CA2261775A1 - Isolated spinach rubisco large subunit n-methyltransferase - Google Patents

Abstract

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Description

CA 0226177~ 1999-01-28 W O 98tO4116 PCTrUS97113095 TSOLATED SPnNACH RUB~SCO LARGE SUBUNrr N-~fETHYLTRANSFERASE

S

ACKNOWLEDGEMENT OF GOVERNMEN~ SUPPORT
This invention was made with Government support under Grant No. DE-FG05-92ER26075, awarded by the Department of Energy. The Govermnent may have certain rights in this invention.

BACKGROUND OF THE INVENTION
1. Field of the Invention This invention relates to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) ~N-methyltransferase (protein methylase ~II or Rubisco LSMT). This enzyme catalyzes methylation of the ~-amine of 15 lysine-14 in the large subunit of Rubisco. Many plant species contain methylated Lys-14 in the LS of Rubisco but some do not (i.e., a des(methyl) lysyl residue in the LS). In addition, the present invention relates to a gene and full-length cDNA clones for Rubisco LSMT. The present invention further relates to transgenic plants and methods of producing same which have the Rubisco LSMT
20 gene inserted into the DNA. This invention also relates to a four amino acid insert (WVQQ) which inactivates the enzymatic activity of Rubisco LSMT and therehy accounts for the subsequent absence of trimethyllysine-14 in the LS of Rubisco.

2. Des.lil lion of the Related Art Protein methylation is a widespread and common post-translational modification catalyzed by several different protein methyltransferases (Paik et al., "Protein methylation," in Freeflm~n et al. (eds), The Enzymology of Posttranslational Modifications of P~oteins, vol. 2, pp. 187-228, Academic Press, London (1985)). Proteins which contain trimethyllysyl residues include CA 0226177~ 1999-01-28 W O 98tO4116 PCTrJS97113095 cytochrome c (Cessay et al., "The relationship between the trimethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae," Int. J.
Biochem. 26(5):721-734 (1994); Cessay et al., "Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria, " Int. J.Biochem. 23(7,8): 761-768 (1991); DiMaria et al., "Cytochrome c specific methylase from wheat germ," Bioc~emistry 21:1036-1044 (1982); Farooqui et al., "Effect of Methylation on the Stability of Cytochrome c of Saccharomyces cerevisiae in vivo~" J. Biol. Chem. 256(10):5041-5045 (1981); and Farooqui et al., "In vivo studies on yeast cytochrome c methylation in relation to protein synthesis, "J. Biol. Chem. 255(10):4468-4473 (1980)), calmodulin (Han et al., "Isolation and kinetic characterization of the calmodulin methyltransferase fromsheep brain," Biochemistry 32:13974-13980 (1993); and Rowe et al., "Calmodulin N-methyltransferase, " J. Biol. Chem. 261(15):7060-7069 (1986)), histone-H1 (Sarnow et al., "A histone H4-specific methylllall~reldse properties,specificity and effects on nucleosomal histones," Biochim. Biophys. Acta 655:349-358 (1981); and Tuck et al., "Two histone H1-specific protein-lysine N-methyltransferases from Euglena gracilis, " J. Biol. Chem. 260(11):7114-7121 (1985)), and ribosomal prot~ s (Chang et al., "Purification and properties of a ribosomal protein methylase from Escherichia coli Q13," Biochemistry 14(22):4994-4998 (1975); Lobet et al., "Partial purification and characterization of the specific protein-lysine N-methyll,all~relase of YL32, a yeast ribosomal protein," Biochim. Biophy. Acta 997:224-231 (1989)). However, the biological function of post-translational protein methylation in all but a few systems remains obscure. Trimethyllysine can serve as a metabolic precursor to carnitine (Paik et al., "Carnitine biosynthesis via protein methylation," TIBS 2: 159-162 (1977)), while carboxyl methylation of bacterial membrane proteins plays a major role in chemotaxis (Clarke, "Protein carboxyl methylL~ sr~lases: Two distinct classes of enzymes," Ann. Rev. Biochem. 54: 479-506 (1985)). Evidence suggests that methylation of Lys-115 in calmodulin affects certain activities including in vitro NAD kinase activation (Roberts et al., "Trimethyllysine and protein function,"

.. ., .. ~ . ..

CA 0226177~ 1999-01-28 W O 98/04116 PCT~US97/13095 J. Biol. Chem. 261(4):1491-1494 (1986)), and in vivo susceptibility to ubiquitination (Gregori et al., "Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination," J. Biol. Chem. 262(6):2562-2567 (1987);
and Gregori et al., "Specific recognition of calmodulin from Dictyosfeli~lm discoideum by the ATP ubiquitin-dependent degradative pathway," J. Biol.
Chem. 260(9):5232-5235 (1985); but see also Ziegenhagen et al., "Multip}e ubiquitination of calmodulin results in one polyubiquitin chain linked to calmodulin," FEBSLett. 271(1,2):71-75 (1990); and Ziegenhagen et al., "Plant and fungus calmodulins are polyubiqllitin~t~d at a single site in a Ca2+-dependent manner," FEBSLett. 273(1,2):253-256 (1990)). Conflicting reports (Faroo~ui et al., "Effect of Methylation on the Stability of Cytochrome c of Saccharomyces cerevisiae in vivo," J. Biol. Chem. 256(10):5041-5045 (1981); Frost et al., "Cytochrome c methylation," Protein methylation, Ch. 4, pp. 59-76 (1990); and Frost et al., "Effect of enzymatic methylation of cytochrome c on its function and synthesis," Int. J. Biochem. 22(10):1069-1074 (1990); versus Cessay et al., "The relationship between the trimethylation of Iysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae," Int. J. Biochem. 26(5):721-734 (1994); Cessay et al., "Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria," Int. J. Biochem.
23(7,8):761-768 (1991)) also implicate methylation of Lys-77 in cytochrome c as having a role in protein stability, heme incorporation, and mitochondrial transport. A major limitation to elucidating the biological role of Iysine methylation in eukaryotes has been the absence of a protein methylase III gene.
Hence, molecular studies of the physiological and biochemical function performed by methylation of protein bound lysyl residues have been restricted tosite-directed mutational analysis of the methylation site in the target protein (Ceesay et al., "The relationship between the trirnethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae," Int. J. Biochem.
26(5):721-734 (1994); Cessay et al., "Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria, " Int. J. Biochem.

