CA2346259A1 - Means and methods for modulating stomata characteristica in plants - Google Patents

Abstract

Described is the use of nucleic acid molecules encoding subtilisin-like serine proteases and the modulation of the corresponding genes for the production of transgenic plants with altered stomata characteristics. Provided are recombinant DNA molecules comprising such nucleic acid molecules and complements thereof; wherein said nucleic acid molecule(s) are operably linked to regulatory elements allowing the expression of the nucleic acid molecule(s) inplants. Also provided are vectors comprising said recombinant DNA molecules as well as plant cells, plant tissues and plants transformed therewith. In addition, the use of the aforementioned nucleic acid molecules, recombinant DNA molecules and vectors in plant cell and tissue culture, plant breeding and/or agriculture is described, in particular for the production of plants with improved phenotypes.

Description

Means and methods for modulating stomata characteristica in plants The present invention relates to recombinant DNA molecules comprising nucleic acid molecules encoding subtilisin-like serine proteases that are involved in the regulation of stomata! density in plants; wherein said nucleic acid molecules could be operably linked to regulatory elements allowing the expression of the nucleic acid molecules in plants. The present invention also provides vectors comprising said recombinant DNA molecules as well as plant cells, plant tissues and plants transformed therewith. The present invention further relates to the use of the aforementioned recombinant DNA molecules and vectors in plant cell and tissue culture, plant breeding and/or agriculture, in particular for the production of plants with improved traits.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
Stomata! complexes (stomata) are specialised structures in the epidermices of all higher land plants that mediate and control the gas exchange between the internal tissues of the plants and the atmosphere. They consist of two guard cells that flank a central pore. In many plant species these central guard cells are surrounded by morphologically distinct epidermal cells (subsidiary cells).
Usually, more than 90% of the gas exchange between a leaf and the atmosphere (uptake of COz into the leaf and release of H20 vapor) occurs through the stomata!
pores.
The major function of the stomata is to create an optimal balance between sufficient C02 uptake and limited water loss. To achieve this, short term control (in the range of minutes to hours) is exerted on the stomata! conductance by opening and closure of the stomata! pores through turgor driven movement of the guard cells (for review see Zeiger, Farquhar and Cowan (eds.) Stomata! Function, Stanford University Press, Stanford 1987; Willmer and Fricker (eds.) Topics in Functional Biology, 2. Stomata, Second Edition, Chapman and Hall Ltd., London, New York, 1996). Besides these rapid and transient changes, long term modulation of stomatal characteristics occurs which predominantly involve morphological aspects such as presence or absence of stomata in the upper andlor lower leaf epidermis, density of stomata in the leaf epidermices, or the size of the stomata. These features are determined both, by endogenous (genetic) and by exogenous (environmental) factors. Hints towards genetic control were obtained through the observation of a .broad variation of the stomatal density between different species of the same genus, between varieties or cultivars of the same species, or in F1 hybrids. Genetic analysis revealed multigenic, oligogenic, or monogenic control and a high heritability of characters such as stomatal density or size (for review see Jones, In Stomatal Function, Eds. E. Zeiger, G.D.
Farquhar and LR. Cowan, pp. 431-443, Stanford University Press, Stanford 1987). In addition to this endogenous control, stomatal characteristics are also modulated through environmental effects. Thus, air humidity (e.g. Schurmann, 1959, Flora 147, 417-520), light intensity (e.g. Gay and Hurd, 1975, New Phytol. 75, 37-46;
Schoch et al., 1980, J. Exp. Bot. 31, 1211-1216; Rahim and Fordham, 1991, Ann.
Bot. 67, 167-171 ), and C02-concentration (e.g. Woodward, 1987, Nature 327, 617-618; Woodward and Bazzaz, 1988, J. Exp. Bot. 39, 1771-1781; Goodfellow et al., 1997, Tree Physiol. 17, 291-299) were found to affect stomatal density.
In several studies, stomatal density was found to be associated with plant yield (e.g.
Walton, 1974, Can J. Plant Sci. 54, 749-754). Pima cotton varieties selected for high yield upon growth under conditions of high irradiance and artificial irrigation exhibit increased stomatal density associated with increased stomatal conductance and reduced leaf temperature (Cornish et al., 1991, Plant Physiol.
97, 484-489; Lu and Zeiger, 1994, Physiol. Plant. 92, 273-278; Lu et al., 1994, Physiol. Plant. 92, 266-272; Srivastava et al., 1995, Plant Sci. 19, 125-131 ). A
similar relationship between stomatal conductance and yield was observed for a series of bread wheat varieties (Lu et al., 1998, J. Exp. Bot. 49, 453-460).
According to these data, the modulation of stomatal characteristics are of high importance for the improvement of elite cultivars of crop plants. In the area of agriculture and forestry, a major aim is the continuous improvement of the crop plants with respect to higher yielding to provide sufficient food for the growing global population and to ensure the supply of renewable resources.
Traditionally, progress towards higher yielding varieties is attempted through breeding, a very labour and time consuming process to be conducted separately for every relevant plant species. Some progress has already been achieved through the application of genetic engineering to plants, i.e. the introduction and expression of recombinant nucleic acid molecules in plants. Such approaches are advantageous as they can usually be applied to many different plant species.
In EP-A 0 511 979, for instance, the use of a procaryotic asparagine synthetase for expression in plant cells is described that, among other changes, leads to increased biomass production. WO 96/21737 describes yield increases in plants achieved through the expression of de- or non-regulated fructose-1,6-bisphosphatase through enhanced rate of photosynthesis. In WO 96/17069, the enhancement of biomass production in transgenic plants achieved through expression of a polyphosphate kinase from E.coli is described. In contrast to these cases, however, no means for a directed manipulation through genetic engineering of stomatal density or distribution in plants were hitherto available, due to the complete lack of knowlegde about genes that are involved in the control of these stomatal characteristics.
Recently, an Arabidopsis thaliana mutant, R-558, has been isolated after chemical mutagenesis which shows a two to four-fold increase in the stomatal density of all aerial plant organs, in the leaves in particular and the occurrence of ca. 10%
clustered stomata, i.e. stomata placed in direct contact to at least one other stomata (D. Berger, 1997, PhD Thesis Freie Universitat Berlin). Besides a minor change in the length of the pedicelli, no other morphological changes were visible in the mutant plants. The form and size of the leaves as well as the structure of the mesophyll (number of cell layers in palisade and spongy parenchyma, form and size of the mesophyll cells) and the intercellular system (including the substomatal cavities) are unchanged. The increased stomatal density resulted in elevated transpiration (loss of H20) and was associated with increased dry matter content in the leaves which in the wild type was ca. 3% and in the mutant ca.
7%.
It was furthermore shown that the increased stomatal density in the R-558 mutant was associated with increased leaf fresh (+ 15%) and dry (+30%) weight, increasd glucose (+70%), fructose (+65%), and protein (+50%) contents in leaves, and enhanced transpiration and COZ-assimilation (D. Berger, 1997, PhD Thesis, Freie Universitat Berlin) in comparison to the wild type. The mutation which caused the increased stomatal density has been mapped relative to a set of (molecular) genetic markers to a ca. 0.59 cM interval located on the top arm of chromosome of Arabidopsis thaliana (D. Berger, 1997, PhD Thesis, Freie Universitat Berlin).
However, the regulation of stomatal density and distribution in plants is still not fully understood and means that can be used to manipulate stomatal characteristics such as density and distribution that may have applications in several aspects of agriculture were hitherto not available.
Thus, the technical problem underlying the present invention was to comply with the need for means and methods for modulating the stomatal density in plants.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the invention relates to a recombinant DNA molecule comprising (i) a nucleic acid molecule encoding a subtilisin-like serine protease or encoding a biologically active fragment of such a protein, selected from the group consisting of (a) nucleic acid molecules comprising a nucleotide sequence encoding a protein comprising the amino acid sequence as given in SEQ ID
NO: 2, 8, 10 or 12;
(b) nucleic acid molecules comprising a nucleotide sequence as given in SEQ ID NO: 1, 7, 9, or 11;
(c) nucleic acid molecules encoding a protein comprising at feast the D
region, H region, substrate binding site and/or S region of the subtilisin-like serine protease encoded by a nucleic acid molecule of (a) or (b); or (d) nucleic acid molecules hybridizing with the complementary strand of a nucleic acid molecule as defined in any one of (a) to (c);
(e) nucleic acid molecules encoding a protein the amino acid sequence of which is at least 65% identical to the amino acid sequence encoded by a nucleic acid molecule of any one of (a} to (c);

(f) nucleic acid molecules, the nucleotide sequence of which is degenerate as a result of the genetic code to a nucleotide sequence of a nucleic acid molecule as defined in any one of (a) to (e); or (ii) a nucleic acid molecule encoding a mutant non-active or hyper-active form of or an antibody against the subtilisin-like serine protease encoded by a nucleic acid molecule of (i}; or (iii) a nucleic acid molecule which specifically hybridizes with a nucleic acid molecule of (i) or the complementary strand thereof.
The present invention is based on the identification of a new class of genes represented by SDD9 from Arabidopsis thaliana which share common structural motifs, see infra. In one aspect, these genes are preferably involved in the control of stomatal density and/or distribution. The SDD1 gene is mutated in the Arabidopsis thaliana mutant R-558; see Examples 1 to 3. Computer-assisted amino acid sequence analysis of the protein encoded by this gene revealed that it belongs to a family of subtilisin-like serine proteases; see Example 4.
Further representatives of this new class of genes have been cloned from potato; see Example 6.
The terms "subtilisin like-serine protease" and "subtilase" are used interchangeable herein and mean a specific class of serine proteases, called subtilisins or dibasic processing endoproteases. In subtilisins, four regions form the catalytic triad and the substrate binding site and are most highly conserved among subtilisins; see also Example 4 and Figure 7. In the context of the present invention, subtilisin-like serine proteases also mean such proteins which show a homology of at feast 65% to the sequence shown in SEQ ID NOs: 2, 8, 10 or 12.
In the context of the present invention, the term "subtilisin-like serin protease"
preferably is understood to mean proteins comprising one or several of the characteristic motifs depicted in SEQ ID NOS: 13 to 37; see infra.
The substrate binding site preferably comprises the motif VICAR (SEQ ID
NO: 38), more preferably the motif CAAGN (SEQ ID NO: 39), in particular the motif AAGNN {SEQ ID NO: 40) and most preferably the amino acid motif VICAAGNNG (SEQ ID NO: 41 ).

In another preferred embodiment, the nucleic acid molecule of the present invention encodes a protein described above with one or more of the following amino acid sequence motifs: SYHSA (SEQ ID N0: 49), GLSPT (SEQ ID N0: 50), WLKSG (SEQ ID NO: 51 ), FNSSS (SEQ ID N0: 52), ASTAG (SEQ ID N0: 53), AAMDV (SEQ ID NO: 54), WIATI (SEQ ID N0: 55), GPSGL (SEQ ID N0: 56), IAALLH (SEQ 1D NO: 57), KPIMD (SEQ ID NO: 58), VSCHD (SEQ ID N0: 59), YPSIS (SEQ ID NO: 60), SLSYR (SEQ ID N0: 61 ).
In a further preferred embodiment the D, H and/or S region of the subtilise of the present invention comprise one or several of the following characteristic motifs:
D region:
IIGVL {SEQ ID NO: 42) or GVLDT (SEQ ID NO: 43) H region:
THTAST (SEQ ID NO: 44) or S-RDS (SEQ ID N0: 45) or RDS-G (SEQ ID N0:46) S re4ion:
HVSGI (SEQ ID NO: 47) or FTV-SGT (SEQ tD N0: 48) While a function of such proteins in the regulation of stomatal density in plants was hitherto unknown, the present invention for the first time provides evidence that the described nucleic acid molecules encode proteins that are involved in controlling the density and the distribution of stomata in plants; see Examples 3 and 7. Furthermore, it is shown that plants lacking or overexpressing such proteins show altered morphological and physiological features of high agronomic importance.
Thus, the present invention for the first time clearly establishes that stomatal characteristics such as density and distribution can be specifically modulated through the application of genetic engineering techniques and provides extremely useful tools for example to:
(i) generate plants with increased stomatal density and consequently with enhanced C02 assimilation, reduced leaf temperature, enhanced organ such as leaf fresh and dry weight, and enhanced sugar and protein contents in organs such as leaves;
(ii) generate plants with decreased stomatal density and consequently with reduced water loss and thus lower water consumption;

(iii) counteract environmental changes such as raises in atmospheric C02 levels, temperature and irradiation that would cause changes in stomatal density to sub- or supra-optimal levels;
In general a nucleic acid molecule encoding a subtilisin-like serine protease can be derived from any material source, for example, from any organism, preferably plants possessing such molecules, preferably form monocotyledonous or dicotyledonous plants, in particular from any plant of interest in agriculture, horticulture or wood culture, such as crop plants, namely those of the family Poaceae, any starch producing plants, such as potato, maniok, rice, wheat, corn, barley, oat, leguminous plants, oil producing plants, such as oilseed rape, soja, sunflower, linenseed, etc., plants using polypeptide as storage substances, such as soybean, plants using sucrose as storage substance, such as sugar beet or sugar cane, trees, ornamental plants etc. or plants belonging to the family Gramineae.
Furthermore, nucleic acid molecules can be used in accordance with the present invention hybridizing to the above-described nucleic acid molecules and encoding subtilisin-like serine protease. Such nucleic acid molecules can be isolated, e.g., from libraries, such as cDNA or genomic libraries by techniques well known in the art. For example, hybridizing nucleic acid molecules can be identified and isolated by using the above-described nucleic acid molecules or fragments thereof or complements thereof as probes to screen libraries by hybridizing with said molecules according to standard techniques. Possible is also the isolation of such nucleic acid molecules by applying the polymerise chain reaction (PCR) using as primers oligonucleotides derived form the above-described nucleic acid molecules. Nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include fragments, derivatives and allelic variants of the above-described nucleic acid molecules that encode subtilisin-like serine proteases or biologically active fragments thereof. Fragments are understood to be parts of nucleic acid molecules long enough to encode the described protein br a fragment thereof having the biological activity as defined above.
Preferably, said fragment comprises at least one region of subtilisin-like serine protease as defined in section (i) (c) supra.

