fatty acid contents.

Additionally, the sequences disclosed herein, or fragments thereof, can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, mammalian cells, yeast cells, and plant cells (Girke at al. 1998, Plant J. 15:39-48). The resultant knockout cells can then be evaluated for their composition and content in seed storage compounds, and the effect on the phenotype and/or genotype of the mutation. For other methods of gene inactivation include US 6004804 "Non-Chimeric Mutational Vectors" and Puttaraju et al. (1999, "Spliceosome-mediated RNA frans-splicing as a tool for gene therapy" Nature Biotech. 17:246-252).

Example 15 - Analysis of the Impact of SUC 5 Protein on the Production of a Desired Seed Storage Compound

The genomic SUC5 sequence (SEQ ID NO: 1 ) was amplified from genomic Arabidopsis thaliana DNA (ecotype Wassilewskija, Ws) with Phusion High Fidelity Polymerase (Finnzymes, Espoo, Fl) using the oligonucleotides AtSUC5+1-Ascl (5'- GAG AGA GAG AGA GGC GCG CCA TGG GAG CCT TGG AAG CAG AAA G -3') and AtSUC5+2082r-Notl (5'-GAG AGA GAG AGA GCG GCC GCC TAA TGG AAT CCC ATA GCC CCT GAC -3'). The obtained PCR fragments were then cloned into the TOPO PCR Blunt II vector (Life Technologies, Carlsbad, CA, USA) and sequenced using the insert flanking vectors M13-f and M13-r. Error-free clones were directionally cloned into the Gateway entry vector VC- LJB1006-1 (Entry_C vector) behind the USP promoter sequence using the restriction endo- nucleases Ascl and Notl (New England Biolabs, Frankfurt, Germany). The resulting entry vector pEntryC-USPp::SUC5 was then subjected into a Gateway reaction using the Gateway LR Clonase Plus II Enzyme Mix (Life Technologies, Carlsbad, CA, USA) together with the entry vectors VC-LJB2174-3 (Entry_A vector, carrying a Napin promoter sequence) and VC-LLL895-1 (Entry_B vector, carrying a USP promoter sequence) and the destination vector VC-LLL1 164-1 , containing a streptomycin/ spectinomycin resistance gene for bacterial selection and a Imazamox resistance gene (AtAHAS) for transgenic plant selection. The Entry_A and Entry_B vectors did not contain any ORF sequences within the cassettes integrated in the final expression clone and were solely used for providing the attachment sides needed for the Gateway reaction. The resulting expression clone pDEST-USP:SUC5 (Figure 8) was then transformed into the Agrobacterium tumefaciens strain C58 (Deblaere et al. 1985. Nucl. Acids Res 13:4777-88). Arabidopsis thaliana plants (Ws) were transformed using the floral dip method described by Clough and Bent (1998. Plant J. 16, 735-743). Transgenic plants were identified by selecting the germinating seeds of the dipped plants with the herbicide Imazamox. Resistant plants were further tested with PCR using a USP promoter specific forward primer {USP-fwd, 5'- CTG CAG CAA ATT TAC ACA TTG CCA CTA-3') and a SUC5 specific reverse primer {SUC5-rev, 5'-TAC ACT TCA CGA CCC ATC CA-3') for correct insert integration. Seeds from transformed Arabidopsis thaliana T2-plants are analyzed by gas chromatography (GC) for total oil content and fatty acid profile. Arabidopsis (ecotype Wassilewskija, WS) is used to investigate the influence of a sucrose transporter 5 gene (SEQ ID NO 1 ) over-expression on seed storage compound accumulation, see also for more information Table 3.