CA 0226l77~ l999-0l-28 W O 98tO4116 PCTrUS97/13095 2,3(7,8):761-768 (1991); and Roberts et al., "Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants," Proc. Nat. Acad. Sci. USA 89:8394-8398 (1992)). These studies have been inconclusive as to the exact biological role of methylation of the ~-amine of protein bound lysyl residues.
Ribulose-l,S-bisphosphate carboxylase-oxygenase (Rubisco) catalyzes the reduction of atmospheric CO2 during photosynthesis. A great deal is known about the quaternary structure, catalytic mech~ni~m, active site residues, in vivo regulatory mech~ni~m~, and gene ~ ession for this abundant enzyme, see, for 0 example, Andrews et al., "Rubisco: Structure, Mech~ni~m.~, and Prospects for Improvement," in Hatch et al. (eds), 7he Biochemistry of Plants, vol. 10, pp. 131-218. Academic Press, New York (1987); Dean et al., "Structure, evolution, and regulation of rbcS genes in higher plants," Annu. Rev. Plant.
PhysioZ. Plant Mol. Biol. 40: 415-439 (1989); and Mullet, "Chloroplast development and gene expression," Ann~. Rev. Plant. Physiol. Plant Mol. Biol.
3g: 475-502 (1988). Higher plant Rubisco is a hex~3er~rneric protein composed of eight chloroplast-encoded large subunits (referred to herein as "LS") and eight nuclear-encoded small subunits (referred to herein as "SS"). Synthesis of the LSis accompanied by post-translational processing of the N-tçrmin~l domain (Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989); and Mulligan et al., "Reaction-interm~di~te analogue binding by ribulosebisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: The amino-terminal residue of the large subunit is acetylated proline," Proc. Natl. Acad. Sci. USA 85:1513-1517 (1988)). The N-terminal Met-l and Ser-2 are removed and Pro-3 acetylated. Additionally, the LS of Rubisco from tobacco, mll~kmelon, pea, and several other species is post-translationally modified by trimethylation of the ~-amine of Lys-14 (Houtz et al., "Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant CA 0226177~ 1999-01-28 species, " "Plant Physiol. 98: 1170-1174 (1992); Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989)). The enzyme responsible for this latter modification is a highly specific chloroplast-5 localized S-adenosylmethionine (AdoMet):protein (lys) ~N-methylLIall~rel~se ~protein methylase III, Rubisco LSMT, EC 2.1.1.43) (Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989)).
Rubisco LSMT has been affinity purified ~ 8000-fold from pea 10 chloroplasts and identified as a monomeric protein with a molecular mass of ~ 57 kDa (Wang et al., "Affinity Purification of Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase Large Subunit fN-Methyl~dnsr~dse," accepted by Protein Expression and Purification (1995)). Recently, Rubisco LSMT cDNAs have been cloned and sequenced from pea and tobacco (Klein et al., "Cloning 15 and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase," Plant Molecular Biol. 27:249-261 (1995); Ying et al., "Org~ni~tion and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit lN-methyltransferase gene in tobacco, " Plant Molecular Biology (ln press)). The 20 dç~ ce~l amino acid sequences of tobacco Rubisco LSMT has 64.5% identity and 75.3% similarity with the sequence of pea Rubisco LSMT, and both proteins contain several copies of a conserved imperfect leucine-rich repeat motifs (Yinget al., "Org~ni~tion and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit ~N-methyltransferase gene in tobacco,"
25 Plarlt Molec~lar Biology (In press)).
Rubisco LSMT has high specific specificity, methylating only Rubisco ~ and only lysyl residue 14 in the LS. Of many plant species e~min~d several contain methylated Lys-14 in the LS of Rubisco, such as pea and tobacco, but some do not, such as spinach and alfalfa (Houtz et al., "Post-translational 30 modifications in the large subunit of ribulose bisphosphate .

CA 0226177~ 1999-01-28 W O98/04116 PCT~US97113095 carboxylaseloxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989);
Houtz et al., "Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylaseloxygenase from several plant species," Plant Physiol. 98:1170-1174 (1992); and unpublished data).
There has been no explanation for the existence of Lys-14 in the LS of Rubisco in a non-methylated state (i.e., a des(methyl) lysyl residue in the LS). Further, since some plant species, such as spinach, wheat, corn (maize) and lettuce do not contain methylated Lys-14 in the LS of Rubisco (Houtz et al. "Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species, " Plant Physiol.
98:1170-1174 (1992); and unpublished data), it was once assumed that these same plant species did not possess the Rubisco LSMT gene.

SUMMARY OF THE INVENTION
In view of the state of the art as previously described, there thus exists a need in the art for a better underst~n-lin~ of post-translational protein methylation in plants. More specifically, a better underst~n-ling for the molecular basis for the absence of trimethylation-14 in the LS of Rubisco from certain plant species.
It is thus an object of the present invention to provide a Rubisco LSMT
gene.
It is a further object of the present invention to provide the DNA and amino acid sequence for a Rubisco LSMT enzyme.
It is a still further object of the present invention to provide full-length cDNA clones for Rubisco LSMT.
In a first aspect, the present invention relates to a Rubisco LSMT gene which exists in a higher plant with a des(methyl) lysyl residue in the LS of Rubisco. A particularly ~lcrellcd higher plant includes the spinach plant.
In a second aspect, the present invention relates to the DNA and amino acid sequence for a Rubisco LSMT enzyme.

CA 0226177~ 1999-01-28 wo 98/04116 Pcr/uss7/1309s In a third aspect, the present invention relates to a recombinant vector including the Rubisco LSMT gene described above. The vector is suitable for tran~rollllillg higher plants.
In a fourth aspect, the present invention relates to an isolated or 5 recombinant Rubisco LSMT en_yme encoded by the Rubisco LSMT gene described above.
ln a fifth aspect, the present invention relates to a recombinant or transgenic plant transformed with the Rubisco LSMT gene described above.
In a sixth aspect, the present invention relates to a method of inactivating 10 Rubisco LSMT activity which comprises inserting a 4 amino acid sequence insert (WVQQ) into Rubisco LSMT.
In a further aspect, the present invention relates to a method for preventing or redllcing Rubisco LSMT activity in a photosynthesizing plant comprising transforming a photosynth~si7ing plant with a recolllbhlalll vector 15 wherein the vector comprises a Rubisco LSMT gene with the 12 nucleotide insert.
With the foregoing and other objects, advantages and features of the invention that will become heleillaflel a~arellt, the nature of the invention may be more clearly understood by reference to the following detailed description of20 the ple~l-ed embo~1im~nt~ of the invention and to the appended claims.