The term "derivative" means in this context that the nucleotide sequence of these nucleic acid molecules differs from the sequences of the above-described nucleic acid molecules in one or more nucleotide positions and are highly homologous to said nucleic acid molecules. Homology is understood to refer to a sequence identity of at least 50 %, preferably 65% identity, particularly an identity of at least 70 % or 75%, preferably more than 80 % and still more preferably more than 90 or 95% identity. The deviations from the sequences of the nucleic acid molecules described above can, for example, be the result of nucleotide substitution(s), deletion(s), addition(s), insertions) and/or recombination(s) either alone or in combination' that may naturally occur or be produced via recombinant DNA
techniques well known in the art; see for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994).
Homology further means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent. The nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological activity as defined herein. They may be naturally occurring variations, such as subtilisin-like serine protease encoding sequences from other prokaryotes and eukaryotes, respectively, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques; see supra. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants; see supra. For example, the amino acid sequences of plant subtilisin-like serine proteases share significant similarities with bacterial, yeast and mammalian subtilisin-like serine protease; see Example 4. In addition, nucleic acid molecules can be employed in accordance with the present invention that encode homologs or analogs of the above described subtilisin-like serine protease but where otherwise unrelated to those proteins.
Such proteins that do not display significant homologies to common subtilisin-like serine protease can be identified by a person skilled in the art using techniques well known in the art, for example, via complementation of mutant genes, for example, in corresponding mutant plants; see Example 3.
In a further embodiment the term derivative encompasses a nucleotide sequence coding for a protein derived from any one of SEQ ID Nos. 2, 8, 10 or 12 which exhibits a degree of homology, i.e, identity to the protein depicted under SEQ
ID
Nos. 2, 8, 10 or 12 of at least 60%, in particular an homology of at least 70%, preferably more than 80% and still more preferably a homology of more than 90%
and particularly preferred of more than 95% and which exhibits at least one, more preferably at least three, even more preferably of at least five, in particular at least ten and particularly preferred of at least twenty of the peptide motifs selected from the group consisting of a) QTYIV, (SEQ ID NO: 13), b) IVQLH, (SEQ ID NO: 14), c) SSRLL, (SEQ ID N0: 15), d) QTTYS, (SEQ ID NO: 16), e) SSSCN, (SEQ ID NO: 17), f) VLGNG, (SEQ 1D NO: 18), g) GAHIA, (SEQ ID NO: 19), h) FRAME, (SEQ ID NO: 20), i} V1CAA, (SEQ ID NO: 21 ), j) AAGNN, {SEQ ID NO: 22), k) SSVAN, (SEQ ID NO: 23), I) YGESL, (SEQ ID NO: 24), m) GSEFC, (SEQ ID NO: 25), n) CLRGS, (SEQ ID N0: 26), o) RGVNG, (SEQ ID NO: 27), p) PATLIG, (SEQ ID N0: 28), q) IFGGT, (SEQ ID NO: 29), r) PQNLG, (SEQ ID NO: 30), s) VNFTV, (SEQ ID NO: 31 ), t) HVSGI, (SEQ ID NO: 32), u) GFSLN, (SEQ ID NO: 33), v) RRVTN, (SEQ ID NO: 34), w) PNSIY, (SEQ ID NO: 35), x) LSYRV, (SEQ ID NO: 36), and y) SPISV. (SEQ ID NO: 37) The proteins encoded by the various derivatives, variants, homologs or analogs of the above-described nucleic acid molecules may share specific common characteristics, such as molecular weight, immunological reactivity, conformation, etc., as well as physical properties, such as electrophoretic mobility, chromatographic behavior, sedimentation coefficients, pH optimum, temperature optimum, stability, solubility, spectroscopic properties, etc. All these nucleic acid molecules and derivatives can be employed in accordance with the present invention as long as the biological activity of the encoded protein remains substantially unaffected in kind, namely that the protein is capable of modulating stomata density in plants. Any one of the above described nucleic acid molecules, in particular those that represent hyper-active mutant forms of subtilisin-like serine proteases are particular suitable for overexpression in transgenic plants.
These transgenic plants may either possess an endogenous functional subtilisin-like serine protease or they may lack the corresponding genes, e.g. due to mutation.
The nucleic acid molecules mentioned in section (ii) and (iii) are particular useful for the suppression of genes encoding subtilisin-like serine proteases in plants.
Hence, in one embodiment said nucleic acid molecules are preferably of at least 50 nucleotides in length hybridizing specifically with a nucleic acid molecule as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences encoding no or substantially different proteins. In particular stringent conditions mean, e.g., the use of an aqueous solution of 1 % BSA, 1 mM EDTA, 0.5 M NaHP04 pH7.2, 7% SDS and incubation at 65°C. Preferably, stringent hybridization is obtained under the following conditions:
Hybridization buffer:
2 x SSC; 10 x Denhardt's solution (Ficoll 400 + PEG + BSA; ratio 1:1:1 ); 0.1 SDS; 5mM EDTA; 50 mM Na2HP04; 250 Ng/ml herring sperm DNA; 50 N/ml tRNA; or 0.25M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS
Hybridization temperature: T=65 to 68°C
Washing buffer: 0.2 x SSC; 0.1 % SDS
Washing temperature: T=68°C
Such nucleic acid molecules may be used as probes andlor for the control of genie expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 50 nucleotides or more in length. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
The nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of nucleic acid sequences according to the invention. Another application is the use as a hybridization probe to identify nucleic acid molecules hybridizing with a nucleic acid molecule of the invention by homology screening of genomic DNA or cDNA
libraries. Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a nucleic acid molecule as described above can be used for repression of expression of a subtilisin-like serine protease encoding gene, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-A1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a nucleic acid molecule of the invention or part thereof. Selection of appropriate target sites and corresponding ribozymes can be done as described, for example, in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al.
eds Academic Press, Inc. (1995), 449-460. Furthermore, the person skilled in the art is well aware that it is also possible to label such a nucleic acid probe with an appropriate marker for specific applications, such as for the detection of the presence of a nucleic acid molecule of the invention in a sample derived from an organism, in particular plants. .
The above described nucleic acid molecules may either be DNA or RNA or a hybrid thereof. Furthermore, said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
Furthermore, nucleic acid molecules encoding antibodies specifically recognizing a subtilisin-like serine protease or parts, i.e. specific fragments or epitopes, of such a protein can be used for inhibiting the activity of the protein in plants.
These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragmerits etc. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Miistein, 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. Furthermore, antibodies or fragments thereof to the aforementioned peptides 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. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention. For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the effciency of phage antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.
Immunol. Methods 183 (1995), 7-13). Expression of antibodies or antibody-like molecules in plants can be achieved by methods well known in the art, for example, full-size antibodies (During, Plant. Mol. Biol. 15 (1990), 281-293; Hiatt, Nature 342 (1989), 469-470; Voss, Mol. Breeding 1 (1995), 39-50), Fab-fragments (De Neve, Transgenic Res. 2 (1993), 227-237), scFvs (Owen, BiolTechnology 10 (1992), 790-794; Zimmermann, Mol. Breeding 4 (1998), 369-379; Tavladoraki, Nature 366 (1993), 469-472) and dAbs (Benvenuto, Plant Mol. Biol. 17 (1991 ), 865-874) have been successfully expressed in Tobacco, Potato (Schouten, FEES Lett. 415 (1997), 235-241 ) or Arabidopsis, reaching expression levels as high as 6.8% of the total protein (Fiedler, Immunotechnology 3 (1997), 205-216).
In addition, nucleic acid molecules encoding mutant forms of a subtilisin-like serine protease can be used to interfere with the activity of the wild type protein.
Such mutant forms preferably have lost their biological activity as defined above and may be derived from the corresponding subtilisin-like serine protease by way of amino acid deletion(s), substitution(s), andlor additions in the amino acid sequence of the protein. As mentioned above, mutant forms of subtilisin-like serine proteases also encompass hyper-active mutant forms of such proteins which display, e.g. an increased substrate affinity andlor higher substrate turnover of the same.
Furthermore, such hyper-active forms may be more stable in the cell due to the incorporation of amino acids that stabilize proteins in the cellular environment.

These mutant forms may be naturally occurring or genetically engineered mutants;
see also supra.
The recombinant DNA molecule of the invention preferably comprises regulatory sequences allowing for the expression the nucleic acid molecules in plants.
Preferably, said regulatory elements comprise a promoter active in plant cells.
Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in plant cells are well known to those skilled in the art.
These regulatory elements may be homologous or preferably heterologous with respect to the nucleic acid molecule to be expressed and/or with respect to the plant species to be transformed. For example, a preferred regulatory element that can be employed in accordance with the present invention is the SDD1 promoter region as depicted in SEQ ID NO: 5 or part thereof.
Preferably, the promoter region of the SDD1 gene comprising SEQ 1D NO: 6 is employed, which corresponds to the nucleotide sequence of SEQ ID NO. 5 starting at position 839. GUS expression studies show that the promoter of of Arabidopsis thaliana in tissues having mitotic activity shows a high activity. For example, a very strong GUS expression can be found in developing stomata and in primordials, but also a weaker expression in lateral roots. By way of computer-assisted studies different domains could be identified which possibly are responsible for the expression pattern of this promoter. On the one hand, a domain was identified which allows expression in roots, on the other hand several characteristic motifs were identified which are termed Dof-motifs (see, ~
e.g., Yanagisawa and Schmidt, Plant J. 17 (1999), 209-214) and which in the present case possibly allow for an expression in guard cells. These motifs have, e.g., been described in German patent application DE 19904754.5. It is assumed that a deletion of the domain, which possibly mediates the expression in roots and which is located within the first 400bp of SEQ ID N0: 6, advantageously changes the specificity of the promoter. For this reason preferred embodiments of the invention use promoter fragments comprising at the 5'-region a deletion of at least 40n-450bp or of 450-600bp or at the most 900bp.

It is possible for the person skilled in the art to isolate with the help of the coding and regulatory sequences of the invention corresponding genes from other species, for example, potato, tomato, barley, wheat, oat, rye, rice, corn, soja, etc.
This can be done by conventional techniques known in the art, for example, by using the regulatory sequences depicted in SEQ ID N4: 6 as a hybridization probe or by designing appropriate PCR primers. It is then possible to isolate the corresponding promoter region by conventional techniques and test it for ifs expression pattern. For this purpose, it is, for instance, possible to fuse the promoter to a reporter gene, such as GUS, luciferase or green fluorescent protein (GFP) and assess the expression of the reporter gene in transgenic plants.
For example the promoters from the two SDD1 homoloas from ~nlam ~m toberosum described in Example 6 can be isolated by conventional means.
Genomic clones can be amplified, e.g., fragments via long template PCR
(employing for example the EXPAND kit, Boehringer Mannheim), using an upstream oriented SDD1 specific primer and a primer to the Lambda left or right arm sequence. The amplified fragment is sequenced via primer walking until several kb upstream from the transcription start point have been reached, if present on the clone, preferably more than 3 kb. Within the cloned genomic sequence, the transcription start site is determined by standard procedures well known to the person skilled in the art, such as 5'-RACE, primer extension or mapping. To define cis-regulatory elements upstream of the transcription start site (i.e. within the putative promoter region), the respective region is fused to marker genes such as genes encoding GUS or GFP, and 5' deletion derivatives of these construct are generated. They are transformed into suitable plant material', and the expression of the marker gene depending on the remaining upstream sequence (putative promoter) is determined. These techniques are well known to the person skilled in the art.
The regulatory sequences so identified may differ at one or more positions from the above-mentioned regulatory sequence but still have the same specificity, namely they comprise the same or similar sequence motifs, preferably 6 to 10 nucleotides in length, responsible for the above described expression pattern.
Preferably such regulatory sequences hybridize to one of the above-mentioned regulatory sequences, most preferably under stringent conditions. Particularly preferred are regulatory sequences which share at least 85%, more preferably 95%, and most preferably 96-99% sequence identity with one of the above-mentioned regulatory sequences and have the same or substantially the same specificity. Particularly preferred are the regulatory sequences that comprise the above mentioned motifs which allow for an expression in guard cells. Such regulatory sequences also comprise those which are altered, for example by nucleotide deletion(s), insertion(s), substitution(s), addition(s), andlor recombination(s) and/or any other modifications) known in the art either alone or in combination in comparison to the above-described nucleotide sequence.
Methods for introducing such modifications in the nucleotide sequence of the regulatory sequences of the invention are well known to the person skilled in the art. It is also immediately evident to the person skilled in the art that further regulatory elements may be added to the regulatory sequences of the invention.
For example, transcriptional enhancers andlor sequences which allow for induced expression of the regulatory sequences of the invention may be employed.
The possibility exists to modify the regulatory sequences as described above or sequence motifs thereof by,~e.g., nucleotide replacements which do not affect the overall structure or binding motif of the regulatory sequence so that it remains capable of conferring the gene expression pattern as described above. Such regulatory sequences may be derived from subtilase genes of potato although other plants may be suitable sources for such regulatory sequences as well.
Furthermore, the nucleotide sequences can be compared using appropriate computer programs known in the art such as BLAST, which stands for Basic 'Local Alignment Search Tool {Altschul, 1997; Altschul, J. Mol. Evol. 36 (1993), 290-390;
Altschul, J. Mol. Biol. 215 (1990); 403-410), to search for local sequence alignments. BLAST produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologues.
In general, regulatory elements employed in accordance with the present invention comprise a promoter active in plant cells. To obtain expression in all tissues of a transgenic plant, preferably constitutive promoters are used, such as the 35S
promoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689). In order to achieve expression in specific tissues of a transgenic plant it is possible to use tissue specific promoters (see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251 ).
Known are also promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley etc.
Inducible promoters may be used in order to be able to exactly control expression. An example for inducible promoters are the promoters of genes encoding heat shock proteins. Further useful promoters are described in the prior art; see, e.g.:
a) inducible promoters:
described in WO 93121334 (=alcAlaIcR system), WO 90/08826, WO 96/37609.
b) promoters active in photosynthetically active tissue:
the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451), the Ca/b promoter (see e.g. US-A-5,656,496; US-A-5,639,952; Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU
promoter (see e.g. US-A-5,034,322; US-A-4,962,028) as well as the STL1 promoter (Eckes et al., Mol. Gen Genet. 205 (1986), 14-22).
c) promoters and cis-active elements mediating expression in guard cells:
- DE 19904754.5 - truncated AGPase promoter (Mullet-Rober et al., Plant Cell 6 (1994), 601-612) - Rhal promoter (Terryn et al., Plant Cell 5 (1993), 1761-1769) d) promoters mediating expression in meristematic tissue:
- wheat histone H4 promoter (Bifgin et al., Plant Science 143 (1999), 35-- rice PCNA promoter (Kosugi et al., Plant J. 7 (1995), 877-886 - wheat histone H2B promoter (Yang et al., Plant Mol. Biol. 28 (1994), cyc07-promoter (Ito et al., Plant Mol. Biol. 24 (1994), 863-878.
Also microspore-specific regulatory elements and their uses have been described (W096/16182). Furthermore, the chemically inducible Tet-system rnay be employed (Gatz, Mol. Gen. Genet. 227 (1991 ); 229-237). Further suitable promoters are known to the person skilled in the art and are described, e.g., in Ward (Plant Mol.
Biol. 22 (1993), 361-366). The regulatory elements may further comprise transcriptional andlor translational enhancers functional in plants cells. A
plant translational enhancer often used is, e.g., the CaMV omega sequences andlor the inclusion of an intron (Intron-1 from the Shrunken gene of maize, for example) that has been shown to increase expression levels by up to 100-fold. (Maiti, Transgenic Research 6 (1997), 143-156; Ni, Plant Journal 7 (1995), 661-676).
Furthermore, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability. The termination signals usually employed are from the Nopaline Synthase gene or from the CaMV 35S RNA gene.
In a preferred embodiment of the recombinant DNA molecule of the invention, the subtilisin-like serine protease is derived from plants. Preferably, said plants are monocotyledonous or dicotyledonous plants such as those mentioned hereinbefore. A particular preferred embodiment of said plant is Arabidopsis.
The present invention also relates to vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain at least one recombinant DNA molecule according to the invention. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989).
Alternatively, the recombinant DNA molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
Advantageously the above-described vectors of the invention comprises a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hpt, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan;
hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl.
Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamara, Biosci. Biotechnol.
Biochem. 59 (1995), 2336-2338). Useful scorable marker are also known to those skilled in the art and are commercially available. Advantageously, said marker is a gene encoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J.
Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or f3-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a vector of the invention. As described above, various selectable markers can be employed in accordance with the present invention.
Advantageously, selectable markers may be used that are suitable for direct selection of transformed plants, for example, the phophinothricin-N-acetyltransferase gene the gene product of which detoxifies the herbicide L-phosphinothricin (glufosinate or BASTA); see, e.g., De Block, EMBO J. 6 (1987), 2513-2518 and Droge, Plants 187 (1992), 142-151.
The present invention, also relates to host cells comprising a recombinant DNA
molecule or vector of the invention. Host cells include prokaryotic and eukaryotic cells such as E. coli and yeast, respectively.
The recombinant DNA molecules according to the invention are in particular useful for the genetic manipulation of plant cells, plant tissue and plants in order to obtain plants with modified, preferably with improved or useful phenotypes as described above. Thus, the present invention relates to a method for the production of transgenic plants with altered stomata characteristics compared to wild type plants comprising the introduction of a recombinant DNA molecule of the invention into the genome of a plant, plant cell or plant tissue.
Methods for the introduction of foreign DNA into plants as well as the selection and regeneration of transgenic plants from plant cells and plant tissue are also well known in the art. These include, for example, the transformation of plant cells, plant tissue or plants with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A
164 575), injection, electroporation, biolistic methods like particle bombardment and other methods known in the art. The vectors used in the method of the invention may contain further functional elements, for example "left border"- and "right border"-sequences of the T-DNA of Agrobacterium which allow for stable integration into the plant genome. Furthermore, methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e.
the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example cotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161; Peng, Plant Mol.
Biol. 27 (1995), 91-104) andlor by using systems which utilize enzymes capable of promoting homologous recombination in plants (see, e.g., W097108331;
Bayley, Plant Mol. Biol. 18 (1992), 353-361 ); Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991 ), 6373-6378). Methods for the preparation of appropriate vectors are described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV
3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res.
12( 1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471;
Koncz, Plant Mol. Biol. 20 (1992), 963-976; Koncz, Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 22; EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example if a phenotype conferred by said strain is desired.
Methods for the transformation using biolistic methods are well known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technoiogy 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1, 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY
(1995).
The transformation of most dicotyledonous plants is possible with the methods described above. But also for the transformation of monocotyledonous plants several successful transformation techniques have been developed. These include the transformation using biolistic methods as, e.g., described above as well as protoplast transformation, electroporation of partially permeabilized cells, introduction of DNA using glass fibers, etc. Transgenic plant tissue and plants can be regenerated by methods well known in the art. There are various references in the relevant literature dealing specifically with the transformation of maize (cf. e.g.
W095/06128, EP 0 513 849; EP 0 465 875). In EP 292 435 a method is described by means of which fertile plants may be obtained starting from mucousless, friable granulous maize callus. In this context it was furthermore observed by Shillito et al., BioITechnology 7 (1989), 581 that for regenerating fertile plants it is necessary to start from callus-suspension cultures from which a culture of dividing protoplasts can be produced which is capable to regenerate to plants. After an in vitro cultivation period of 7 to 8 months Shillito et al. obtain plants with viable descendants which, however, exhibited abnormalities in morphology and reproductivity.
Prioli and Sondahl, Biotechnology 7 (1989), 589 have described how to regenerate and to obtain fertile plants from maize protoplasts of the Cateto maize inbreed Cat 100-1. The authors assume that the regeneration of protoplasts to fertile plants depends on a number of various factors such as the genotype, the physiological state of the donor-cell and the cultivation conditions. With regard to rice various transformation methods can be applied, e.g. the transformation by agrobacterium-medicated gene transfer (Hiei et al., Plant J. 6 (1994), 271-282; Hiei et al., Plant Mol.