Table 3: A table of the function of the SUC 5 protein

Total fatty acid content of seeds of control and transgenic plants are measured with bulked seeds (usually 5 mg seed weight) of a single plant. Two different types of controls are used: Untransformed Ws wt plants, that are grown under exactly the same conditions as the transformed plants and BPS empty (without SUC 5 protein gene of interest) binary vector construct (GB1 ). Seeds from transgenic and control plants are sown on potting soil (65% peat, 25% washed sand, 10% clay granulate), stratified in the dark for 3 days at 4°C and then transferred to the growth chamber. Plants are grown under short day conditions (8 h light/16 h dark cycles) for 4 weeks and then long day conditions (16 h light/8 h dark cycles at 180-200 moles nr² sec- ¹) at 22°C with 60% relative humidity. After induction of flowering, inflorescences are framed with Plexiglas tubes. Plants are watered during seed development and ripe seeds are collected not before 10 weeks after sowing. Seeds are filtered and stored at room temperature in the dark at low humidity until fatty acid analysis. The sucrose transporter 5 gene expression is driven by a seed specific USP promoter. The p values (as obtained by simple t-test) reveal significant increases in at least 2 independent transgenic events in the T3 seed gen- eration of at least 5% respectively. The results suggest that sucrose transporter 5 over- expression with a seed specific promoter allows the manipulation of total seed oil content.

Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims to the invention disclosed and claimed herein.

Example 16 - Analysis of the Impact of the SUC 5, the GPT1 and the NTT1 Proteins on the Production of a Desired Seed Storage Compound

The genomic SUC5 sequence (SEQ ID NO: 1) was amplified from genomic Arabidopsis thaliana DNA (ecotype Wassilewskija, Ws) with Phusion High Fidelity Polymerase (Finnzymes, Espoo, Fl) using the oligonucleotides AtSUC5+1-Ascl (5'- GAG AGA GAG AGA GGC GCG CCA TGG GAG CCT TGG AAG CAG AAA G -3') and AtSUC5+2082r-Notl (5'-GAG AGA GAG AGA GCG GCC GCC TAA TGG AAT CCC ATA GCC CCT GAC -3'). The obtained PCR fragments were then cloned into the TOPO PCR Blunt II vector (Life Technologies, Carlsbad, CA, USA) and sequenced using the insert flanking vectors M13-f and M13-r. Error-free clones were directionally cloned into the Gateway entry vector VC- LJB1006-1 (Entry _C vector) behind the USP promoter sequence using the restriction endo- nucleases Ascl and Notl (New England Biolabs, Frankfurt, Germany). The resulting entry vector pEntryC-USPp::SUC5 was then subjected into a Gateway reaction using the Gate- way LR Clonase Plus II Enzyme Mix (Life Technologies, Carlsbad, CA, USA) together with the entry vectors GPT1 [EntrA-pUSP] (Entry_A vector, carrying the GPT1 (SEQ ID NO: 83) cDNA sequence under control of the USP promoter) and NTT1 [EntrB-pUSP] (Entry_B vector, carrying the NTT1 (SEQ ID NO: 84) cDNA sequence under control of the USP promoter sequence) and the destination vector VC-LLL1 164-1 , containing a streptomycin/ spectino- mycin resistance gene for bacterial selection and a Imazamox-resistance gene (AtAHAS) for transgenic plant selection. The resulting expression clone pDEST- USP:GPT/USP:NTT/USP:SUC5 (Figure 9) was then transformed into the Agrobacterium tumefaciens strain C58 (Deblaere et al. 1985. Nucl. Acids Res 13:4777-88). Arabidopsis thaliana plants (Ws) were transformed using the floral dip method described by Clough and Bent (1998. Plant J. 16, 735-743). Transgenic plants were identified by selecting the germinating seeds of the dipped plants with the herbicide Imazamox. Resistant plants were further tested with PCR using a USP promoter specific forward primer (USP-fwd, 5'- CTG CAG CAA ATT TAC ACA TTG CCA CTA -3') and a SUC5 specific reverse primer (SUC5- rev, 5'- TAC ACT TCA CGA CCC ATC CA -3') for correct insert integration. Plants success- fully transformed with the pDEST-USP:GPT/USP:NTT/USP:SUC5 are designated below as BioOI3-plants.