BRIEF DESCRIPTION OF T~IE DRAWINGS
Figure lA illustrates the genomic org~ni7.~tion and restriction map of rbcMT-S. Exons are shown as heavy black bars, introns as horizontal lines, and the auxon is inrlic~ted by an arrow.
2~ Figure lB is a diag~ ic represçnt~tion of the S3~ and S40 cDNAs with coding regions as heavy black bars, untr~n~ d regions as open bars and the auxon as a shaded bar.
Figure lC shows Probe I, which is a 1056-bp S~I fragment with the 12-bp auxon, and Probe II, which is a riboprobe for the RNAase protection assay CA 0226177~ 1999-01-28 W O 98/04116 PCTrUS97/13095 which results in only one 775-nt fragment protected by S40 mRNA, and two 306-nt and 457-nt fragments protected by S38 rnRNA.
Figure lD depicts the strategy for PCR cloning and joining different cDNA fr~mPnts. The restriction enzymes labeled with stars were used to ligate S corresponding fr~m~nt~. Abbreviations for restriction sites: B, BglI; E, EcoRI;
S, SacI; Sc, ScaI; Sf, SfiI; Sp, Spel and X, XbaI.
Figure 2 shows the nucleotide se~uence of the rbcMT-S and the corresponding de~ ced amino acid sequences. Introns are printed in lower case letters and exons in upper case letters. The putative start and stop codons are 10 underlined. The 12 nucleotides and corresponding 4 amino acids representing the auxon sequence are in(lic~ted by bold italic letters. The de~luced polypeptide for the S38 cDNA is underneath the one for the S40 cDNA that contains the auxon. The oligonucleotide primers for sequencing, PCR and RACE, are inrliç~ted by arrows above the nucleotide sequence. The primers labeled with a 15 star are derived from the conserved regions of pea and tobacco Rubisco LSMTs.Figure 3 is a comparison of the cledllce~l amino acid sequences of S38, S40, with tobacco and pea Rubisco LSMTs. Identical residues are indicated by vertical lines and similar residues by colons. Gaps introduced to n~a~ e ~lignment are indicated by dashes. Potential N-glycosylation sites are shown in 20 bold. ~eucine-rich repeat-like motifs are underlined. The four amino acid sequence, WVQQ, d~duced from the 12-nt auxon is shown in bold italic letters.
The conserved peptide sequences, from which the primers are derived to clone the rbcMT-S, are intliç~tecl by arrows.
Figure 4 illustrates alternative splicing of intron M of rbcMT-S mRNA.
25 The top portion shows the sequence of intron III and fl~nking regions. Shown below are the two types of rnRNAs (S40 and S38) produced by alternative splicing. When the second 3'splice site is utilized, the 12-nt auxon is retained to produce S40 mRNA (center), which encodes a 55.5 kD polypeptide. If the f*st 3'splice site is utili~ed, the auxon is absent and S38 rnRNA is produced (bottom), 30 which encodes a 55.0 kD polypeptide.

... ,.. ~, . ... ,.. .. ,~ .. . . .

CA 0226177~ 1999-01-28 W O 98104116 PCTrUS97/13095 Figure 5 is an analysis of the spinach genomic DNA. An aliquot of 20 ~g of spinach genomic DNA was digested with ScaI and Eco~ respectively, electrophoresed on a 0.7~ agarose gel and processed for DNA gel-blot analysis by hybridization to the rbcMT-S cDNA probe labeled with digoxigenin-UTP. A
5 rbcMT-S cDNA clone in BlueScript II KS(+) digested with EcoRI corresponding to one copy was used for copy number reconstitution.
Figure 6 shows expression of both S38 and S40 mRNA in spinach leaves.
RNase protection assays using a 785-nt antisense riboprobe designed to protect a775-nt of the S40 mRNA from nt-455 to nt-1229, and a 306-nt and 457-nt of the S38 mRNA from nt-455 to nt-760 and from nt-761 to nt-1217 respectively, were carried out. Lanes 1, 2, 3, 4 and 5 are 2.5, 5, 10, 20 and 20 llg of spinach leave total RNA. After hybridization all but lane 5 were digested with 1:100 dilution of RNases. Lane 5 was digested with a 1:50 dilution of RNases (Ambion).
Figure 7A is a Western blot analysis of S-40, S-38, P-55 and P-55-174 mRNAs expressed in E. coli. Lane 1, standard markers; lanes 2 and 3, S-40;
lanes 4 and ~, P-55; lanes 6 and 7, P-55-174; lanes 8 and 9, S-38; lanes 2, 4, 6and 8, soluble protein; lanes 3, 5, 7 and 9, insoluble protein.
Figure 7B is a bar graph representing Rubisco LSMT activity from the different constructs corresponding to the lanes in Figure 7A.

DETAILED DESCRIPIION OF THE INVENTION
The present invention relates to a Rubisco LSMT gene, its DNA and amino acid sequence encoding therefor, cDNA clones thereof, and a four amino acid sequence insert which inactivates the enzymatic activity of Rubisco LSMT.
In the present application, naturally occurring amino acid residues in peptides are abbreviated as recomm~n(led by the IUPAC OIUB Biochemical Nomen~ re Commission as follows: Phenyl~l~ninP is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Norleucine is Nle;
Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr CA 0226177~ 1999-01-28 W O 98/04116 PCTrUS97/1309~-or T; Alanine is Ala or A; Tyrosine is Tyr of Y; Histidine is His or H;
Ghlt~min~ is Gln or Q; Asparagine is Asn or N, Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C;
Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is 5 any amino acid.
Synthetic or non-naturally occurring amino acids refer to amino acids which do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein. Preferred synthetic amino acids are the D-amino acids of naturally occurring L-amino acids as well as non-naturally 10 occurring D and L amino acids represented by the formula H2NCHR'COOH, wherein R' is: (1) a lower alkyl group; (2) a cycloalkyl group of from 3 to 7 carbon atoms; (3) a heterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen;

(4) an aromatic or arylalkyl residue of from 6 to 15 carbon atoms optionally 1~ having from 1 to 3 substituents on the aromatic nucleus selected from the group consisting of hydroxyl, lower alkoxy, amino, and carboxyl; (5) alkylene-Y
where alkylene is an alkylene group of from 1 to 7 carbon atoms and Y is selected from the group consisting of hydroxy, amino, cycloalkyl of from 3 to 7 carbon atoms, heterocyclic of from 3 to 7 carbon atoms and 1 to 2 heteroatoms 20 selected from the group con~i~ting of oxygen, sulfur and nitrogen, and -C(O)R2 where R2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl; (6) alkylene-S(O)nR5 where n is 1 or 2, and R5 is a lower alkyl or lower alkylene.
2~ Particularly p~ ed synthetic amino acids include, by way of example, the D-amino acids of na~urally occurring L-amino acids, L-l-napthyl~l~ninf~, L-2-naphthyl~l~nin~, L-cyclohexyl~l~ninP, L-2-amino isobutyric acid, the sulfoxide and sulfone derivatives of methionine, and the lower alkoxy derivatives of methionine.