WO 00/22144 PCT/EP99/0?633 Biol. 35 (1997), 205-218; Park et al., J. Plant Biol. 38 (1995), 365-371 ), protoplast transformation (Datta in "Gene transfer to plants", I. Potrykus, G.
Spangenberg (Eds.), Springer-Verlag Berlin Heidelberg (1995), pages 66-75; Datta et al., Plant Mol. Biol. 20 (1992), 619-629; Sadasivam et al., Plant Cell Rep. 13 (1994), 396), the biolistic approach (Li et al., Plant Cell Rep. 12 (1993), 250-255;
Cao et al., Plant Cell Rep. 11 (1992), 586-591; Christou, Plant Mol. Biol. (1997), 197-203) and electroporation (Xu et al., in "Gene transfer to plants", I. Potrykus, G.
Spangenberg (Eds.), Springer-Verlag Berlin Heidelberg (1995}, pages 201-208.
Once the introduced DNA has been integrated in the genome of the plant cell, it usually continues to be stable there and also remains with the descendants of the originally transformed cell. It usually contains a selectable marker which confers resistance against biozides or against an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricine etc. to the transformed plant cells.
The individually selected marker should therefore allow for a selection of transformed cells against cells lacking the introduced DNA.
The transformed cells grow in the usual way within the plant (see also McCormick et al., Plant Cell Rep. 5 (1986), 81-84). The resulting plants can be cultivated in the usual way and cross-bred with plants having the same transformed genetic heritage or another genetic heritage. The resulting hybrid individuals have the corresponding phenotypic properties.
Two or more generations should be grown in order to ensure whether the phenotypic feature is kept stably and whether it is transferred. Furthermore, seeds should be harvested in order to ensure that the corresponding phenotype or~other properties will remain.
In general, the plants, plant cells and plant tissue which can be modified with a recombinant DNA molecule or vector according to the invention can be derived from any desired plant species. They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture, such as crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.-), vegetable plants such as tomato, melon, banana, chicoree, salad, cabbage or potato, tobacco, alfalfa, clover, oil producing plants (e.g. oilseed rape, sunflower, peanut, soybean, etc.), cotton, sugar beet, linseed, flax, millet, hemp, sugar cane, leguminous plants (e.g. beans, peas etc.), wood producing plants, preferably trees, etc.
Thus, the present invention relates also to transgenic plant cells which contain a nucleic acid molecule as defined above or a recombinant DNA molecule or vector according to the invention wherein the nucleic acid molecule is foreign to the transgenic plant cell.
By "foreign" it is meant that the nucleic acid molecule is either heterologous with respect to the plant cell, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the plant cell but located in a different genomic environment than the naturally occurring counterpart of said nucleic acid molecule. This means that, if the nucleic acid molecule is homologous with respect to the plant cell, it is not located in its natural location in the genome of said plant cell when stably integrated into the genome, in particular it is surrounded by different genes. In this case the nucleic acid molecule may be either under the control of its own promoter or under the control of a heterologous promoter. The nucleic acid molecule, vector or recombinant DNA molecule according to~the invention which is present in the plant cell may either be integrated into the genome of the plant cell or it may be maintained in some form extrachromosomally.
In one aspect the present invention relates to a transgenic plant cell comprising stably integrated into the genome a recombinant DNA molecule of the invention or a vector of the present invention or obtainable according to the method of the invention wherein the expression of the nucleic acid molecule results in an increased expression or activity of subtilisin-like serine proteases in transgenic plants compared to wild-type plants.
The term "increase in activity" in the context of the present invention is understood to mean an increase in the expression of endogenous genes coding for a protein of the invention andlor an increase of the amount of the protein of the invention in the cells.
The increase in expression can for example be determined by measuring the amount of transcripts encoding the protein of the invention, e.g., by Northern-blot analysis, preferably by the more sensitive NASBA method (as e.g. described by Leone et al., Journal of Virological Methods 66 (1997), 19-27; Leone et al., Nucleic Acid Res. 26 (1998), 2150-2155; Nakahara et al., Nucleic Acid Res. 26 (1998), 1854-1856) or by RT-PCR. Preferably, an increase in this context means an increase of the amount of transcripts encoding subtilises as compared to corresponding cells which are not genetically modified by at least 5%, more preferably by at least 20%, in particular by at least 50%, and most preferably by at least 400%.
Preferably, the increased expression or activity of subtilisin-like serine proteases in transgenic plants results in decreased stomata density, see, e.g., Example 7.
The increase of the amount of the protein of the invention can for example be determined by Western-blot analysis. Preferably, an increase in this context means an increase of the amount of the protein of the invention as compared to corresponding cells which are not genetically modified by at least 5%, more preferably by at least 20%, in particular by at least 50%, and most preferably by at least 400%.
Alternatively, a plant cell having a nucleic acid molecule encoding a subtilisin-like serine protease present in its genome can be used and modified such that said plant cell expresses the endogenous gene corresponding to this nucleic acid molecules under the control of heterologous promoter and/or enhancer elements.
The introduction of the heterologous promoter and mentioned elements which do not naturally control the expression of a nucleic acid molecule encoding a subtilisin-like serine protease using, e.g., gene targeting vectors can be done according to standard methods, see supra and, e.g., Hayashi, Science 258 (1992), 1350-1353; Fritze and Walden, Gene activation by T-DNA tagging. In Methods in Molecular biology 44 (Gartland, K.M.A. and Davey, M.R., eds).
Totowa: Human Press (1995), 281-294) or transposon tagging (Chandlee, Physiologic Plantarum 78 (1990), 105-115). Suitable promoters and other regulatory elements such as enhancers include those mentioned hereinbefore.
Furthermore, the present invention relates to transgenic plants or plant tissue comprising plant cells of the invention or obtainable by the above described method Preferably, the transgenic plant of the invention displays a decreased stomata density, lower conductance of stomata andlor the water consumption is lowered compared to wild type plants.

Methods for determining stomatal density, leaf conductance and water consumption comprise the following:
- Method for determining stomatal density by means of taking copies using clear nail varnish as described in Sachs et al., Annals of Botany 71 (1993), 237-243.
- Method for determining conductance as described in Muschak et al., Photosynthetica 33 (1997), 455-465.
- Methods for determining water consumption are known to the person skilled in the art.
Preferably, the transgenic plant of the invention displays one or more of the following phenotypes:
a) stomatal density: reduced by at least 2%, preferably by at least 5%, more preferably by at least 10%, most preferably by at least 30%;
b) conductance reduced by at feast 2%, preferably by at least 5%, more preferably by at least 10%, most preferably by at least 25%;
c) water consumption reduced by at least 1 %, preferably by at least 3%, more preferably by at least 5%, most preferably by at least 10%;
as compared to a corresponding wild type plant.
In another aspect, the present invention relates to a transgenic plant cell which contains stably integrated into the genome a recombinant DNA molecule of the invention or part thereof, a vector of the present invention or obtainable according to the method of the invention, wherein the presence, transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis or the activity of subtilisin-like serine proteases in transgenic plants compared to wild type plants.
Usually the activity will be reduced by at least 10%, preferably by at least 30%, more preferably by at least 70%, most preferably by at least 100%. Methods of how to determine a decrease in activity as well as the definition of the term "activity" have been mentioned in the above. As it appears to be the case that, even minor changes in the amount of expression can have some effect on the phenotype of the plant methods such as NASBA analysis and RT-PCR which are considerably more sensitive in place of the Northern-blot analysis are employed for the analysis of the transgenic plant.

Preferably, said reduction is achieved by an antisense, sense, ribozyme, co-suppression in vivo mutagenesis andlor dominant mutant effect. Therefore, DNA
molecules encoding an antisense RNA which is complementary to transcripts of a DNA molecule of the invention are also the subject matter of the present invention, as well as these antisense molecules. Thereby, complementarity does not signify that the encoded RNA has to be 100% complementary. A low degree of complementarity is sufficient, as long as it is high enough in order to inhibit the expression of a protein of the invention upon expression in plant cells. The transcribed RNA is preferably at least 90% and most preferably at least 95%
complementary to the transcript of the nucleic acid molecule of the invention.
In order to cause an antisense-effect during the transcription in plant cells such DNA
molecules have a length of at least 15 bp, preferably a length of more than 100 by and most preferably a length or more than 500 bp, however, usually less than bp, preferably shorter than 2500 bp.
The invention further relates to DNA molecules which, during expression in plant cells, lead to the synthesis of an RNA which in the plant cells due to a cosupression-effect reduces the expression of the nucleic acid molecules of the invention encoding the described protein. The invention also relates to RNA molecules encoded thereby. The principle of the cosupression as well as the production of corresponding DNA sequences is precisely described, for example, in W090112084, Jorgensen, Trends Biotechnol. 8 (1990), 340-344; Niebel et al. (Curr. Top.
Microbiol.
Immunol. 197 (1995), 91-103; Flavell et al. (Curr. Top. Microbiol. Immunol.

(1995), 43-36; Palaqui and Vaucheret, Plant. Mol. Biol. 29 (1995), 149-159;
Vaucheret et al., Mol. Gen. Genet. 248 (1995), 311-317; de Borne et al., Mol.
Gen.
Genet. 243 (1994), 613-621 and in other sources.
Such DNA molecules preferably encode an RNA having a high degree of homology to transcripts of the nucleic acid molecules of the invention. It is, however, not absolutely necessary that the coding RNA is translatable into a protein.
In a further embodiment the present invention relates to DNA molecules encoding an RNA molecule with ribozyme activity which specifically cleaves transcripts of a DNA molecule of the invention as well as these encoded RNA molecules.