Seeds from transgenic BioOI3 plants of the T2 generation and control plants were sown on potting soil (65% peat, 25% washed sand, 10% clay granulate), stratified in the dark for 3 days at 4°C and then transferred to the growth chamber. Plants were grown under short day conditions (8 h light/16 h dark cycles) for 4 weeks and then long day conditions (16 h light/8 h dark cycles at 180-200 pinoles nr² sec-¹) at 22°C with 60% relative humidity. After induction of flowering, inflorescences were framed with Plexiglas tubes. Plants were watered during seed development and ripe seeds were collected not before 10 weeks after sowing. Seeds were filtered and stored at room temperature in the dark at low humidity until fatty acid analysis.

Seeds from at least 10 different plants of the T2 generation of each independent transgenic BioOI3 line and from 10 wt plants (ecotype Ws) were analyzed by gas chromatography (GC) for total oil content and fatty acid profile as described in Example 1 (Analysis of TAG and fatty acids). Total TAG content (Figure 1 1 ) and fatty acid composition of the TAGs (Fig- ure 10) were measured. A significant increase in total TAG levels of at least 5% could be detected for 2 independent lines (p<0.05 for BioOI3-1 and BioOI3-15, Figure 1 1 ). Total TAG contents within individual plants from independent BioOI3 lines show a Gaussian distribution (Figure 13). Moreover, when TAG content is plotted against the number of T2 plants from the BioOI3 lines and the analyzed wt plants, a peak shift towards higher TAG content was observed for the distribution of BioOI3 plants. Figure 13 shows such histograms for the 3 BioOI3 lines with the highest increase in total TAG (lines BioOI3-1 , BioOI3-4 and BioOI3- 15). When the fatty acid (FA) composition of the TAGs was analyzed, more significant increases of individual FAs could be detected for almost all independent BioOI3 lines. In particular, highly significant increases in pg/mg seeds could be detected for the FAs C20:0 (increase from 6.27±0.39 to 7.36±0.34, p<0.001 ), C20:1 [1 1] (increase from 56.94±4.66 to 67,30±5.70, p<0.001 ), C20:2 (increase from 5.25±0,39 to 7.03±0.60, p<0.001 ), C22:0 (in- crease from 0.43±0.05 to 0.66±0.06, p<0.001 ) and C22:1 (increase from 4.12±0.31 to 5.78±0.41 , p<0.001 ) in 12 independent BioOI3 lines. Figure 12 shows the FA composition of the 3 BioOI3 lines with the highest overall TAG increase. Interestingly, such significant increases could only be measured for C20 or longer fatty acids. These FAs are being elongated in the plant cytosol, whereas elongation of fatty acids until C18 takes place in the plastids. This specific increase in FAs elongated in the cytosol becomes also apparent, when the weight percentages (weight %) of the individual FAs are calculated (Table 4). In wt plants the weight % of TAG FAs elongated in the plastids (up to C18) is 76.6% against 23.4% for the TAG FAs elongated in the cytosol. In BioOI3 lines, this percentage is shifted in favor of cytosolic elongated FAs (72.6 % FA elongated in the plastids against 27.4% FA elongated in the cytosol). The most distinct increase in weight % is calculated for eicosenoic acid [C20:1 (1 1 )], which accounts for an increase of 3.1 weight % from 16.1 % to 19.2%.

Table 4:

Example 17

Sequence Alignment of AtSUCs

In the alignment shown in Figure 14 the nine disaccharide transporters from Arabidopsis thaliana share 25.3% identical positions (black/ white) and 77.0% consensus positions (grey/ white). For seven SUCs sucrose transport activity has been reported (SUC1/2: Sauer and Stolz, 1994, SUC3: Barker et al., 2000, Meyer et al., 2000, SUC4: Weise et al., SUC5: Ludwig et al., 2000, SUC8/9: Sauer et al., 2004). SUC6 and SUC7 are encoded by pseudogenes and no functional transport properties have been described for the corresponding proteins (Sauer et al., 2004). SUC mediated transport of biotin has only been shown for SUC5 and the SUC2 homolog from Plantago major, PmSUC2 (Ludwig et al, 2000) so far and not been published for the remaining SUCs. The high degree of sequence and structural homology between SUC5 and the other members of the SUC family in Arabidopsis suggests nevertheless that biotin transport is a feature of other SUCs as well. Overexpression of single SUC family members in seed as disclosed in Figure 14 increases TAG content in the seed.