CA 0226177~ 1999-01-28 W O 98/04116 PCTAUS97tl3095 ~ "Peptide mimetics" are also encomp~e~l by the present invention and include peptides having one or more of the following modifications:
peptides wherein one or more of the peptidyl [-C(O)NH-] linkages (bonds) have been replaced by a non-peptidyl linkage such as carbamate linkage ~-5 OC(O)N <], phosphon~te linkage, ~mid~te linkage, sulfonarnide linkage, andsecondary amine linkage or with an alkylated peptidyl linkage [C(O)NR6- where R6 is a lower alkyl], peptides wherein the N-te~ s is derivatized to a -NR7R8 group, to a -NC(o)R7 group where R7 and R8 are independently selected from hydrogen and 10 lower alkyls with the proviso that R7 and R8 are both not hydrogen, to a succinimide group, to a benzyloxycarbonyl-NH-(CBZ-NH-) group, to a benzyloxycarbonyl-NH- group having from 1 to 3 substin~ent~ on the phenyl ring selected from the group con~i~ting of lower alkyl, lower alkoxy, chloro, and bromo, peptides wherein the C terminus is derivatized to > C(O)R9 where R9 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, and NRI~R'' where R10 and R'l are independently selected from the group consisting of hydrogen and lower allyl.
Other abbreviations are as follows: aa, amino acid(s); auxon, auxiliary 20 exon; bp, base pair(s); nt, nucleotide(s); Rubisco LSMT, Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit tN-methyltransferase; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction Although the present invention is described with respect to spinach, it will 25 be appreciated that the techniques employed herein are applicable to other plants species which contain a des(methyl) forrn of Rubisco with regards to trimethylation of Iysyl residue 14 in the large subunit (LS). Examples of such plant species include alfalfa, wheat, corn (maize) and lettuce.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit 30 (LS) ~N-methylllallsr~lase (referred to herein as "Rubisco LSMT") catalyzes CA 0226l77~ l999-0l-28 W O98/04116 PCTrUS97/13095 methylation of the ~-amine of lysine-14 in the LS of Rubisco. Rubisco is the world's most abundant protein, and serves as the only significant link between the inorganic and organic carbon pools in the Earth's biosphere by catalyzing the reduction of atmospheric carbon dioxide to carbohydrates during photosynthesis.
5 Perturbations of Rubisco activity translate directly into similar changes in plant growth and yield. Thus, there is ~ignifi~nt interest in the art in the potentialmanipulation and control of Rubisco activity through genetic engineering.
However, the complexity and m~ imeric nature of Rubisco have proven to be substantial obstacles to achieving this goal, which have not yet been 10 overcome. Rubisco LSMT provides an opportunity for the selective manipulation of Rubisco activity through changes in the structure and stability of the N-terminal region in the LS, an area known to be essential for catalytic activity. Rubisco LSMT is a highly specific enzyme which is found to interact only with Rubisco and does not interact with any other protein in the plant cell.
15 Since Rubisco catalyzes the reduction of atmospheric CO2 during photosynthesis, Rubisco and Rubisco LSMT are critical to the plant cell for viability.
Furthermore, the exceptionally tight and specific nature of the h~ d~lion between Rubisco LSMT and des(methyl) forms of Rubisco creates the possibility for the development of novel synthetic polypeptide herbicides, whose target is the 20 in vivo interaction between Rubisco LSMT and Rubisco, whose specificity crosses a group of plant species related only by the presence of Rubisco LSMT, and whose target protein has no homologue in the entire animal kingdom.
Finally, this same affinity of Rubisco LSMT for des(methyl) forms of Rubisco also creates the possibility for the site and protein specific delivery of compounds 25 into the chloroplast and to Rubisco, for the potential manipulation of Rubisco activity andlor stability.
Ribulose bisphosphate carboxylase/oxygenase (Rubisco) from spinach (Spinach oleracea) is a des(methyl) form of Rubisco with regards to trimethylation of lysyl residue 14 in the large subunit (LS). In inves~ig~ting the 30 molecular basis for the absence of trimethylation-14 in the LS of spinach .. . ... . .. . . .