Ribozymes are catalytically active RNA molecules capable of cleaving RNA
molecules and specific target sequences. By means of recombinant DNA
techniques it is possible to alter the specificity of ribozymes. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of two different groups of ribozymes. The first group is made up of ribozymes which belong to the group I intron ribozyme type. The second group consists of ribozymes which as a characteristic structural feature exhibit the so-called "hammerhead"
motif. The specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule.
In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a DNA molecule of the invention, for example a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA
sequences which are homologous to sequences encoding the target protein. Sequences encoding the catalytic domain may for example be the catalytic domain of the satellite DNA of the SCMo virus (Davies et al., Virology 177 (1990), 216-224 and Steinecke et al., EMBO J. 11 (1992), 1525-1530) or that of the satellite DNA
of the TobR virus (Haseloff and Gerlach, Nature 334 (1988), 585-591 ). The DNA
sequences flanking the catalytic domain are preferably derived from the above-described DNA molecules of the invention. The expression of ribozymes in order to decrease the activity in certain proteins in cells is also known to the person skilled in the art and is, for example, described in EP-A1 0 321 201. The expression of ribozymes in plant cells was, for example, also described, in Feyter et al.
(Mol.
Gen. Genet. 250 (1996), 329-338).
In a preferred embodiment this reduction is effected by means of an antisense effect. For this purpose the DNA molecules of the invention or parts thereof are linked in antisense orientation with a promoter ensuring the transcription in plant cells and possibly with a termination signal ensuring the termination of the transcription as well as the polyadenylation of the transcript. In order to ensure an WO 00/22144 PC'TIEP99/07633 efficient antisense effect in the plant cells the synthesized antisense RNA
should exhibit a minimum length of 15 nucleotides, preferably of at least 100 nucleotides and most preferably of at least 500 nucleotides. Furthermore, the DNA sequence encoding the antisense RNA should be homologous with respect to the plant species to be transformed. However, DNA sequences exhibiting a high degree of homology to DNA sequences which are present in the cells in endogenic form may also be used, preferably with a homology of more than 95%. To inhibit gene expression of the nucleic acid molecule of the invention, preferably DNA
molecules are used that show a homology, i.e. identity to the nucleotide sequences of SEQ ID NO: 1, 7, 9 or 11 of at least 90%, more preferably at least 93%, in particular at least 95% and most preferably at least 98%.
In a further embodiment the reduction of the amount of proteins encoded by the DNA molecules of the invention is effected by a ribozyme effect. The basic effect of ribozymes as well as the construction of DNA molecules encoding such RNA
molecules have already been described above. In order to express an RNA with ribozyme activity in transgenic cells the above-described DNA molecules encoding a riboyzme are linked with DNA elements which ensure the transcription in plant cells, particularly with a promoter and a termination signal. The ribozymes synthesized in the plant cells lead to the cleavage of the mRNA encoding the subtilisin-like serine proteases described above.
Furthermore, the subtiiisin-like serine protease activity in the plant cells of the invention can also be decreased by the so-called "in vivo mutagenesis" also called "chimeraplasty", for which a hybrid RNA-DNA oligonucleotide ("chimeroplast") is introduced into cells by transformation of cells (Zhu et al., Proc.
Natl. Acad. Sci. 96 (1999), 8768-8773, Kipp et al., poster session at the Stn International Congress of Plant Molecular Biology, September 21-27, 1997, Singapore; Dixon and Arntzen, meeting report on "Metabolic Engineering in Transgenic Plants", Keystone Symposia, Copper Mountain, C0, USA, TIBTECH 15 (1997), 441-447; W095/15972; Kren et al., Hepatology 25 (1997), 1462-1468;
Cole-Strauss et al., Science 273 (1996), 1386-1389).
Part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous subtilisin-like serine protease, in comparison to the nucleic acid sequence of the endogenous subtilisin-like serine protease it displays, however, a mutation or contains a heterologous region which is surrounded by the homologous regions. By means of base pairing of the homologous regions of the RNA-DNA oligonucleotide and of the endogenous nucleic acid molecule followed by a homologous recombination the mutation contained in the DNA component of the RNA-DNA oligonucleotide or the heterologous region can be transferred to the genome of a plant cell. This results in a decrease of the activity.
In addition, the present invention relates to transgenic plants or plant tissue comprising the above described plant cells of the invention.
In a preferred embodiment the transgenic plant displays increased stomatal density, higher conductance of stomata and/or higher content of sugars and protein in plant leaves or other tissue or organs, compared to wild type plants. An increase in the stomatal density is understood to refer to an elevated content of stomata in all aerial plant organs, preferably in the leaves of plants of the present invention in the order of at least about 10% compared to the corresponding non-transformed wild type plant, which already provides for beneficial effects on the vitality of the plant such as, e.g., improved dry matter. Advantageously, the stomatal density is increased by at least about 50%, preferably by more than about 75%, particularly preferred at least about more than 100% and still more preferably more than about 200%. With respect to a decrease in the stomatal density due to the increased expression or activity of subtilisin-like serine proteases according to the invention in the transgenic plant of the invention, the stomatal density is decreased by at least 2%, preferably by more than 5%, particularly preferred at least about more than 10%, and still more preferably more than about 30%. Preferably, the transgenic plant of the invention shows a yield increase, preferably with respect to a harvestable part of the plant.
The term "yield increase" in the present context is understood to mean preferably an increase in production of ingredients, in particular soluble sugars andlor proteins andlor biomass, in particular if measured in fresh or dry weight per plant.
An increase in protein and/or sugar content in this context means that the protein content in the plant cells of the invention is increased by at least 5%, preferably by at least 20%, in particular by at least 50% and most preferably by at least 75% as compared to plant cells of wild type plants that are not modified andlor the sugar content is increased by at least 5%, preferably by at least 25%, in particular by at least 50% and most preferably by at least 75% as compared to plant cells of wild type plants that are not modified.
Methods for determining sugar and protein content are known to the person skilled in the art.
The term "yield increase" means an increase of dry weight by least 3%, preferably by at least 10%, in particular by at least 20% and most preferably by at least 30%
and/or an increase in fresh weight by least 2%, preferably by at least 5%, in particular by at least 10% and most preferably by at least 20%.
In yet another aspect, the invention also relates to harvestable parts and to propagation material of the transgenic plants according to the invention which contain transgenic plant cells described above, i.e. at least one recombinant DNA
molecule or vector according to the invention and/or which are derived from the above described plants. Harvestable parts can be in principle any useful parts of a plant, for example, leaves, ~ stems, flowers, fruit, seeds, roots etc.
Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
In addition, the present invention relates to a kit comprising the recombinant DNA
molecule or the vector of the invention. The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic plant cells, plant tissue or plants.
The kit of the invention may advantageously be used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool. The parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art. The kit or its ingredients according to the invention can be used in plant cell and plant tissue culture, for example in agriculture. The kit of the invention and its ingredients are expected to be very useful in breeding new varieties of, for example, plants which display improved properties such as those described herein.
Thus, the present invention also relates to a method for the production of a transgenic plant comprising an increased yield and/or increased stomatal density compared to wild type plants, wherein (a) a plant cell is genetically modified by the introduction of a foreign nucleic acid molecule the presence of which or the expression of which results in a decreased activity of a subtilise;
(b) a plant is regenerated from the cell prepared according to step (a); and (c) further plants, if any, are generated from the plant prepared according to step (b).
Likewise, the present invention relates to a method for the production of a transgenic plant having a decreased water consumption and/or decreased stomatal density compared to wild type plants wherein (a) a plant cell is genetically modified by the introduction of a foreign nucleic acid molecule the presence of which or the expression of which results in an increased activity of a subtilise;
(b) a plant is regenerated from the cell prepared according to step (a); and (c) further plants, if any, are generated from the plant prepared according to step (b).
Furthermore, the present invention relates to use of at least one nucleic acid molecule encoding and/or regulating the expression of a subtilisin-like serine protease, a nucleic acid molecule hybridizing with such a nucleic acid molecule, a nucleic acid molecule encoding a product that interferes with the expression or activity of subtilisin-like serine proteases in plants, or a recombinant DNA
molecule or vector of the invention in the production of transgenic plants for increasing yield, and/or increasing stomatal density, and/or increasing leaf fresh andlor dry weight, andlor increasing leaf dry matter content, andlor increasing sugar content in leaves, and/or increasing protein content in leaves, andlor increasing C02-assimilation, and/or sustaining photosynthesis (prevention of photoinhibition) under conditions of high irradiance (see Example 1 ), andlor changing the water consumption of plants, and or couteracting the consequences of changing environmental contitions with respect to stomatal density by the inhibition or stimulation of a subtilisin-like serine protease encoding gene.
Preferably such nucleic acid molecules are derived from plant genes encoding subtilases. Modulation of the activity of these genes leads to several morphological and physiological changes that are useful for the engineering of improved plants for agriculture, wood culture, or horticulture. Furthermore, the above described nucleic acid molecules and the recombinant DNA molecules and vectors according to the invention may be useful for the alteration or modification of piant/pathogene interaction. The term "pathogen" includes, for example, bacteria, viruses and fungi as well as protozoa. The plants, plant tissue and plant cells of the invention as well as harvestable parts and propagation of such plants can be used for the preparation of feed and food or additives therefor.
Deposit One plasmid produced and used within the scope of the present invention was deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures) (DSMZ) in Braunschweig, Federal Republic of Germany, which is recognized as an international depository, in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
On October 4, 1999 the following plasmid pAH 14158 was deposited at the German Collection of Microorganisms and Cell Cultures, (DSMZ) (Deposit number): DSM

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on the Internet, for example under http://www.ncbi.nlm.nih.gov/PubMedlmedline.html. Further databases and addresses, such as http:/lwww.ncbi.nlm.nih.gov/, http:llwww.infobiogen.fr/, http:Ilwww.fmi.chlbiologylresearch tools.html, http:/lwww.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.lycos.com. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
The Figures show:
Figure 1: Top: Release of water vapour (transpiration) from leaves of the wildtype (wt) and the R-558 mutant (R-558) at irradiances of 300 NE
,(Nmol m-2 s-') and 1200 NE (Nmol m-2 s-') of white light measured by an infrared gas analyzer.
Bottom: Net uptake of C02 (assimilation) into leaves of the wildtype and the R-558 mutant (R-558) at irradiances of 300 pE (Nmol m-a s~') and 1200 NE (Nmol m'2 s-') of white light measured by an infrared gas analyzer.
Figure 2: Schematic representation of the SDD1 protein marked with the amino acid positions bordering the putative pre- and the pro-sequence and the positions of the four invariant amino acids (D, H, N, S) found in all known subtilisins. Furthermore, the consequence of the mutation present in the R-558 mutant is indicated which converts the R codon at amino acid position 492 into a stop codon leading to the formation of a C-terminally truncated protein lacking the essential serine residue at position 552 (S552).
Figure 3: Schematic representation of the plasmid pG-SDD9 Fragment A: 7067 by Sall - EcoRV subfragment of the BAC
IGF20D22 that includes the 2328 by coding region of SDD9 in addition to 2 kb upstream DNA (promoter) and 2.8 kb downstream DNA was inserted into the Sall and Smal sites of the vector pBIB-Hyg.
Vector: pBIB-Hyg (Becker, 1990, Nucleic Acids Res. 18, 203).

Figure 4: Schematic representation of the plasmid p35S-SDD1 Fragment A: 35S promoter of the Cauliflower Mosaic Virus;
(Gardner et al., 1981, Nucleic Acids Res. 9, 2871-2888) Fragment B: 2328 nucleotides coding region of the SDD1 gene (SEQ ID No. 1 ) Fragment C: polyadenylation signal of the gene 3 from the T-DNA of the Ti-plasmid pTi ACH 5 (Gielen et al., 1984, EMBO J.
3, 835-846) Vector: pBIB-Hyg (Becker, 1990, Nucleic Acids Res. 18, 203).
Figure 5: Sequence alignments in the four highly conserved domains, the D
region, the H region, the substrate binding site, and the S region of the subtiiisins Subtilisin BPN' (Wells et al. 1983, Nucleic Acids Res.
11, 7911-7925), the KEX2 of yeast (Mizuno et al. 1988, Biochem.
Biophys. Res. Commun. 156, 246-254), the human FURIN/PACE
(Wise et al. 1990, Proc. Natl. Acad. Sci USA 87, 9378 - 9382), the human PC1/PC3 (Seidah et al., 1991, Mol. Endocrinol. 5, 111 - 122;
Smeekens et al., 1991, Proc. Natl. Acad. Sci. USA 88, 340 - 344), the CUCUMISiN from Cucumis melo (Yamagata et al., 1994, J. Biol.
Chem. 269, 32725 - 32731 ), LeP69 from Lycopersicon esculentum (Tomero et al., 1996, Proc. Natl. Acad. Sci. USA 93, 6332 - 6337), the AG12 from Alnus glutinosa (Ribeiro et al., 1995, Plant Cell 7, 785 - 794) and of SDD1. The positions of the invariant amino acids are marked with *. Identical amino acids present at corresponding positions in the different subtilisins are highlighted with black boxes.
Figure 6: Schematic representation of the plasmid p35S-a,SDD9 Fragment A: 35S promoter of the Cauliflower Mosaic Virus;
(Gardner et al., 1981, Nucleic Acids Res. 9, 2871-2888) Fragment B: 2079 by - fragment (position 74 - 2153 according to the sequence shown in SEQ ID No. 1 ) of the SDD9 gene inserted in antisense orientation to the 35S
promoter.
Fragment C: polyadenylation signal of the gene 3 from the T-DNA of the Ti-plasmid pTi ACH 5 (Gielen et al., 1984, EM80 J.
3, 835-846) Vector: pBIB-Hyg (Becker, 1990, Nucleic Acids Res. 18, 203).
Figure 7: Amino acid comparison of three different subtilases;
at subp1=Subtilase from Arabidopsis thaliana, see SEQ ID No. 2, st subp1=Subtilase from Solanum tuberosum, see SEQ ID No. 8;
st subp2=Subtilase from Solanum tuberosum, see SEQ ID No. 12.
The Examples illustrate the invention:
Example 1: HZO transpiration and COZ assimilation are increased in the Arabidopsis thaliana R-558 mutant particularly under conditions of high irradiance Arabidopsis thaliana R-558 mutant plants and corresponding wildtype plants (wt) were grown until bolting in soil (Einheitserde Typ P I Einheitserde Typ T /
sand: 2 /
1 I 1 ) under standard culture conditions in a climatised growth chamber at 16 h photoperiod (180 Nmol m-2 s' fluorescent light; lamp type: TLD36WI840 and TLD36WI830, Philips, Hamburg, Germany) with day and night temperature and relative humidity of 20°C, 60% relative humidity and 16°C, 75%
relative humidity, respectively. Single leaves (n=10) were clamped into a gas exchange measurement chamber of an infrared gas analyzer (Walz, Effeltrich, Germany) with H20 release from as well as C02 uptake into the leaves were measured according to the procedure described by Muschak et al. (Photosynthetica 33, 45~-465, 1997}. As shown in Figure 1, the leaves of the mutant plants showed increased transpiration of H20 and increased assimilation (net uptake) of COZ
under low light (300 Nmol m-2 s'') and under high light (1200 Nmol m'2 s'') conditions applied during the measurements. Enhancement of C02 assimilation, being almost double in the R-558 mutant in comparison to the wild type, was most prominent under the high light conditions which caused a depression of C02 assimilation in the wild type in comparison to the low light conditions (photoinhibition).
Example 2: Isolation of the SDDl gene through map-based gene cloning The genetic locus affected by the mutation in the R-558 mutant has previously been mapped to the top arm of the Arabidopsis thaliana chromosome 1 to an interval of approximately 0.59 cM bordered by the molecular markers IGF-20G19LE and IGF-2513RE (D. Berger, 1997, PhD Thesis Freie Universitat Berlin).
Two clones of the Arabidopsis thaliana genomic IGF-BAC library (Mozo et al., 1998, Mol. Gen. Genet. 258, 562-570), IGF20D22 and IGF21 M11, which fully cover this region, were sequenced by the SPP consortium (see http:/lsequence-www.stanford.edu/ara/SPP.html) as part of the Arabidopsis genome initiative (Bevan et al., 1997; Plant Cell 9, 476-478; http:llgenome-www.stanford.edu/Arabidopsislagi.html). The 0.59 cM region was thus identified to cover 113 kb of genomic DNA sequence. In order to identify the SDD1 gene corresponding to the mutant locus, this region was scanned for mutations by application of the restriction SSCP- (single strand conformational polymorphism) technique (Dean and Gerrard, 1991, BioTechniques 10, 332 - 333; Iwahana et al., 1992, BioTechniques 12, 64 - 66) which for this purpose was adapted for the use in plants. This approach is novel and has not been applied for mutation scanning in plants before. Thus, 57 DNA fragments of 2-kb each, were separately PCR
amplified from total DNA of wild type and R-558 plants and after digestion with Alu I and/or Hinf I they were analysed through polyacrylamid gel electrophoresis as described by Dean and Gerrard, 1991 (BioTechniques 10, 332 - 333) and Iwahana et al., 1992 (BioTechniques 12, 64 - 66). A single SSCP was detected that discriminated between the two genotypes and which upon sequencing of the corresponding DNA fragments was shown to be caused by a single C/G -> TIA
mutation (Seq. ID No. 1; Seq. ID No. 3). This mutation introduced a premature stop codon into an ORF of a predicted gene spanning 2328 by that encoded for a deduced polypeptide of 775 amino acids (Fig. 2; Seq. ID No. 2; Seq. ID No. 4;
Genbank Accession AC002411; http:llpgec-genome.pw.usda.pov/F20D22.anno.html#anchorl 2).
Example 3: Genetic complementation of the R-558 mutant by Agrobacterium tumefaciens - mediated DNA-transfer In order to confirm the idenfiity of the 2328 by DNA sequence (Seq. ID No. 1;) as the protein coding region of the SDD1 gene defective in the R-558 mutant, genetic complementation experiments were performed with the introduction of a wild type DNA-copy into the R-558 mutant through Agrobacterium tumefaciens - mediated genetic transformation.
Two plasmids were generated for this purpose:
Plasmid pG-SDD1' (Fig. 3) carries the 7067 by Sall - EcoRV subfragment of the BAC IGF20D22 that includes the 2328 by coding region of SDD1 in addition to 2 kb upstream DNA (promoter) and 2.8 kb downstream DNA was inserted into the Sall and Smal sites of the l'-DNA vector pBIB-Hyg (Becker, 1990, Nucleic Acids Res. 18, 203).
The second plasmid, p35S-SDD1 (Fig. 4), harbours the three fragments, A, B, C, inserted into the pBIB-Hyg vector (Becker, 1990, Nucleic Acids Res. 18, 203).
Fragment A, which was inserted between the EcoRl and Sacl restricition sites in the polylinker of pBIB-Hyg, includes the 35S promoter of the Cauliflower Mosaic Virus (CaMV) comprising the nucleotides 7146 through 7464 as described by Gardner et al. (Nucleic Acids Res. 9, 2871-2888, 1981 ). Fragment C contains the polyadenylation signal of the gene 3 from the T-DNA of the Ti-plasmid pTi ACH