Example 18

TAG Content of BioOil4 (PSUC5::SUC5) Plants

Total TAG content of dry seeds obtained from wt (white bar) and transgenic BioOil4 plants (black bars) is shown in Figure 15. Total TAG content is shown in g/mg dry seeds for 1 1 independent transgenic BioOil4 lines. Error bars represent measurements of seeds from 10 different plants per line. An average increase of +4.7% TAG was observed throughout all BioON4 plants. The BioON4 line 4-16 showed an increase of +9.5 % TAG. In BioOil4 plants, the SUC5 gene is under the control of its endogenous promoter. The endogenous SUC5 promoter is disclosed in Figure 18. Figure 18 shows the SUC5 promoter sequence as used in constructs used for construction of above BioOil4 plants. Sequences highlighted in grey or dark grey show primers used for amplification of the promoter sequence from genomic Arabidopsis DNA.

Example 19

Expression of AtBIOI and AtCACIA in AtSUC5 over-expressing plants

qPCR measurements of AtSUC5-, AtBIOI - and AtCAC1A-mRNA levels in siliques (10 DAF) of Wild-type (Wt, white bar) and selected BioOil3 and Biooil4 lines (black bars). In the SUC5-overexpressing BioOil3 and BioOil4 lines, elevated levels of SUC5 mRNA were followed by an increased amount of BI01 -mRNA coding for the biotin synthesis enzyme BI01 and of CAC1A-mRNA coding for the biotin binding subunit of ACCase, BCCP2. These data strongly suggest that increased SUC5 activity induces enzymes necessary for fatty acid elongation. Expression of TRANSLOCASE INNER MEMBRANE 44-2 (ΑΠΊΜ44-2) was used as internal reference gene for determination of relative expression (Kleindt et al., 2010). Example 20

Content of Total and Free Biotin in Seeds of Wt and SUC5 Over-expressing Plants

Total and free biotin levels were measured in fully developed seeds of Wild-type plants (Wt) and plants expressing the SUC5 gene either under the control of the USP-promoter

(PUSP::SUC5 = BioOil3-lines) or under the control of its own promoter (PSUC5::SUC5 = BioOil4-lines). Biotin levels were measured using the FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins from Molecular Probes (Life Technologies). Levels of free biotin were obtained by homogenizing seed tissue in aqueous solution, total biotin levels (i.e. free biotin plus protein bound biotin) were obtained by hydrolyzing homogenized seed tissue in 2N sulphuric acid. Results show an elevation of total biotin content in the seeds of SUC5-overexpressing plants as shown in Figure 17. Moreover, decrease in free biotin content and therefore an elevated total/free biotin ratio in these plants indicate a higher incorporation of free biotin in target proteins like ACCase in SUC5-overexpressing plants. This correlates with the elevated amount of mRNA coding for the BCCP2 subunit of ACCase presented in Figure 16.

Example 21

SUC5 expression during seed development

SL/C5-mRNA levels were measured by qPCR in developing seeds harvested from siliques of wt or SL/C5-overexpressing lines (BioOil3, BioOil4) at the indicated days after flowering (DAF). ACTIN2 (ACT2) was used as internal reference gene for the determination of relative expression. SUC5 mRNA levels are strongly elevated in the SL/C5-overexpressing lines BIOOH3 and BIOOH4 in contrast to the wt (Figure 19). SUC5 expression in BioOil3 exceeds wt peak expression of SUC5 already at 4-DAF. In BioOil4, SUC5 expression is also increased already at 4-DAF and stays high until 8-DAF, a time when SUC5 expression in the wt has declined to almost zero.

Example 22

Sucrose and raffinose content in ripe seeds of wt, BioOil3 and BioOM.

Both sucrose and raffinose levels are unaltered between seeds (>21 -DAF) of wt and the SL/C5-overexpressing lines, indicating that elevated sucrose import during seed development in BioOil3 and BioOM plants rather leads to higher TAG synthesis than to greater carbohydrate storage - see Figure 20.