wo 98104116 PCT/USg7/1309 Rubisco, the inventor has isolated and sequenced two full-length cDNAs (S40 and S38) and the gene for spinach Rubisco LSMT (rbcMT-S). This discovery was quite unexpected since it was once thought ~ha~ spinach did not possess the Rubisco LSMT gene because it contained a des(methyl) Iysyl residue in the LS of 5 Rub sco. The gene for spinach Rubisco LSMT, covering all 6 exons and 5 introns, has an ur~ tion similar to the tol~acco Rubisco LSMl gene (rbcMT-T). Southern blot analy iis of spinach genomic DNA shows that the rbcMT-S is present as a single copy. The deduced amino acid sequence from the rbcMT-S
cDNAs shows 60% and 62% identity with the amino acid sequences of pea an~
10 tobacco Rubisco LSMT, respectively.
Moreover, the particular sequence disclosed herein for the .plnach Rubisco LSMT gene may be used to deter.~ine the particu'dr sequence in other photosynthesi7.ing plants. The sequence of the gene nlay be used as a probe to screen cDNA or genomic DNA librari~s fr~m other plants and, due tO the 15 expected homology between the gene ~equences in the various plant species, the particular sequence f )r the Ruhisco LSMT gene in other species may then be found.
In a ~..rther aspect, the present invention relates to a recombinant or transge;lic plant transformed with the F~ubisco LSMT gene described above. The 20 methods employed for transforming the plants are generally known in the art.
For example, the transforrnation method described in Bechtold et al, Planta Agrobacterium Mediated Gene Transfer By Infiltration of Adult Ar~hidopsis Thaliana Plants, C.R. Acad. Sci., Paris 316:1194-1199 (1993) and Valvekens et al, "Agrobacterium tumefaciens-mediated transformation of Arabiclopsis Ihaliana 25 root explants by using kanamycin selection," Proc. Natl. Acad. Sci. USA
85:5536-5540 (1988), may be used in the method of the presen~ invention.
To achieve the present invention, a ii~ length cDNA cione was isolated by the present inventor lltili7.ing polymerase chain reacti(~n (PCR)-based technology and conventional bacteriophage library screening. PCR techniques 30 are disclosed, for example, in Klein et al, "Cloning and Developmental Expression of the Sucrose-Phosphate-Synthase Gene Frcm Spinach," Planta 190:498-510 (1993); in Ampli-Taq PCR kit by Perkin Elmer - Cetus, Emeryville, CA); and in the m~mlf~r.tllrer's instruction manual. Bacteriophage library screening is described, for example, in Gantt et al, "Transfer of ~p122 to the Nucleus Greatly Preceded its loss from the Chloroplast and Invol~,red the Gain of an Intron," F,MBO J. 10:3073-3078 (1('91), and in the inforrnation provided by the manufacturer of the screening membrane (Stratagene, I,a Jolla, CA).
A cDNA of the Rubisco LSMT gene from spinac!~ was isolated and studies of Rubisco LSMT gene expression initi~ted. Utilizing amino acid 10 sequence information derived from purified peptic polypeptide fr~m~nt~ from proteolyzed Rubisco LSMT, a full-length cDNA-of Rubisco LSMT was obtained.
The cDNA of Rubisco LSMT, rbcMT, was used to ex~mine organ-specific and developmental parameters affecting rbcMT gene ~ essi~
The present specification details the purification of peptic fra~ments from 15 spinach RU'DjSCO I SMT and a PCR-based cloning strategy for isolating a full-length cDNA. A similar strategy was previously utilized to obtain a full-length cDNA of sucrose-phosphate synthase from spinach (Klein et al, "Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach,"
Planta. 190:498-510 (1993)) and to obtain the cDNA of the Rubisco LSMT gene 20 from pea and from tobacco. The protein sequence information obtained from peptic fragments pennitted the confirm~tion of clones encoding for Rubisco LSMT. Hence, a molecular probe of the spinach Rubisco LSMT gene was rapidly obtained thereby pennitting identification of protein and nucleotide sequence, and characleri2alion of its gene ~plession.
The a nino acid sequence deduced from tne S40 cDNA, as described in the Examples and in Figures 2, 3 and:~, contains a 4-arnino acid insert (WVQQ) located near the center of the ~rotein, which is a consequence of alternative 3'mRNA splicing and inclusion of 12 nucleotides from the 3'end of intron III.
For example, the 4-amino acid sequence was determined to be a 12 nucleotide 30 insert (TGGGTGCAACAG). Bacterial e),~ression of the S40 cDNA using a pET

CA 0226177~ 1999-01-28 Wo 98/04116 PCT/USg7/13095 expression vector resulted in the synthesis of a protein with no detectable activity. Furthermore, engineering of the 4-amino acid insert from the S40 cDNA into the corresponding position in pea Rubisco LSMT resulted in a complete loss of enzyme activity. This technique of inserting the 4-amino acid insert to inactivate the LSMT could also be used in other species having RubiscoLSMT, for example, in tobacco, tomato, potato, pepper, legumes, soy beans, cucumbers, melons and gourds. The methods employed for inserting the 4-amino acid insert into the Rubisco LSMT are generally known in the art. The alternative 3'mRNA splicing, therefore, resulted in the inactivation of the S40 10 LSMT. This is one molecular rationale for the absence of trimethyllysine-14 in the LS of spinach Rubisco.
Catalytically inactivated forms of Rubisco LSMT can act as competitive ligands to prevent or reduce methylation at Lys-14. Therefore, transgenic plantscan be constructed which carry full-length copies of the Rubisco LSMT with the 15 4-amino acid insert. Since the Rubisco LSMT enzyme is e~enti~l for Rubisco activity, the down-regulation the enzyme's activity would be expected to be lethal to the plant since it would be unable to catalyze net CO2 fixation during photosynthesis. Accordingly, the present invention provides a method for preventing or reducing Rubisco LSMT activity in a photosynthesizing plant.
20 This method, and variations of this method, could thus be used as a herbicide to selectively elimin~te or reduce photosynthesizing plants.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

W O 98/04116 PCTrUS97/1309S

EXAMPLES
Example 1 Plant growth Spinach (Spinacea oleracea L. cv. Melody) plants were cultured in 5 ProMix~ soil media in a greenhouse at approximately 20~C with a natural light photoperiod during the winter season (Lexington, Kentuclcy).

F,Y~mp'e 2 Cloning and sequencing of rbcMT-S cDNAs The two rbcMT-S cDNAs were obtained by RT-PCR (reverse 10 transcription-polymerase chain reaction) and RACE (rapid amplification of cDNA
ends). For RT-PCR, 5 ,ug of total RNA isolated from spinach leaves using Trizol (GIBCO/BRL) was reverse-transcribed with an oligo d(T)~7 primer. The resulting first-strand cDNA product was amplified by PCR with Taq polymerase (GIBCO/BRL) using a forward primer (SF-8), and a reverse primer (SR-2). The 15 SF-8 and SR-2 primers were synthesized corresponding to conserved peptide se~uences between pea (Klein et al., "Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyllldllsrelase~ " Plant Molecular Biol. 27:249-261 (1~95)) and tobacco (Yinget al., ''Ol~ n and characterization of the ribulose-1,5-bisphosphate 20 carboxylasetoxygenase large subunit ~N-methyltransferase gene in tobacco,"
Plant Molecular Biology (In press)) Rubisco LSMTS. The SF-8 sequence, including an EcoRI site and encoding the peptide WAFGILRSRA, is 5'CGA
TGG GCA TTT GGA ATT CTC AGA TCA AGG GC. The SR-2 sequence, including a BgllI site and encoding the peptide ERRLKDLGLA, is 5'GGC CAA
25 GGC CAA GAT CTT TAA GCC TCC TTT C. Conditions for PCR were 35 cycles of: 94~C 1 min, 50~C 1 min, 72~C 1.5 min and final extension 72~C 10 min. The PCR product was di~ested with EcoRI and BglII, and gel-purified.