(Gielen et al., EMBO J. 3, 835 - 846, 1984), nucleotides 11749 through 11939 which was isolated as Pvu !I - Hind III fragment from the plasmid pAGV 40 (Herrera-Estrella et al., Nature 303, 209 - 213, 1983) and which, after addition of a Sph I linker to the Pvu II restriction site, was inserted into the Sph I and Hind III
restriction sites of pBIB-Hyg. The resulting intervening plasmid was called pBIN-AR-Hyg. Fragment B covers the 2328 nucleotides coding region of the SDD1 gene (Seq. ID No. 1 ) that was amplified by PCR from the BAC IGF20D22 and provided with Asp718 and Xbal linker sequences and which was inserted into the Asp718 and Xbal restriction sites of pBIN-AR-Hyg.
Both plasmids were separately introduced into Agrobacterium tumefaciens according to the procedure described by Hofgen and Willmitzer (Nucleic Acids Res. 16, 9877, 1988) and the corresponding T-DNAs were stably introduced into the R-558 mutant by Agrobacterium tumefaciens - in planta transformation following the method described by Bechtold et al. (Comet. Rend. Acad. Sci.
316, 1194 - 1199, 1993). Transformed seedlings selected for antibiotic (Hygromycin) resistance were grown to maturity and' tested for the expression of mutant or wildtype phenotypes by microscopic examination of rosette leaves cleared with 80 % ethanol.

Table 1: Analysis of stomatal density and distribution in the abaxial epidermices of cotyledons and leaves of wild type (wt), mutant (R-558) and transgenic mutant (R-558 / G-SDDI; R-558 /355-SDDI) Cotyledon Primary Leaf Plant Single Clustered Single Clustered Density StomataAStomatabn' Stomata"Stomatabn' (no.~mm'1 wt #1 100 0 % 58 100 0 % 163 97.0 % %

wt #2 100 0 % 40 100 0 % 174 124.3 % %

wt #3 100 0 % 51 98.9 1.1 176 125.7 % % %

wt #4 100 0 % 49 100 0 % 160 114.3 % %

wt #5 100 0 % S 100 0 % 166 I 18.6 % 8 %

R-S.i8 #I 61 % 39 % 136 86 % 14 % 492 351.4 R-5~8 #2 56 % 44 % 62 90.1 9.9 421 300.7 % %

R-S.iB #3 54 % 46 % 137 89.6 10.4 395 282.1 % %

R-8 #4 58 % 42 % 109 93.9 6.1 409 292.1 % %

R-558 #5 60 % 40 % 85 91.6 8.4 403 287.9 % %

R-558 / G-SDDI 100 0 % 47 I 00 0 % 279 199.3 # 1 % %

R-5~8 / G-SDDI 100 0 % 53 83.4 16.6 181 161.6 #2 % % %

R-S.iB l G-SDDI 100 0 % 52 100 0 % 139 99.3 #3 % %

R-558 / G-SDDI 96.4 3.6 % 55 100 0 % 195 139.3 #4 % %

R-558 / G-SDDI 100 0 % 53 100 0 % 180 128.6 #5 % %

R-558 / G-SDDI 100 0 % 55 100 0 % 169 120.7 #6 io %

R-558 / G-SDDI 100 0 % 53 100 0 % 163 116.4 #7 % %

R-5~8 / G-SDDI 96.8 3.2 % 62 100 0 % 285 203.6 #8 % %

R-558 / G-SDDI 100 0 % 37 100 0 % 98 70.0 #9 % %

R-558 / G-SDDI 96.7 3.3 % 60 100 0 % 199 142.1 # 10 ,'0 %

R-558 / 355-SDDI82 % 18 % 66 100 0 % 167 119.3 # ( %

R-S.S8 / 355-SDDI100 0 % 34 100 0 % 123 87.9 #2 % %

R-558 / 35S-SDDI93 % 7 % 55 100 0 % 168 120 #3 %

R-5581355-SDDI 74 % 26 % 80 100 0 % 136 97.1 #4 %

R-558 / 35S-SDDI100 0 % 45 100 0 % 122 87.1 #5 % %

R-S.i8 / 355-SDDI80 % 20 % 70 100 0 % 195 139.3 #6 %

R-558 / 355-SDDI53 % 47 % 131 98 % 2 % 200 142.9 #7 R-558 / 35S-SDDI91 % 9 % 68 100 0 % 123 87.9 #8 %

R-,i58 / 355-SDDI80 % 20 % 90 95.1 4.9 123 87.9 #9 % %

R-5581355-SDDI 94 % 6 % 65 100 0 .'0 108 77.1 #10 %

° Stomata separated from other stomata by at least one epidermal cell.
b Stomata placed in direct contact to at least one other stoma. ° Number of stomata sampled.
SUBSTITUTE SHEET (RULE 26) As shown in Table 1, 7 out of 10 and 2 out of 10 transformants harbouring the T-DNAs of the pG-SDD1 or the p35S-SDD1 plasmids, respectively, showed a wildtype phenotype on cotyledons with respect to the appearanceiabsence of clustered stomata. 7 transformants carrying the T-DNA of p35S-SOD1 showed an intermediate phenotype in cotyledons due to inappropriate expression of the transgene in this organ. In primary leaves, all 10 transformants harboring the T-DNA of pG-SDD1 and all 10 transformants carrying the T-DNA of p35S-SDD1 showed a strong reduction in stomatal density and/or the fraction of clustered stomata as compared to the R-55'8 mutant. These data unequivocally demonstrated the identity of the 2325 by DNA fragment as the coding region of the SDD 1 gene.
Example 4: Analysis of the SDD~ nucleotide sequence and SDD1 amino acid sequence The analysis of the SDD1 nucleotide and derived amino acid sequences was performed using the GCG 8:1 and BLAST 2.0 computer programs (see: Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711; Altschul et al. 1997, Nucleic Acids Res. 25, 3389-3402).
The derived amino acid sequence of SDD1 shows significant identity I
similarity to known members of a specific class of serine proteases, called subtilisins or dibasic processing endoproteases. In subtifisins, four regions form the catalytic triad and the substrate binding site and are most highly conserved among subtilisins, prominent representatives of which are the bacterial SUBTILISIN
BPN' (Wells et al. 1983, Nucleic Acids Res. 11, 7911-7925), the KEX2 of yeast (Mizuno et al. 1988, Biochem. Biophys. Res. Commun. 156, 246-254}, the human furin/PACE (GenBank, Acc. No. X17094) and PC11PC3 (Seidah et al., 1991, Mol.
Endocrinol. 5, 111 - 122; Smeekens et al., 1991, Proc. Natl. Acad. Sci. USA
88, 340 - 344). Several genes from plants encoding subtilises have been isolated such as CUCUMISIN from Cucumis meio (Yamagata et al., 1994, J. Biol. Chem.
269, 32725 - 32731 ), P69 from Lycopersicon esculentum (Tornero et al., 1996, Proc. Natl. Acid. Sci. USA 93, 6332 - 6337), or AG12 from Alnus glutinosa (Ribeiro et al., 1995, Plant Cell 7, 785 - 794). The in vivo functions of these plant enzymes, however, are hitherto unknown. In the four conserved regions, SDD1 displays highest sequence similarity to the subtilisins listed above and contains the four characteristic invariant amino acids present in all subtilisins hitherto known (Fig. 5). This unequivocally proves the belonging of SDD1 to this class of endoproteases. The amino acid sequence motif VICAAGNNG within the substrate binding site, however, is unique and distinguishes SDD9 from all other known subtilisins. The mutation present in the R-558 mutant creates a premature stop codon leading to the formation of a C-terminally truncated protein which lacks the essential S-domain containing the catalytically active serine residue (Fig.
2).
Example 5: Modulation of stomata) density in plants through modulation of SDD9 expression by genetic engineering The usefulness of the SDD1 gene for the creation of plants with various different levels of increased or decreased stomata) densities through modulation of the degree of SDD9 gene expression was shown by the analysis of further transgenic plants. 4 out of 10 of the transgenic plants carrying the aforementioned (Example 4) T-DNA of p35S-SDD1 and 1 out of 10 of the transgenic plants carrying the aforementioned (Example 4) T-DNA of pG-SDD~ showed lower stomata) density than the corresponding wildtype plants analyzed in parallel (Table 1 ).
Furthermore, SDD9 antisense inhibition studies were performed:.To this end, the plasmid p35S-a.SDD1 was generated which contains an antisense-gene construct called '35S-aSDD1' (Fig. 6). A 2079 by - fragment (position 74 - 2153 according to the sequence shown in seq. ID 1 ) of the SDD9 gene was PCR-amplified and subcloned into the pCR 2.1 - vector (Invitrogen, Leek The Netherlands). Using the flanking Asp718 (3') and Xbal (5') restriction sites, the 2 kb SDD9 - fragment was cut from the pCR 2.1 - vector and inserted into the Asp718 and Xbal sites of the pBIN-AR-Hyg. vector (see example 3), thus placing it in antisense orientation to the CaMV 35S - promoter.
The plasmid p35S-aSDD1 was introduced into Agrobacterium tumefaciens according to Hofgen and Willmitzer (Nucleic Acids Res. 16, 9887, 1988) and was used to generate transgenic Arabidopsis thaliana plants through application of the procedure described by Schmidt and Willmitzer (Plant Cell Rep. 7, 583-586, 1988). Among the transgenic plants carrying the T-DNA of p35S-a.SDD9 thus generated, individuals with increased stomatal density were obtained.
It was thus demonstrated that through the application of genetic engineering techniques, a gene encoding a subtilisin-like serine protease, can be used to generate plants with various different levels of decreased or increased stomatal densities brought about by the modulation of the expression of said gene.
Example 6: Cloning of two SDD1 homologs from Solanum tuberosum A 2328 by fragment representing the complete SDD1 coding region from Arabidopsis was amplified from the clone IGF20D22 of the Arabidopsis thaliana genomic IGF-BAC library (Mozo et al., 1998, Mol. Gen. Genet. 258, 562-570) via PCR and was used as a radiolabeled probe (Random Primed DNA Labeling Kit, Boehringer PJlannheim) in the screening procedure.
Plaque lifting was performed on 1.6 x 1 O6 pfus of a genomic library from Solanum tuberosum L. line AM8015793 (Liu et al., 1991, Plant Mol. Biol. 17, 1139-1154), using Hybond N filters (Amersham).
After pre-hybridization for 4 h at 42° C in buffer A (5 x SSC, 0,5 %
BSA, 5 x Denhardt, 1 % SDS, 40 mM phosphate buffer, pH 7.2, 100 mg/l herring sperm DNA, 25 % formamid) filters were hybridized to the radiolabeled probe (see above).
After 8 h of hybridization the filters were washed 3 times for 20 min at 50°C in a buffer containing 3 x SSC and 0.5 % SDS. X-ray film exposure was usually performed for 14h.
Ten strongly hybridizing phage plaques were rescreened and purified to homogenity. Phage DNA was prepared according to the method described by Patterson & Dean 1987 (NAR Vo1.15 (15), 6298).
According to their restriction patterns and Southern analysis, using the radiolabeled SDD1 PCR fragment as a probe, phages were divided into two different classes. In case of class I a 3864 by hybridizing EcoRI/Sall fragment was subcloned into pMCSS (EcoRI/Sall) (Mo Bi Tec, Gottingen), yielding plasrriid pAH10158. In case of class II a ~4.5 kb hybridizing Scal- fragment was subcloned in pMCSS (Mo Bi Tec, Gottingen), yielding plasmid pAH 14158. Both of the plasmids were subjected to DNA-sequencing analysis and contained the nucleotide sequences, coding for SDD9 homologs from Solarium tuberosum P1gen (SEQ ID NO: 7), P1 - corresponding to P1gen without the intron - (SEQ ID
N0: 9), and P2 (SEQ ID NO: 11 ) The class I a fragments are characterized by the presence of introns, class Ila fragments by the absence of introns. The amino acid sequences encoded by P1 and P2 are shown in SEQ ID NOS: 8, 10 and 12, respectively, and compared to that of SDD1 in Figure 7.
Example 7: Overexpression of subtilase in tobacco A genomic fragment of 2.3 kb was amplified by PCR tram the genome of A.
thaliana var. C24, and Asp 7181 Xba I (the primers comprised these sequences) and cloned into the vector pBinAR Hyg (Hofgen and Willmitzer (1990), Plant Sci.
66: 221-230).
Agrobacteria GV2260 (Deblaere (1985), Nucl. Acid Res. 13: 4777-4788) were transformed by way of heat shock, and tobacco plants were infected cv. SNN and regenerated (Rosahl et al., (1987) EMBO J. 6: 1155-1159).
In total, 24 plants were examined as to their RNA expression. For this, RNA
from leaf plants was prepared according to Logemann et al. (1987) Anal. Biochem.
163: 16-20) in tissue culture (22°C, 50% atmospheric humidity, 2000 Lux, 16h/8h light rhythm). About 10Ng RNA were loaded onto a denaturating gel (Lehrach et al., (1977) Biochem. 16: 4743-4751 ) and thereupon transferred onto a positively charged membrane Hybond N+ (Amersham Buchler, Braunschweig) via capillary transfer. Thereafter, the RNA was fixed onto the membrane via heat fixation (2h180°C), washed in 2XSSC for a short period of time (2-3 min), prehybridized for at least 1 h at 65°C (0.25M Na-P buffer pH 7.2; 1 % BSA, 7% SDS and 10mM
EDTA) and hybridized overnight at 65°C with a radioactively labeled probe (Feinberg, and Vogelstein, (1983) Anal. Biochem. 132: 6-13). After two washes with 2XSSC at 65°C for 30 min each the filter was exposed on an X-ray filter overnight at -80°C.
In total, 24 plants were analyzed by way of "Northern technique", of which 12 could be classified as positive, 9 being classified as strong and 3 as weaker expressers. Copies using clear nail varnish were prepared from these plants as well as from the wild type, 5 leafs from different plants of the wild type were analyzed. For doing so, 5 areas each were counted and stomatal density was determined as stomata/mm2. When doing so, it was surprisingly found that stomata) density on the adaxial leaf surface was about twice as high as on the abaxial surface; see table 2.
Table Microscopicalysis an of stomata) density WTladaxial PL-Nr. Stomata/m m2 T! 75 79 76 73 62 MV: 56 STD: 6,13 WT/abaxial PL-Nr. ~ Stomata/m m2 ' MV: 112 STD: 21,79 AR

SubtJadaxial PI. Nr. Stomata/m m2 MV: 19 STD: 2,72 AR

SubtJabaxial PI. Nr. Stomata/m m2 22 58 47 62 __ 56 46 37 ' 97 95 100 93 77 mean MV: 63 STD: 9, value WT
AR

Subt.

adaxial56 abaxial112 STD standard = deviation MV - mean value WT - wi ~d type AR - transgenic plant After microscopic analysis of the copies of the transgenic plants which were taken by using clear nail varnish, a decrease in stomatal density by about 50% on both leaf sides could be detected. The hypostomatic phenotype, however, is also found in the transgenic plants; see table 2.
No differences could be found between strong and weak "expressers"; thus even a minor increase of SDD1 activity seems to be sufficient to result in a phenotype.