Example 23

Uptake of sucrose and biotin by wt, BioOil3 and BioOM embryos.

Siliques (8-DAF) from wt and SUC5 overexpressing BioOil3 and BioOM plants were collected and dissected under the binocular with fine forceps. Developing seeds were selected and zygotic embryos in the upturned U stage were transferred into 25 mM sodium phosphate buffer pH 7.0. For every uptake experiment 50 embryos were incubated at 22 °C in 200 μΙ solution containing 25 mM NaHP0 (pH 5.5) and 2 mM CaCI₂ added with the radiolabeled substrate at the indicated concentration. Incubation time was 6 h for biotin and 90 min for sucrose. After incubation samples were filtered on glass microfibres filters 696 (VWR, Darmstadt, Germany) and washed with an excess of distilled H₂0. Incorporation of radioactivity was determined by scintillation counting.

Bars and errors in Figure 21 (A) and (B) represent mean values and standard deviations from 3 independent measurements.

(D) Uptake of 2 mM [ ⁴C]-sucrose into wt, BioOH3 and BioOM embryos at 8-DAF. The sucrose taken up per embryo after 90 min is given in pmoles.

(E) Uptake of 10 μΜ [ ⁴C]-biotin into wt and BioOM embryos at 8-DAF. The biotin taken up per embryo after 6 h is given in fmoles.

(F) Isolated wt, BioOil3 and BioOM embryos at 8-DAF used for uptake measurements with radiolabeled biotin or sucrose. Bars are 250 pm. REFERENCES

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Claims

873

2013/024121 89 PCT/EP2012/065958

A method of producing a transgenic plant having an increased level of fatty acids in the seed comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence, generating from the plant cell the transgenic plant, analyzing the production of fatty acids in the seed of the transgenic plant, and selecting a transgenic plant having an increased level of fatty acids as compared to a corresponding untransformed wild type variant of the plant, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence as defined in SEQ ID NO:1 ;

The method of producing a transgenic plant according to claim 1 , wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence comprises a polynucleotide having at least 90% sequence identity with the polynucleotide sequence of a), b) or c).

The method of producing a transgenic plant according to claim 1 and 2, wherein the seed- specific promoter is the USP promoter.

The method of producing a transgenic plant according to claim 1 and 2, wherein the seed- specific promoter is the SUC5 promoter.

A method of increasing the level of the total fatty acids in the seed of a plant comprising, transforming a plant cell with an expression vector comprising a nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence, generating from the cell a transgenic plant, and selecting a transgenic plant having an increased level of fatty acids as compared to a corresponding untransformed wild type variety of the plant, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence as defined in SEQ ID NO:1 ;

b) a polynucleotide sequence encoding a polypeptide as defined in SEQ ID NO:2; PF 71873

WO 2013/024121 90 PCT/EP2012/065958 c) a polynucleotide sequence encoding a polypeptide having at least 70 % sequence identity with the polypeptide as defined in SEQ ID NO: 2;

A method of increasing the level of the total fatty acids in the seed of a plant according to claim 5, wherein the nucleic acid sequence encoding a sucrose transporter 5 polypeptide sequence comprises a polynucleotide sequence having at least 90% sequence identity with the polynucleotide sequence of a), b) or c).

A method of increasing the level of the total fatty acids in the seed of a plant according to claim 5 and 6, wherein the seed-specific promoter is the USP promoter.

A method of increasing the level of the total fatty acids in the seed of a plant according to claim 5 and 6, wherein the seed-specific promoter is the SUC5 promoter.

A transgenic plant with increased total fatty acids content in the seed of the plant as compared to a wild type variety of the plant comprising a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence as defined in SEQ ID NO:1 ;

A transgenic plant with an increased total fatty acids content in the seed of the plant as compared to a wild type variety of the plant comprising a polypeptide encoding the polypeptide as described by SEQ ID NO: 2 or a polypeptide having at least 70% sequence identity with the polypeptide as defined by SEQ ID NO: 2.

1 1. Transgenic seed produced by a transgenic plant according to any of claims 9 and 10.