CA 0226177~ 1999-01-28 W O 98/04116 PCT~US97/1309~

The purified fragment was cloned into Bluescript 11 KS(+) vector (Stratagene) for sequencing. After sequencing, this clone was clecign~t~d as S25' (Fig. lD).
For 5'RACE, reverse-transcription was the same as described above except for using an rbcMT-S-specific primer (SR-3, Fig. 2) anchored in the mid-5 coding region and followed by poly d(C)-tailing as described in Ying et al., ''Isolation and characterization of xnov, a Xenopus laevis ortholog of the chicken nov gene," Gene 171:243-248 (1996)). The resulting dC-tailed products were amplified using a nested primer (SR-5) which included a XbaI restriction site, and a poly (dG/dI)-cont~ining oligonucleotide (AP-2, 5'GCT AAG CTT CTA
10 GAG CTC GGI IGG GII GGG lIG G, SacI). The PCR products were digested with ScaI and XbaI, gel-purified and cloned into Bluescript II KS(+) vector for sequencing. After sequen~ing, two different clones were identified, one with a 12-bp auxiliary exon (auxon) ~lesign~t~rl as S40' and another without the auxon designated as S38'.
For 3'RACE, 5 ~g of total RNA from spinach leaves was reverse-transcribed with an adapter-primer (AP-1, 5'GGC CAC GCG TCG ACT AGT
ACT (T)l6). Amplification by PCR was as described above except for using the AP-1 and spinach specific primer (SF-9). The PCR product was cloned into pCR-Scrip Direct SK(+) vector (Stratagene) for sequencing, designated as S2' (Fig. lD).
Two to five independent clones were chosen for sequencing from each of the above constructs. Both strands of each clone were sequenced by the dideoxy chain terrnination method (Sanger et al., "DNA sequencing with chain-termin~ting inhibitors," Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) using Sequenase (US Biochemical) and 35S-dATP (NEN) with M13 reverse and -40 primers. In addition, 18 to 27-mer oligonucleotides synthesized according to sequence information obtained were used directly as prirners for further sequencmg.

W O 98/04116 PC~r~US97/1309', Both full-length S38 and S40 cl~NAs were obtained by ligation of clones S2' and S25' to S38' and S40', accordingly, based on restriction sites within the overlapped regions (Fig. lD).

F,Y~mple 3 Isolation and Southern analysis of the rbcM~-S
The rbcMT-S gene was cloned by PCR. Spinach nuclear DNA was isolated using Floraclean (BiolO1, Inc.). Approximately 100 ng of the nuclear DNA was amplified by PCR with Taq polymerase (GIBCO/BRL) using a forward primer (SF-1) and a reverse primer (SRT1). The PCR product was 10 cloned into pCR-Script SK(+) for sequencing and restriction mapping.
For Southern analysis, spinach nuclear DNA was digested with EcoRI or ScaI, electrophoresed on a 0.7% agarose gel and transferred onto nylon membranes (MSI) (Sambrook et al., Moleclclar Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), 2nd Ed. (1989)). The 15 DNA blot was hybridized with the cDNA probe I (SfrI fr~gmen~, 1056-bp long, Fig. lC) labeled with digoxigenin-UTP according to the procedure provided by the manufacturer (Boehringer Manrlheim).

Fx~mrle 4 Genetic engineering of the (12-bp) auxon into the pea LSMT
A 5'end-trlmr~t~cl pea LSMT cDNA cloned in pET-23d (P-55) (Cheng and Houtz, unpublished data) was digested with KpnI which generated a 802-bp fragment I and a 4300-bp fragment H which were gel-purified. The purified 802-bp fragment was self-ligated and then amplified by Taq polymerase with a forward primer ~P-F, 5'AGT CCC GGG TGC AAC AGA TTA ACC ACA GTG
25 CAG GAG TTA C, SmaI. Note: 12 nucleotides, including one in the reverse primer, are in bold italic letters and consist of the auxon) and a reverse primer (P-R, 5'AGT TTT AAA GGT CTG CCA TTG GAA CCA C, DraI) at 35 cycles of: 94~C 1 min, 56~C 1 min, 72~C 40 sec and final extension 72~C 10 min. The ~ ~ , . . .

CA 0226177~ 1999-01-28 PCR product was digested with SmaI and DraI, and self-religated. The circular DNA was digested with KpnI, ligated into KpnI-fragment I, and transformed into DH5~ cells (BRL/GIBCO). After screening 180 colonies, two of them (designated as P-55-84, and P-55-174) were selected for sequencing to confirm that the 12-bp auxon ~vas engineered into the P-55 and no other mutation was caused by PCR. The full-length encoding regions of S40 and S38 cDNA were also cloned into the pET-23d E. coli expression vector (~le~ign~d as S-40 and S-38 respectively).

Example 5 f~Nase protection assay The ~nti~n~e riboprobe (probe II) was made by transcribing a rbcMT-S
cDNA clone 210-1 (which contained a 775-bp EcoR~-SacI fragment with the 12-bp auxon and was linearized by EcoRI, Fig. lC) with T7 RNA polymerase, (cY-32P)UTP (800 Ci/mmol, 10 mCilml) and cold NTP. Probe III generated a 775-nt which was fully protected by the S40 mRNA but only partially protected by the S38 mRNA. The 2.5, 5, 10, 20 and 20 ,ug of total RNA isolated from spinach leaves were hybridized with lxlOs cpm of the probe II according to the m~mlfacturer's instructions (Ambion).

Example 6 Rubisco LSMT activity assay and western blot analysis Individual clones (S-40, S-38, P-55 and P-55-174) in pLysS host cells were cultured at 37~C for 3.5 hrs in 5 ml LB broth with 50 ,ug/ml carbenicillin and 35 ,ug/ml chlorophenicol and in~uced by the addition of IPTG to the growing cells at a final concentration of 0.5 mM. After induction cell cultures were ~ 25 continued for 2.5 hrs at 25~C. After induction the cells were harvested by centrifugation at 5000xg for 5 min at 4~C, washed twice with deionized water, and resuspended in 100 ~l of buffer (50 mM TRIS-K+, pH 8.2, 5 mM McCl2, 1 mM EDTA) with prot~ ase inhibitors (1 mM PMSF, 10 ~g/ml leupeptin) and wo 98/04116 PcTruss7/l30s5 frozen at -80~C. The activity of Rubisco LSMT was determined as described previously (Wang et al., "Affinity purification of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyl~lal~Çclase," Prot. Expr. Pur.