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atg cccaaacct ttctttctc tgcattatc tttcttcta ttttgt 48 gaa Met ProLysPro PhePheLeu CysIleIle PheLeuLeu PheCys Glu tct tcgtcagag atcctgcag aagcagact tacattgtt cagctt 96 tct Ser SerSerGlu IleLeuGln LysGlnThr TyrIleVal GlnLeu Ser cat aatagcgaa accgetaaa acctttgcc tcaaagttt gattgg 144 cct His AsnSerGlu ThrAlaLys ThrPheAla SerLysPhe AspTrp Pro cat tcttttctc caagaagcg gttttaggt gttgaagaa gaagag 192 ctt His SerPheLeu GlnGluAla ValLeuGly ValGluGlu GluGlu Leu gaa ccttcttct cgacttctc tactcctat ggctctgcg attgaa 240 gag Glu ProSerSer ArgLeuLeu TyrSerTyr GlySerAla IleGlu Glu gga getgetcag ttgactgaa tcagaagcc gagatactg agatat 288 ttt Gly AlaAlaGln LeuThrGlu SerGluAla GluIleLeu ArgTyr Phe tca gaagttgtt gcagtgaga cctgaccat gttcttcag gttcaa 336 cct Ser GluValVal AlaValArg ProAspHis ValLeuGln ValGln Pro acc tactcttac aagttcttg ggactcgac ggttttgga aactcc 384 act Thr TyrSerTyr LysPheLeu GlyLeuAsp GlyPheGly AsnSer Thr ggt tggtctaaa tctcggttt ggtcaaggc acaattatc ggcgtg 432 gta Gly TrpSerLys SerArgPhe GlyGlnGly ThrIleIle GlyVal Val ctt gat act gga gtt tgg cct gaa agt cct agc ttt gac gat acc gga 480 Leu Asp Thr Gly Val Trp Pro Glu Ser Pro Ser Phe Asp Asp Thr Gly atg cct tcg att cca cgg aaa tgg aaa ggg att tgc caa gaa gga gaa 528 Met Pro Ser Ile Pro Arg Lys Trp Lys Gly Ile Cys Gln Glu Gly Glu agt ttc agt tct tcg agc tgt aac cgg aag cta atc ggt get aga ttc 576 Ser Phe Ser Ser Ser Ser Cys Asn Arg Lys Leu Ile Gly Ala Arg Phe ttc atc aga gga cac cgt gtc get aat tca cca gag gaa tca cca aac 624 Phe Ile Arg Gly His Arg Val Ala Asn Ser Pro G1u Glu Ser Pro Asn atg cct cgt gaa tac att tcc gca aga gat tca acg gga cac ggg act 672 Met Pro Arg Glu Tyr Ile Ser Ala Arg Asp Ser Thr Gly His Gly Thr cac acc gcc tca aca gtt ggt gga tcc tct gtt tcg atg gcg aat gtt 720 His Thr Ala Ser Thr Val Gly Gly Ser Ser Val Ser Met Ala Asn Val ctt ggc aat gga get ggt gtg get cgt ggg atg get cct gga get cac 768 Leu Gly Asn Gly Ala Gly Val Ala Arg Gly Met Ala Pro Gly Ala His att gca gtc tat aaa gtc tgt tgg ttc aat ggt tgt tac agc tct gac 816 Ile Ala Val Tyr Lys Val Cys Trp Phe Asn Gly Cys Tyr Ser Ser Asp att cta gca get ata gat gta gcg att caa gat aaa gtc gat gtt ctt 864 Ile Leu Ala Ala Ile Asp Val Ala Ile Gln Asp Lys Val Asp Val Leu tcg ctt tcc ctt ggc ggt ttc cct att cct ttg tat gat gac aca atc 912 Ser Leu Ser Leu Gly Gly Phe Pro Ile Pro Leu Tyr Asp Asp Thr Ile gcc att gga aca ttc cga gcc atg gaa cgc ggt ata tct gta atc tgt 9'00 Ala Ile Gly Thr Phe Arg Ala Met Glu Arg Gly Ile Ser Val Ile Cys gca get ggt aac aac ggt cca atc gaa agc tct gtt gca aac aca get 1008 Ala Ala Gly Asn Asn Gly Pro Ile Glu Ser Ser Val Ala Asn Thr Ala cct tgg gtc tca acc att ggc gca ggc acg ctt gat cga aga ttt ccc 1056 Pro Trp Val Ser Thr Ile Gly Ala Gly Thr Leu Asp Arg Arg Phe Pro get gtg gtc aga tta gcc aac gga aag ctt ctc tat gga gag tca ttg 1104 Ala Val Val Arg Leu Ala Asn Gly Lys Leu Leu Tyr Gly Glu Ser Leu tat ccg gga aaa ggt ata aag aat gcc ggg aga gag gtt gag gtg att 1152 Tyr Pro Gly Lys Gly Ile Lys Asn Ala Gly Arg Glu Val Glu Val Ile tac gtc aca gga gga gat aaa gga agt gag ttc tgt ttg aga ggg tca 1200 Tyr Val Thr Gly Gly Asp Lys Gly Ser Glu Phe Cys Leu Arg Gly Ser cttcca agagaagaa atccgaggc aaaatggtg atttgt gatcgcgga 1248 LeuPro ArgGluGlu IleArgGly LysMetVal IleCys AspArgGly gtcaat ggaagatcg gagaaagga gaagcggtt aaagaa getggagga 1296 ValAsn GlyArgSer GluLysGly GluAlaVal LysGlu AlaGlyGly gttgca atgatctta gccaataca gagatcaac caagaa gaagattct 1344 ValAla MetIleLeu AlaAsnThr GluI1eAsn GlnGlu GluAspSer attgac gttcatctc ttaccaget acattgatt ggttac actgagtca 1392 IleAsp ValHisLeu LeuProAla ThrLeuTle GlyTyr ThrGluSer gtcctt ctgaagget tatgttaat gccacggtg aaacca aaggcgcgg 1440 ValLeu LeuLysAla TyrValAsn AlaThrVal LysPro LysAlaArg ataatt tttggtggt acggtgatt gggaggtca cgagca ccggaggtg 2488 IleIle PheGlyGly ThrValIle GlyArgSer ArgAla ProGluVal getcag ttttcaget cgaggaccg agtttagcc aatcct tcgatacta 1536 AlaGln PheSerAla ArgGlyPro SerLeuAla AsnPro SerIleLeu aaaccg gatatgatt getccggga gtcaatatc attgcg gettggcct 1584 LysPro AspMetIle AlaProGly ValAsnIle IleAla AlaTrpPro caaaat ctaggacca accggactt ccttatgat tcaaga agagttaac 1632 GlnAsn LeuGlyPro ThrGlyLeu ProTyrAsp SerArg ArgValAsn ttcact gtaatgtca ggaacttca atgtcttgt ccacat gttagcgga 1680 PheThr ValMetSer GlyThrSer MetSerCys ProHis ValSerGly atcact getcttatc cggtctgca tacccgaac tggtct ccagetgca 1728 IleThr AlaLeuIle ArgSerAla TyrProAsn TrpSer ProAlaAla atcaaa tccgcattg atgacaaca gcggatttg tacgat cgtcaaggg 1776 IleLys SerAlaLeu MetThrThr AlaAspLeu TyrAsp ArgGlnGly aaagcg ataaaggat ggtaacaaa ccagccggt gtgttt gcgattgga 1824 LysAla IleLysAsp GlyAsnLys ProAlaGly ValPhe AlaIleGly gcaggg catgtgaat ccgcaaaag gcgataaac ccggga ttggettac 1872 AlaGly HisValAsn ProGlnLys AlaIleAsn PrcGly LeuValTyr aacatt caaccagtg gattacata acttacctc tgcact cttggattc 1920 AsnIle GlnProVal AspTyrIle ThrTyrLeu CysThr LeuGlyPhe acaaga tcagatatt ttagcaatc actcataag aacgtg agctgcaat 1968 ThrArg SerAspIle LeuAlaIle ThrHisLys AsnVal SerCysAsn gga ata ttg cgg aaa aac ccg ggt ttt agt ctc aat tac ccg tcg ata 2016 Gly Ile Leu Arg Lys Asn Pro Gly Phe Ser Leu Asn Tyr Pro Ser Ile gcc gtg att ttc aaa cgt ggc aag act acg gag atg atc aca agg cgt 2064 Ala Val Ile Phe Lys Arg Gly Lys Thr Thr Glu Met Ile Thr Arg Arg gtc act aac gtt ggg agt cct aac tcg ata tac tca gtg aat gtc aag 2112 Val Thr Asn Val Gly Ser Pro Asn Ser Ile Tyr Ser Val Asn Val Lys getccagag gggatcaaa gttattgtc aatcctaag agacttgtg ttc 2160 AlaProGlu GlyIleLys ValIleVal AsnProLys ArgLeuVal Phe aaacacgtg gatcagacg ctgagctat agagtatgg tttgtattg aag 2208 LysHisVal AspGlnThr LeuSerTyr ArgValTrp PheValLeu Lys aagaaaaac agaggaggg aaggtgget agctttgca caagggcag ttg 2256 LysLysAsn ArgGlyGly LysValAla SerPheA1a GlnGlyGln Leu acttgggtc aactctcat aatctgatg cagcgagtt agaagtcca atc 2304 ThrTrpVal AsnSerHis AsnLeuMet GlnArgVal ArgSerPro Ile tctgtaacc ttgaagact aactga 2328 SerValThr LeuLysThr Asn <210> 2 <211> 775 <212> PRT
<213> Arabidopsis thaliana <400> 2 Met Glu Pro Lys Pro Phe Phe Leu Cys Ile Ile Phe Leu Leu Phe Cys Ser Ser Ser Ser.Glu Ile Leu Gln Lys Gln Thr Tyr Ile Val Gln Leu His Pro Asn Ser Glu Thr Ala Lys Thr Phe Ala Ser Lys Phe Asp Trp His Leu Ser Phe Leu Gln Glu Ala Vat Leu Gly Val Giu Glu Glu Glu Glu Glu Pro Ser Ser Arg Leu Leu Tyr Ser Tyr Gly Ser Ala Ile Glu Gly Phe Ala Ala Gln Leu Thr Glu Ser Glu Ala Glu Ile Leu Arg Tyr Ser Pro Glu Val Val Ala Val Arg Pro Asp His Val Leu Gln Val Gln Thr Thr Tyr Ser Tyr Lys Phe Leu Gly Leu Asp Gly Phe Gly Asn Ser Gly Val Trp Ser Lys Ser Arg Phe Gly Gln Gly Thr Ile Ile Gly Val Leu Asp Thr Gly Val Trp Pro Glu Ser Pro Ser Phe Asp Asp Thr Gly Met Pro Ser Ile Pro Arg Lys Trp Lys Gly Ile Cys Glr_ Glu Gly Glu Ser Phe Ser Ser Ser Ser Cys Asn Arg Lys Leu Ile Gly Ala Arg Phe Phe Ile Arg Gly His Arg Val Ala Asn Ser Pro Glu Glu Ser Pro Asn Met Pro Arg Glu Tyr Ile Ser Ala Arg Asp Ser Thr GIy His Gly Thr His Thr Ala Ser Thr Val Gly Gly Ser Ser Val Ser Met Ala Asn Val Leu Gly Asn Gly Ala Gly Val Ala Arg Gly Met Ala Pro Gly Ala His Ile Ala Val Tyr Lys Val Cys Trp Phe Asn Gly Cys Tyr Ser Ser Asp Ile Leu Ala Ala Ile Asp Val Ala Ile Gln Asp Lys Val Asp Val Leu Ser Leu Ser Leu Gly Gly Phe Pro Ile Pro Leu Tyr Asp Asp Thr Ile Ala Ile Gly Thr Phe Arg A1a Met Glu Arg Gly Ile Ser Val Ile Cys Ala Ala Gly Asn Asn Gly Pro Ile Glu Ser Ser Val Ala Asn Thr Ala Pro Trp Val Ser Thr Ile Gly Ala Gly Thr Leu Asp Arg Arg Phe Pro Ala Val Val Arg Leu Ala Asn Gly Lys Leu.Leu Tyr Gly Glu Ser Leu Tyr Pro Gly Lys Gly Ile Lys Asn Ala Gly Arg Glu Val Glu Val Ile Tyr Val Thr Gly Gly Asp Lys Gly Ser Glu Phe Cys Leu Arg Gly Ser Leu Pro Arg Glu Glu Ile Arg Gly Lys Met Val Ile Cys Asp Arg Gly Val Asn Gly Arg Ser Glu Lys Gly Glu Ala Val Lys Glu Ala Gly Gly Val Ala Met Ile Leu Ala Asn Thr Glu Ile Asn Gln Glu Glu Asp Ser Ile Asp Val His Leu Leu Pro Ala Thr Leu Ile Gly Tyr Thr Glu Ser Val Leu Leu Lys Ala Tyr Val Asn Ala Thr Val Lys Pro Lys Ala Arg Ile Ile Phe Gly Gly Thr Val Ile Gly Arg Ser Arg Ala Pro Glu Val Ala Gln Phe Ser Ala Arg Gly Pro Ser Leu Ala Asn Pro Ser Ile Leu Lys Pro Asp Met Ile Ala Pro Gly Val Asn Ile Ile Ala Ala Trp Pro Gln Asn Leu Gly Pro Thr Gly Leu Pro Tyr Asp Ser Arg Arg Val Asn Phe Thr Val Met Ser Gly Thr Ser Met Ser Cys Pro His Val Ser Gly Ile Thr Ala Leu Ile Arg Ser Ala Tyr Pro Asn Trp Ser Pro Ala Ala Ile Lys Ser Ala Leu Met Thr Thr Ala Asp Leu Tyr Asp Arg Gln Gly Lys A1a Ile Lys Asp Gly Asn Lys Pro Ala Gly Val Phe Ala Ile Gly Ala Gly His Val Asn Pro Gln Lys Ala Ile Asn Pro Gly Leu Val Tyr Asn Ile Gln Pro Val Asp Tyr Ile Thr Tyr Leu Cys Thr Leu Gly Phe Thr Arg Ser Asp Ile Leu Ala Ile Thr His Lys Asn Val Ser Cys Asn Gly Ile Leu Arg Lys Asn Pro Gly Phe Ser Leu Asn Tyr Pro Ser Ile Ala Val Ile Phe Lys Arg Gly Lys Thr Thr Glu Met Ile Thr Arg Arg Val Thr Asn Val Gly Ser Pro Asn Ser Ile Tyr Ser Val Asn Val Lys Ala Pro Glu Gly Ile Lys Val Ile Val Asn Pro Lys Arg Leu Val Phe Lys His Val Asp Gln Thr Leu Ser Tyr Arg Val Trp Phe Val Leu Lys Lys Lys Asn Arg Gly Gly Lys Val Ala Ser Phe Ala Gln Gly Gln Leu Thr Trp Val Asn Ser His Asn Leu Met Gln Arg Val Arg Ser Pro Ile Ser Val Thr Leu Lys Thr Asn <210>