6:528-536 (1995)).
For Western analysis protein extracts plepared as described above were separated by SDS-PAGE (15% acrylamide) and ~ sr~lled to PVDF-membranes (Millipore Corp). The membranes were probed with antibody raised against the precursor forrn of pea Rubisco LSMT expressed in E. coli.

F.~r~mrle 7 0 Isolation of rbcMT-S cDNA.
The high homology between pea and tobacco Rubisco LSMT enabled the inventor to design Rubisco LSMT-specific primers for amplifying a 7B6-bp fragment (S25', Fig. lD) from a spinach first-strand cDNA pool reverse-transcribed from total RNA isolated from spinach leaves. Cloning and sequencing of the 786-bp fragment showed that it was a truncated rbcMT-S
cDNA which lacked 5' and 3'ends. The rem:~ining 5' and 3' sequences were obtained by 5' and 3' RACE, respectively (for review see Forhman, "RACE:
rapid amplification of cDNA ends," In PCR protocols: a guide to methods and applications, pp.28-38, Innis et al., eds. Ac:~çmic Press, San Diego (1990)).
For 5'RACE using an rbcMT-S-specific primer (SR-3, Fig. 2) for first-strand cl~NA synthesis and a second nested gene-specific antisense primer (SR-5,Fig. 2) for PCR amplification, resulted in the ide~tifi~tion of two 5'RACE
products (836-bp and 848-bp fr~gmçnts) after sequencing, one with a 12-bp insertion designated as S40', and the other without the insertion desi~n:~tç~l as S38'. In the region where the ~'RACE products and the PCR product (S25') have sequence in common, complete sequence identity was observed and 118-bp overlapped in the cDNA sequences excepting the 12-bp insertion in S40' (Fig.
2).

, CA 0226177~ 1999-01-28 W O 98/04116 PCT~US97/1309 For 3'RACE using an adapter-primer (AP-1) for first-strand cDNA
synthesis and also as a reverse primer, and SF-9 as the rbcMT-S-specific primer for PCR amplification, a single 761-bp PCR product was obtained. Se~uence analysis confirm~ the identity of the 3'RACE product as encoding the predicted 5 3' portion of the rbcMT-S protein including the 3' untr~ncl~ted region (Fig. lD, Fig. 2). Given these overlapping clones, the inventor was able to assemble the two cDNA sequences (S40 and S38) of the rbcMT-S as shown in the Fig. lB and Fig. 2.
Both rbcMT-S cDNAs contain a 5' leader of 31-nt and encode for proteins of 495-aa (S40) and 491-aa (S38) with predicted molecular mass of 55.5 kD for S40 and 55.0 kD for S38, which are similar to that of pea (55.0 kD) and tobacco (56.0 kD) (Fig. 3). The dedl~cecl rbcMT-S proteins contain four potential N-linked glycosylation site which fit the consensus sequence Asn-Xaa-Ser/Thr (NXS/T), one of which is conserved in the pea and tobacco Rubisco 15 LSMTs (Fig. 3), and like that of pea and tobacco, they also contain five imperfect copies of a motif similar to leucine-rich repeats (LR~) (Fig. 3) (Kobeet al., "The leucine-rich repeat: a versatile binding motif," Trends Biochem.
Sci., 19:415-21 (1994)).

Example 8 20 Characterization of rbcMl-S.
The rbcMT-S covering the entire coding region was cloned and sequenced in the overall length of 3144-bp (Fig. 2). Comparison of the genomic DNA and cDNA sequences allowed the precise location of the six exons and five introns tobe mapped (Fig. lA). It has the similar genomic org~ ion of the tobacco 25 Rubisco LSMT gene (rbcMT-T). The size of the exons is fairly constant while that of the introns is quite variable. Intron III of rbcMT-S occurs at a position corresponding to the 12-bp insertion in the rbcMT-S S40 cDNA (Fig. 2). An identical 12-bp sequence was found to be present at the 3'end of the intron.
Ex~min~tinn of the DNA sequence of this intron and fl~nking regions suggested CA 0226177~ 1999-01-28 W O 98104116 PCT~US97/13095 that either of two 3'splice sites (separated by the 12-bp sequence) is utilized during splicing of the rbcMT-S transcripts. Thus, as illustrated in ~igure 4, when the intron III sequence is completely removed, S38 mRNA encoding a 55.0 l~D polypeptide is produced. However, if splicing occurs at the alternative site, 5 S40 mRNA that retains a 12-nt portion of the 3'end of the intron III is generated, and subsequently a 4-amino acid longer polypeptide of 55.5 kD is produced.
A sequence comparison between the rbcMT-S gene and a Drosoph~la tra gene (O'Neil et al., "Interspecific comparison of the transformer gene of Drosophila reveals an unusually high degree of evolutionary divergence,"
Genetics 131:113-128 (1992)) which has been studied for alternative 3'splicing events (Mckeown, "Alternative mRNA splicing," Annu. Rev. Cell Biol. 8:133-155 (1992)) shows two striking TC-rich regions of primary sequence homology between these genes ((~ l l' l l l CTC and T(~ l l l l l CCTTGTTCCT for rbcMT-S,and T(~ l l l l l GTT and l l l l l l l l CTC for tra) in the region preceding the lS regulated splice site of both genes, and what is likely to be the regulated splice site of rbcMT-S.
Southern blot analysis suggests that the rbcMT-S is a single copy gene.
Figure 5 shows hybridization of probe I of a 32P-labeled rbcMT-S cDNA
fragment (Fig. lC) to spinach genomic DNA digested with EcoRI and ScaI.
20 Probe I ~l~t~c~ed a predicted major 2424-bp EcoRI fragment. Additionally, a predicted 876-bp and two other ScaI fr~gn~ent~ were also detectell (Fig. S). Theintensity of the signals in each lane is equivalent to a single copy standard (Croy et al., "Plant Nucleic Acids," In: Croy, R.R.D. (eds.) Plant Molecular Biology, pp. 21-48. BIOS Scientific Publishers ~ imite~, Oxford (199x)) on the left side 25 of the blot. Therefore, we conclude that rbcMT-S is a single copy gene in the spinach genome as rbcMT-T is in the tobacco genome.

... .. . . ..