<211> 328 <212>
DNA

<213> dopsis ana Arabi thali <220>

<221>
CDS

<222> 1)..(1473) ( <400>

atggaa cccaaacct ttctttctc tgcattatc tttcttcta ttttgt 48 MetG1u ProLysPro PhePheLeu CysIleIle PheLeuLeu PheCys tcttct tcgtcagag atcctgcag aagcagact tacattgtt cagctt 96 SerSer SerSerGlu IleLeuGln LysGlnThr TyrIleVal GlnLeu catcct aatagcgaa accgetaaa acctttgcc tcaaagttt gattgg 144 HisPro AsnSerGlu ThrAlaLys ThrPheAla SerLysPhe AspTrp catctt tcttttctc caagaagcg gttttaggt gttgaagaa gaagag 192 HisLeu SerPheLeu GlnGluAla ValLeuGly ValGluGlu GluGlu gaagag ccttcttct cgacttctc tactcctat ggctctgcg attgaa 240 GluGlu ProSerSer ArgLeuLeu TyrSerTyr GlySerAla IleGlu ggattt getgetcag ttgactgaa tcagaagcc gagatactg agatat 288 GlyPhe AlaAlaGln LeuThrGlu SerGluAla GluIleLeu ArgTyr tcacct gaagttgtt gcagtgaga cctgaccat gttcttcag gttcaa 336 SerPro GluValVal AlaValArg ProAspHis ValLeuGln ValGln accact tactcttac aagttcttg ggactcgac ggttttgga aactcc 384 ThrThr TyrSerTyr LysPheLeu GlyLeuAsp GlyPheGly AsnSer ggtgta tggtctaaa tctcggttt ggtcaaggc acaattatc ggcgtg 432 GlyVal TrpSerLys SerArgPhe GlyGlnGly ThrIleIle GlyVal cttgat actggagtt tggcctgaa agtcctagc tttgacgat accgga 480 LeuAsp Thr.GlyVal TrpProGlu SerProSer PheAspAsp ThrGly atgcct tcgattcca cggaaatgg aaagggatt tgccaagaa ggagaa 528 MenPro SerIlePro ArgLysTrp LysGlyIle CysGlnGlu GlyGlu agtttc agttcttcg agctgtaac cggaagcta atcggtget agattc 576 SerPhe SerSerSer SerCysAsn ArgLysLeu IleGlyAla ArgPhe ttcatc agaggacac cgtgtcget aattcacca gaggaatca ccaaac 624 PheIle ArgGlyHis ArgValAla AsnSerPro GluGluSer ProAsn atgcct cgtgaatac atttccgca agagattca acgggacac gggact 672 Met Pro Arg Glu Tyr Ile Ser Ala Arg Asp Ser Thr Gly His Gly Thr cac acc gcc tca aca gtt ggt gga tcc tct gtt tcg atg gcg aat gtt 720 His Thr Ala Ser Thr Val Gly Gly Ser Ser Val Ser Met Ala Asn Val ctt ggc aat gga get ggt gtg get cgt ggg atg get cct gga get cac 768 Leu Gly Asn Gly Ala Gly Val Ala Arg Gly Met Ala Pro Gly Ala His att gca gtc tat aaa gtc tgt tgg ttc aat ggt tgt tac agc tct gac 816 Ile Ala Val Tyr Lys Val Cys Trp Phe Asn Gly Cys Tyr Ser Ser Asp att cta gca get ata gat gta gcg att caa gat aaa ytc gat gtt ctt 864 Ile Leu Ala Ala Ile Asp Val Ala Ile Gln Asp Lys Val Asp Val Leu tcg ctt tcc ctt ggc ggt ttc cct att cct ttg tat gat gac aca atc 912 Ser Leu Ser Leu Gly Gly Phe Pro Ile Pro Leu Tyr Asp Asp Thr Iie gcc att gga aca ttc cga gcc atg gaa cgc ggt ata tct gta atc tgt 960 Ala Ile Gly Thr Phe Arg Ala Met Glu Arg Gly Ile Sea Val Ile Cys gca get ggt aac aac ggt cca atc gaa agc tct gtt gca aac aca get 1008 Ala Ala Gly Asn Asn Gly Pro Ile Glu Ser Ser Val Ala Asn Thr Ala cct tgg gtc tca acc att ggc gca ggc acg ctt gat cga aga ttt ccc 1056 Pro Trp Val Ser Thr Ile Gly Ala Gly Thr Leu Asp Arg Arg Phe Pro get gtg gtc aga tta gcc aac gga aag ctt ctc tat gga gag tca ttg 1104 Ala Val Val Arg Leu Ala Asn Gly Lys Leu Leu Tyr Gly Glu Ser Leu tat ccg gga aaa ggt ata aag aat gcc ggg aga gag gtt gag gtg att 1152 Tyr Pro Gly Lys Gly Ile Lys Asn Ala Gly Arg Giu Val Glu Val Ile tac gtc aca gga gga gat aaa gga agt gag ttc tgt t~g aga ggg tca 1200 Tyr Val Thr Gly Gly Asp Lys Gly Ser Glu Phe Cys Leu Arg Gly Ser ctt cca aga gaa gaa atc cga ggc aaa atg gtg att tgt gat cgc gga 1248 Leu Pro Arg Glu Glu Iie Arg Gly Lys Met Val Ile Cys Asp Arg Gly 405 410 4~5 gtc aat gga aga tcg gag aaa gga gaa gcg gtt aaa gaa get gga gga 1296 Val Asn Gly Arg Ser Glu Lys Gly Glu Ala Val Lys Glu AIa Gly Gly gtt gca atg atc tta gcc aat aca gag atc aac caa gaa gaa gat tct 1344 Val Ala Met Ile Leu Ala Asn Thr Glu Ile Asn Gln Glu Glu As_b Ser att gac gtt cat ctc tta cca get aca ttg att ggt tac act gag tca 1392 Ile Asp Val His Leu Leu Pro Ala Thr Leu Iie Gly Tyr Thr Glu Ser gtc ctt ctg aag get tat gtt aat gcc acg gtg aaa cca aag gcg cgg 1440 Val Leu Leu Lys Ala Tyr Val Asn Ala Thr Val Lys Pro Lys Ala Arg ata att ttt ggt ggt acg gtg att ggg agg tca tgagcaccgg aggtggctca 1493 Ile Ile Phe Gly Gly Thr Val Ile Gly Arg Ser gttttcagct cgaggaccga gtttagccaa tccttcgata ctaaaaccgg atatgattgc 1553 tccgggagtc aatatcattg cggcttggcc tcaaaatcta ggaccaaccg gacttcctta 1613 tgattcaaga agagttaact tcactgtaat gtcaggaact tcaatgtctt gtccacatgt 1673 tagcggaatc actgctctta tccggtctgc atacccgaac tggtctccag ctgcaatcaa 1733 atccgcattg atgacaacag cggatttgta cgatcgtcaa gggaaagcga taaaggatgg 1793 taacaaacca gccgg~gtgt ttgcgattgg agcagggcat gtgaatccgc aaaaggcgat 1853 aaacccggga ttggtttaca acattcaacc agtggattac ataacttacc tctgcactct 1913 tggattcaca agatcagata ttttagcaat cactcataag aacgtgagct gcaatggaat 1973 attgcggaaa aacccgggtt ttagtctcaa ttacccgtcg atagccgtga ttttcaaacg 2033 tggcaagact acggagatga tcacaaggcg tgtcactaac gttgggagtc ctaactcgat 2093 atactcagtg aatgtcaagg ctccagaggg gatcaaagtt attgtcaatc ctaagagact 2153 tgtgttcaaa cacgtggatc agacgctgag ctatagagta tggtttgtat tgaagaagaa 2213 aaacagagga gggaaggtgg ctagctttgc acaagggcag ttgacttggg tcaactctca 2273 taatctgatg cagcgagtta gaagtccaat ctctgtaacc ttgaagacta actga 2328 <210> 4 <211> 491 <212> PRT
<213> Arabidopsis thaliana <400> 4 Met Glu Pro Lys Pro Phe Phe Leu Cys Ile Ile Phe Leu Leu Phe Cys Ser Ser Ser Ser Glu Ile Leu Gln Lys Gln Thr Tyr Ile Val Gln Leu His Pro Asn Ser Glu Thr Ala Lys Thr Phe Ala Ser Lys Phe Asp Trp His Leu Ser Phe Leu Gln Glu Ala Val Leu Gly Val Glu Glu Glu Glu Glu Glu Pro Ser Ser Arg Leu Leu Tyr Ser Tyr Gly Ser Ala Ile Glu Gly Phe Ala Ala Gln Leu Thr Glu Ser Glu Ala Glu Ile Leu Arg Tyr Ser Pro Glu Val Val Ala Val Arg Pro Asp His Val Leu Gln Val Gln Thr Thr Tyr Ser Tyr Lys Phe Leu Gly Leu Asp Gly Phe Gly Asn Ser Gly Val Trp Ser Lys Ser Arg Phe Gly Gln Gly Thr Ile Ile Gly Val Leu Asp Thr Gly Val Trp Pro Glu Ser Pro Ser Phe Asp Asp Thr Gly Met Pro Ser Ile- Pro Arg Lys Trp Lys Gly Ile Cys Gln Glu Gly Glu Ser Phe Ser Ser Ser Ser Cys Asn Arg Lys Leu Ile Gly Ala Arg Phe Phe Ile Arg Gly His Arg Val Ala Asn Ser Pro G1u Glu Ser Pro Asn Met Pro Arg Glu Tyr Ile Ser Ala Arg Asp Ser Thr Gly His Gly Thr His Thr Ala Ser Thr Val Gly Gly Ser Ser Val Ser Met Ala Asn Val Leu Gly Asn Gly Ala Gly Val Ala Arg Gly Met Ala Pro Gly Aia His Ile Ala Val Tyr Lys Val Cys Trp Phe Asn Gly Cys Tyr Ser Ser Asp Ile Leu Ala Ala Ile Asp Val Ala Ile Gln Asp Lys Val Asp Val Leu Ser Leu Ser Leu Gly G1y Phe Pro Ile Pro Leu Tyr Asp Asp Thr Ile Ala Ile Gly Thr Phe Arg Ala Met Glu Arg Gly Ile Ser Val Ile Cys Ala Ala Gly Asn Asn Gly Pro Ile Glu Ser Ser Val Ala Asn Thr Ala Pro Trp Val Ser Thr Ile Gly Ala Gly Thr Leu Asp Arg Arg Phe Pro Ala Val Val Arg Leu Ala Asn Gly Lys Leu Leu Tyr G1y Giu Ser Leu Tyr Pro Gly Lys Gly Ile Lys Asn Ala Gly Arg Glu Val Glu Val Ile Tyr Val Thr Gly Gly Asp Lys Gly Ser Glu Phe Cys Leu Arg Giy Ser Leu Pro Arg Glu Glu Ile Arg Gly Lys Met Val Ile Cys Asp Arg Gly Val Asn Gly Arg Ser Glu Lys Gly Glu Ala Val Lys Glu Ala Gly Gly Val Ala Met Ile Leu Ala Asn Thr Glu Ile Asn Gln Glu Glu Asp Ser ~1 Ile Asp Val His Leu Leu Pro Ala Thr Leu Ile Gly Tyr Thr Glu Ser Val Leu Leu Lys Ala Tyr Val Asn Ala Thr Val Lys Pro Lys Ala Arg Ile Ile Phe Gly Gly Thr Val Ile Gly Arg Ser <210> 5 <211> 1970 <212> DNA
<213> Arabidopsis thaliana <400> 5 gtcgactttg attcaagctt tgtttcgatt gattgagcca actgctggaa aaattactat 60 tgacaacatt gacatttctc aaattggtct tcatgatctt cgtagtcgcc ttgggattat 120 acctcaagat cctacattat ttgaaggaac aatccgagca aatcttgacc cacttgaaga 180 acattcagat gataaaatct gggaggtatc tataaatatg ttgtttgata ctcttatctt 240 gtttatgttt tagacactaa actctgagat ttggagtttg attctagaga tttacccaac 300 tttgctgaca ggcgcttgat aaatcccagc ttggagacgt tgttagagga aaagacctaa 360 aacttgactc tccaggtaac tatttacatc aaaagtcctc tctttttccc gggtcttttt 420 cgcttctttc tactgatttt tttggctcaa aaccgagtag tactggaaaa tggagataac 480 tggagtgtag ggcagagaca gcttgtgtca cttggacgag cattactgaa acaagccaaa 540 atacttgttc ttgatgaagc aacagcatcg gttgacacag caacagacaa tctgatccag 600 aagataatca gaacagagtt tgaagactgc acggtctgca ccattgctca ccggatacca 660 actgttatag acagtgacct agttttggtt ctcagcgacg gttagtctca tacaaattaa 720 aaacatggat ctttcttcat attactcgtc gtcttttggg agaattcaat gtttatgttt 780 atggtttgtt gcaggtagag tagcagagtt tgatactcct gcacggctat tagaagacaa 840 atcatcgatg ttcttgaaat tggtaacaga atactcctca agatctactg gaatccctga 900 attatgatcc tccatgttaa aaattcagtt tagggggttt cttttctcaa gaggatataa 960 aagaactgat atgtgacaaa agcttaaggt ctaaagtaag agagagtttt ccacagggtt 1020 taagaaaaga aaaagcatga aaggatgcca aaatctccgc gcttaaaaaa ctttgggttt 1080 aaatctcttc tgtcgaacat tgggagaaac tttttttgta tggaacagtt agtttctttg 1140 gttttcatgt ttatcaataa actgcaaaaa caaacaaaga ttagagtaga aactactaat 1200 cattcgtcat catccctcaa gtgtgatctg tattgggctt attataagtg taatcagtac 1260 tgggcttata agttgtacag agcccaatat aacaatatct gtacgacgtc gttttgttgt 1320 gacgtagtac ccgatgacaa aacgaggcgt gttgagcgtg cgtgtataaa tgcacgtagt 1380 gaacgaacac taaccacgct gctgcattta attcctttct caacggtcgt attttcctat 1440 tcaaggcttt aactaagttt aatagtatgt ttttaaaaaa atacatatat ttggccaatg 1500 gttgattatt aagttattgt taaatgaatt ttctttggct tggtaaaact tcaatggaaa 1560 ccaaataaat ttcaaaatct tttgttgctg aaaaaggcta cacaaactac tatacgcaat 1620 aaaacctaac cataacatct cgtgaacaag gaaaaaaaaa ggaaaaacag agacacagag 1680 acaaggcaga caaaaaagcc ttacaagtga aaaaccttaa acagcttcag ttaatatagt 1740 tcatgtctta gaaaaaaata aaagagaaga agctcctacc tctgcaacat acaaaggata 1800 ctcgtgaggc agagctctta ctcttcacga gcttcatcat cttcttcctt agactccaaa 1860 tcccaggttt tgaattctct tcattcttct ttaaataccc tattcttctt cttctctaaa 1920 accatgcact tcaactacac taaaccaact ccttttttta actctctcca 19?0 <210> 6 <211> 1140 <212> DNA
<213> Arabidopsis thaliana <400> 6 aaatcatcga tgttcttgaa attggtaaca gaatactcct caagatctac tggaatccct 60 gaattatgat cctccatgtt aaaaattcag tttagggggt ttcttttctc aagaggatat 120 aaaagaactg ataLgtgaca aaagcttaag gtctaaagta agagagagtt ttccacaggg 180 tttaagaaaa gaaaaagcat gaaaggatgc caaaatctcc gcgcttaaaa aactttgggt 240 ttaaatctct tctgtcgaac attgggagaa actttttttg tatggaacag ttagtttctt 300 tggttttcat gtttatcaat aaactgcaaa aacaaacaaa gattagagta gaaactacta 360 atcattcgtc atcatccctc aagtgtgatc tgtattgggc ttattataag tgtaatcagt 420 actgggctta taagttgtac agagcccaat ataacaatat ctgtacgacg tcgttttgtt 480 gtgacgtagt acccgatgac aaaacgaggc gtgttgagcg tgcgtgtata aatgcacgta 540 gtgaacgaac actaaccacg ctgctgcatt taattccttt ctcaacggtc gtattttcct 600 attcaaggct ttaactaagt ttaatagtat gtttttaaaa aaatacatat atttggccaa 660 tggttgatta ttaagttatt gttaaatgaa ttttctttgg cttggtaaaa cttcaatgga 720 aaccaaataa atttcaaaat cttttgttgc tgaaaaaggc tacacaaact actatacgca 780 ataaaaccta accataacat ctcgtgaaca aggaaaaaaa aaggaaaaac agagacacag 840 agacaaggca gacaaaaaag ccttacaagt gaaaaacctt aaacagcttc agttaatata 900 gttcatgtct tagaaaaaaa taaaagagaa gaagctccta cctctgcaac atacaaagga 960 tactcgtgag gcagagctct tactcttcac gagcttcatc atcttcttcc ttagactcca 1020 aatcccaggt tttgaattct cttcattctt ctttaaatac cctattcttc ttcttctcta 1080 aaaccatgca cttcaactac actaaaccaa ctcctttttt taactctctc caatggaacc 1140 <210> 7 <211> 3865 <212> DNA
<213> Solanum tuberosum <220>
<221> exon <222> (3)..(551) <220>
<221> intron <222> (552)..(966) <220>
<221> exon <222> (967)..(1654) <220>
<221> intron <222> (1655)..(1737) <220>
<221> exon <222> (1738)..(2222) <220>
<221> intron <222> (2223)..(2485) <220>
<221> exon <222> (2486)..(3252) <220>
<221> CDS
<222> (3)..(551) <220>
<221> CDS
<222> (90'7)..(1653) <220>
<221> CDS
<222> (1737)..(2222) <220>