CA 0226l77~ l999-0l-28 W O 98/04116 PCTrUS97/1309S

Example 9 The rbcMT-S mRNA presen~ in vivo and E. coli expression in vitro.
To determine whether both S38 and S40 mRNA are present in the spinach leaves, total RNA from spinach leaves was subjected to an RNase protection 5 analysis using probe II directed toward the middle region of both S38 and S40 mRNAs (Fig. lC), where the auxon is present in S40 rnRNA. Probe II was designed to protect a single fragment (775-nt) of S40 mRNA and two fr~gment~
(306-nt and 457-nt) of S38 mRNA. Figure 6 shows that S38 mRNA is 20 fold more than S40 mRNA in spinach leaves based on qu~ntit~tive analysis with a PhosphorImager 445SI (Molecular ~ynamic). S40 mRNA is very low in abun-l~nre but detectable when high concentrations of total RNA are used.
However, S38 and S40 rnRNAs are lm-letect~hle. in spinach roots, stems, and flowers by RNase protection assay (data not shown).
In vitro bacterial expression of the S40 cDNA (S-40) using a pET
expression vector did yield a protein (Fig 7A) at detectable levels but with n(lçtect~hle activity (Fig. 7B). Furthermore, engin~ering of the 4 amino acid insert encoded by the 12-bp auxon into the corresponding position in pea RubiscoLSMT (P-55), and bacterial expression of the engineered pea Rubisco LSMT (P-55-174, Fig. 7A) demonstrated that the 4 amino acid insert resulted in complete inactivation of pea Rubisco LSMT activity (Fig. 7B). Therefore, alternative 3'mRNA splicing may result in the inactivation of S40 LSMT. Investigation of the merll~ni~m for inactivation of S38 LSMT is still under way. For some unknown reason, bacterial expression of S38 cDNA (S-38) has been unsuccessful (Fig. 7A).
While the invention has been described and illustrated herein by r~relences to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material, combinations ofmaterial, and procedures selected for that purpose. Numerous variations of such details can be implied and will be appreciated by those skilled in the art.

WO 98/04116 PCT/USg7/13095 Furthermore, all of the publications, patents and patent applications cited herein are incorporated by r~re~nce in their entirety.

Claims (34)

CLAIMS:

1. An isolated Rubisco LSMT gene, wherein said gene is derived from a plant with a des(methyl) lysyl residue in the LS of Rubisco.

2. The isolated gene of Claim 1, wherein said plant is spinach.

3. The isolated gene of Claim 2, wherein said spinach is Spinacia oleracea.

4. The isolated gene of Claim 1, wherein said gene encodes a cDNA
having amino acid sequence S38 or S40, as set forth in Figure 2.

5. The isolated gene of Claim 1, wherein said gene encodes a cDNA
having nucleotide sequence S38 or S40, as set forth in Figure 2.

6. A recombinant vector comprising the isolated Rubisco LSMT gene of Claim 1.

7. The recombinant vector of Claim 6, wherein said isolated Rubisco LSMT gene is a spinach Rubisco LSMT gene.

8. The recombinant vector of Claim 6, wherein said gene encodes a cDNA having amino acid sequence S38 or S40, as set forth in Figure 2.

9. The recombinant vector of Claim 6, wherein said gene encodes a cDNA having nucleotide sequence S38 or S40, as set forth in Figure 2.

10. The recombinant vector of Claim 6, wherein said vector is capable of transforming a plant.

11. An isolated Rubisco LSMT enzyme which is encoded by the Rubisco LSMT gene of Claim 1.

12. The isolated enzyme of Claim 11, wherein said isolated Rubisco LSMT gene is a spinach Rubisco LSMT gene.

13. The isolated enzyme of Claim 11, wherein said gene encodes a cDNA having amino acid sequence S38 or S40, as set forth in Figure 2.

14. The isolated enzyme of Claim 11, wherein said gene encodes a cDNA having nucleotide sequence S38 or S40, as set forth in Figure 2.

15. A recombinant Rubisco LSMT enzyme encoded by the Rubisco LSMT gene of Claim 1.

16. The recombinant enzyme of Claim 15, wherein said isolated Rubisco LSMT gene is a spinach Rubisco LSMT gene.

17. The recombinant enzyme of Claim 15, wherein said gene encodes a cDNA having amino acid sequence S38 or S40, as set forth in Figure 2.

18. The recombinant enzyme of Claim 15, wherein said gene encodes a cDNA having nucleotide sequence S38 or S40, as set forth in Figure 2.

19. A method for expressing a Rubisco LSMT gene in a plant, comprising transforming a plant with the isolated Rubisco LSMT gene of Claim 1.

20. The method of Claim 19, wherein said isolated Rubisco LSMT
gene is a spinach Rubisco LSMT gene.

21. The method of Claim 19, wherein said gene encodes a cDNA
having amino acid sequence S38 or S40, as set forth in Figure 2.

22. The method of Claim 19, wherein said gene encodes a cDNA
having nucleotide sequence S38 or S40, as set forth in Figure 2.

23. The method of Claim 19, wherein said plant is a photosynthesizing plant.

24. A recombinant plant transformed with the Rubisco LSMT gene of Claim 1.

25. The recombinant plant of Claim 24, wherein said isolated Rubisco LSMT gene is a spinach Rubisco LSMT gene.

26. The recombinant plant of Claim 24, wherein said gene encodes a cDNA having amino acid sequence S38 or S40, as set forth in Figure 2.

27. The recombinant plant of Claim 24, wherein said gene encodes a cDNA having nucleotide sequence S38 or S40, as set forth in Figure 2.

28. The recombinant plant of Claim 24, wherein said plant is a photosynthesizing plant.

29. A fragment of the isolated gene of Claim 1, having the amino acid sequence of S40, wherein said fragment has the amino acid sequence: WVQQ

30. The fragment of Claim 29, wherein said fragment has the nucleotide sequence: TGGGTGCAACAG.

31. A method of inactivating Rubisco LSMT activity, comprising inserting the fragment of Claim 29 into Rubisco LSMT.

32. The method of Claim 31, wherein said fragment has the nucleotide sequence: TGGGTGCAACAG.

33. A method for preventing or reducing Rubisco LSMT activity in a photosynthesizing plant, comprising transforming a photosynthesizing plant with a recombinant vector wherein said vector comprises a Rubisco LSMT gene with the fragment of Claim 29.

34. The method of Claim 33, wherein said fragment has the nucleotide sequence: TGGGTGCAACAG.