<221> CDS
<222> (2486)..(3253) <400> 7 ga att ctg ttc aac ccc ttt aaa tac ccc cat caa att ata tca aca 47 Ile Leu Phe Asn Pro Phe Lys Tyr Pro His Gln Ile T_le Ser Thr aac att cca tta ttc aac ttc aaa tat aat tca atg gaa ctc aat ttc 95 Asn Ile Pro Leu Phe Asn Phe Lys Tyr Asn Ser Met Glu Leu Asn Phe caa ttc tat ttt ctc tgt ttt cta ctc tgt ttt att ccc ctg cta caa 143 Gln Phe Tyr Phe Leu Cys Phe Leu Leu Cys Phe Ile Pro Leu Leu Gln get caa aat ttg caa act tat ata gta caa tta cat cca caa cat gca 191 Ala Gln Asn Leu Gln Thr Tyr Ile Val Gln Leu His Pro Gln His Ala tca aca aga acc cct ttt agt tct aaa ttt cag tgg cac ctt tca ttt 239 Ser Thr Arg Thr Pro Phe Ser Ser Lys Phe Gln Trp His Leu Ser Phe ctt gaa aat ttc aca aac att cca tta ttc aac ttc aaa tat att caa 287 Leu Glu Asn Phe Thr Asn Ile Pro Leu Phe Asn Phe Lys Tyr Ile Gln tgg aac tca att cca att cta ttt ctc tgt ttc tac tct gtt tat tcc 335 Trp Asn Ser Ile Pro Ile Leu Phe Leu Cys Phe Tyr Ser Val Tyr Ser cct get aca agc att tcc tca ggt gaa aac tcg agt tct cgc ctt ttg 383 Pro Ala Thr Ser Ile Ser Ser Gly G1u Asn Ser Ser Ser Arg Leu Leu tac tct tac cat tct gca ttt gaa ggt ttt gca gca crt cta tct gaa 431 Tyr Ser Tyr His Ser Ala Phe Glu Gly Phe Ala Ala Leu Leu Ser Glu aat gag cta aag gca ctg aag aaa tcg aat aat gtg tta tca ata tat 479 Asn Glu Leu Lys Ala Leu Lys Lys Ser Asn Asn Val Leu Ser Ile Tyr ccg gag agg aag ctt gag gtt caa aca act tat tct tac aag ttc tta 527 Pro Glu Arg Lys Leu Glu Val Gln Thr Thr Tyr Ser Tyr Lys Phe Leu gga ctt agt cct aca aag gaa ggt atgttacata gtttgtaata tatataaagt 581 G1y Leu Ser Pro Thr Lys Glu Gly tgaggaaaca atgagtcata aggctttatt tattaaatga ctagtctgga ctagagtgtc 641 acacgtagga tttaaacttg tgaattaagg aaaattctta aacccccttt acaactgctc 701 tggagtcatt tcttatgtca atgtgattca acagttcata tataacaaaa aggatttgtc 761 tttaccttgt tttcgaagta aaaatatttc aacgaagggg attcaaccga accccattgg 821 ttcccttcaa ctctgctaat agatattttc tatgtatttc atttaagact tgaattttta 881 agctttaaat tttctatgtt ccctcggagc ctttcntctn acttactttt atactgtctt 941 '~4 tgtatctttt tttttctaat aaggt act tgg tta aag tct gga ttt ggt cga 993 ThrTrp LeuLysSer GlyPheGly Arg ggcgcgatcatt ggagtt cttgatact ggaatttgg ccagaaagt cca 1041 GlyAlaIleIle GlyVal LeuAspThr GlyIleTrp ProGluSer Pro agttttgttgat catgga atgtctcct attccaaag aaatggaaa ggt 1089 SerPheValAsp HisGly MetSerPro IleProLys LysTrpLys Gly ntctgccaagaa ggaaaa aacttcaat tcttcaagt tgcaatcgc aag 1137 XaaCysGlnGlu GlyLys AsnPheAsn SerSerSer CysAsnArg Lys 225 230 , 235 240 cttattggtgca aggttt ttccagata ggacacatg atggcatca aag 1185 LeuIleGlyAla ArgPhe PheGlnIle GlyHisMet MetAlaSer Lys acatcaaaatca atagat tttatggag gattatgta tcacctcga gat 1233 ThrSerLysSer IleAsp PheMetGlu AspTyrVal SerProArg Asp tctcaaggccat ggtaca catacagca tctactgca gggggaget ccc 1281 SerGlnGlyHis GlyThr HisThrAla SerThrAla GlyGlyAla Pro gttccaatggcg agtgtg cttggaaat ggagcagga gaggetcga ggg 1329 ValProMetAla SerVal LeuGlyAsn GlyAlaGly GluAlaArg Gly atggcccctggt getcat atcgcgata tacaaagtt tgttggtct agt 1377 MetAlaProGly AlaHis IleAlaIle TyrLysVal CysTrpSer Ser ggttgttatagt tctgat atacttgca gcaGtggat gtagetatt aga 1425 GlyCysTyrSer SerAsp IleLeuAla AlaMetAsp ValAlaIle Arg gatggagtagac atattg tctctttca attggtggt ttccctgtt cca 1473 AspGlyValAsp IleLeu SerLeuSer IleGlyGly PheProVal Pro ctttatgaggat actatt getattggc agttttcga getatggaa cgt 1521 LeuTyrGluAsp ThrIle AlaIleGly SerPheArg AlaMetGlu Arg ggaatttcagtt atatgt getgcagga aataatggt ccaattcta agt 1569 GlyIleSerVal IleCys AlaAlaGly AsnAsnGly ProIleLeu Ser tcagtagcaaat gagget ccttggatt gccactatt ggtgetagc aca 1617 SerValAlaAsn GluAla ProTrpIle AlaThrIle GlyAlaSer Thr cttgacaggaaa tttcca gcaataatt cagctaggt atgtcacatt 1663 LeuAspArgLys PhePro AlaIleIle GlnLeuG1y ttgtttctta aaatgatatt tcgcgtgttt ccagcctaaa ttatgtgtcc ctcattcata 1723 '15 ttttccaaca ggtaatggc aagtatgtg tatgga gaatccttg tac 1772 ccg AsnGly LysTyrVal Tyr GluSerLeu TyrPro Gly ggcaaacaagttcataat tctcagaaa gttctt gagattgtt tatctc 1820 GlyLysGlnValHisAsn SerGlnLys ValLeu GluIleVal TyrLeu aatgacggtgataatgga agtgaattt tgctta agagggtct ctgcca 1868 AsnAspGlyAspAsnGly SerGluPhe CysLeu ArgGlySer LeuPro agagetaaagtccatgga aaaatcgtt gtatgt gatcgtgga gttaat 1916 ArgAlaLysValHisGly LysIleVal ValCys AspArgGly ValAsn ggaagagcagagaaaggt caagttgtt aaagaa tcaggtggt gttgcc 1964 GlyArgAlaGluLysGly GlnValVal LysGlu SerGlyGly ValAla atgatcctagcaaataca gcagtaaat atggag gaagattct gtggac 2012 MetIleLeuAlaAsnThr AlaValAsn MetGlu GluAspSer ValAsp gtacatgtcctacctgca acattgatt ggtttt gacgaatca attcag 2060 ValHisValLeuProAla ThrLeuIle GlyPhe AspGluSer IleGln ttgcaaagctatatgaac tcaacgcga aaacca acagetcga atcata 2108 LeuGlnSerTyrMetAsn SerThrArg LysPro ThrAlaArg I1eIle tttggaggaacagttata ggaaaatct agtgca cctgetgta gcacaa 2156 PheGlyGlyThrValIle GlyLysSer SerAla ProAlaVal AlaGln ttttcttcaaggggtcca agttttact gatcct tcaattctc aaacct 2204 PheSerSerArgGlyPro SerPheThr AspPro SerIleLeu LysPro gatgtgattgetccaggt cagtttttat tgaaccca ca ttatt 2252 attat AspValIleAlaProGly tagatcatag atagcaatgt gaccaacagg ttagggattc gagccgtgga atctatcatt 2312 gatgcttgaa tcagggtaga ctgcttacat cacacccttc accagaccat gcatgaaaac 2372 gggatgctct ttttatatgc atgtgaaaaa aactttaata aatagtgtaa tgttatgttt 2432 tgaacctata tcttcgtata actcagaatc ttgaatccgc ctctgctcca ggt gtc 2488 Val aac ata att get get tgg ccg caa aat cta ggt cct agt ggc ctg get 2536 Asn Ile Ile Ala Ala Trp Pro Gln Asn Leu Gly Pro Ser Gly Leu Ala gag gat tca aga aga gta aac ttc act gtc tta tca gga act tca atg 2584 Glu Asp Ser Arg Arg Val Asn Phe Thr Val Leu Ser Gly Thr Se. Met get tgt cct cat gtt agt ggc att get gca cta ctc cat tca att cat 2632 AlaCys ProHisVal SerGlyIle AlaAlaLeu LeuHisSer IleHis cctaaa tggtcacca getgcaatc aaatccgcg ctaatgaca actgca 2680 ProLys TrpSerPro AlaAlaIle LysSerAla LeuMetThr ThrAla gacaca acaaaccac caaggaaaa ccaatcatg gatggtgac acacga 2728 AspThr ThrAsnHis GlnGlyLys ProIleMet AspGiyAsp ThrArg getgga cttttcgcc ataggaget ggacatgta aatcctgga agatcc 2776 AlaGly LeuPheAla IleGlyAla GlyHisVal AsnProGly ArgSer gatgat cccggattg atatatgac attaatgca aatgactat atcact 2824 AspAsp ProGlyLeu IleTyrAsp IleAsnAla AsnAspTyr IleThr cacctt tgcactatt ggttacaaa aactctgaa atcc~cagc attact 2872 HisLeu CarsThrIle GlyTyrLys AsnSerGlu IleLeuSer IleThr cacaag aatgttagc tgccacgac gttttacag aaaaacagg ggtttt 2920 HisLys AsnValSer CysHisAsp ValLeuGln LysAsnArg GiyPhe agtctc aattacccc tctatttcc gtaatcttt aaggcagga aaaacg 2968 SerLeu AsnTyrPro SerIleSer Va1IlePhe LysAlaGly LysThr agaaaa atgatcaca aggagagtg acaaatgtg gggagtcct aattca 3016 ArgLys MetIleThr ArgArgVal ThrAsnVal GlySerPro AsnSer atctac tcagttgaa attgtggca ccagaagga gttaaagtg agagtt 3064 IleTyr SerValGlu IleValAla ProGluGly ValLysVal ArgVal aaaccg cgacgtctg gtatttaaa cacgttaat caaagttta agttac 3112 LysPro ArgArgLeu ValPheLys HisValAsn GlnSerLeu SerTyr agagtt tggtttata tcaaggaag agaattggg actcaaagg agaagc 3160 ArgVal TrpPheIle SerArgLys ArgIleGly ThrGlnArg ArgSer tttgca gaaggacaa ttgatgtgg atcaactcc agagataaa taccag 3208 PheAla GluGlyGln LeuMetTrp IleAsnSer ArgAspLys TyrGln aaagtt agaagtcct atttcagtt gcatgggca tcaaagaag tga 3253 LysVal ArgSerPro IleSerVal AlaTrpAla SerLysLys aggctgtgcc tattttcacg tacacggaaa ataggcaggt tacgtgctat aacatatact 3313 actgatagtg taaaaattct ttatactatc agcgtagaca gtacgtaagt ngaatctatt 3373 gttgtagcac caaagtttca ttgtt.ccgaa catttaatta ggattctatt actatattgt 3433 atcagcacca aaagtttcat cgtcttagtt ccaaaaattt gattaagctt ttaagttgca 3493 1i~
ttgtcttagt tccaaatatg ttactataac ttgctgataa aaacgtaaat aattatcaaa 3553 tagggtattg aacattcaaa gaatttagga taaccaaata aacaataaaa aaatgaaaac 3613 acaaaattgt tcctatttag taattttaca aatgtcaatt gttttgagaa ctcatcgatt 3673 ttgcaataaa tttatagaag aacttgtcta ttttagtaaa atatagatca gaatttcctt 3733 gcttgttcaa gtttcattct tttcatttta ttttatttta aaaaaagacc caaagtttgc 3793 ctcataaaag aattttctct tatcatataa gagggagtta gtgctgactt tcctcttatc 3853 atatccgtcg ac 3865 <210> 8 <211> 829 <212> PRT
<213> Solanum tu~erosum <400> 8 Ile Leu Phe Asn Pro Phe Lys Tyr Pro His Gln Ile Ile Ser Thr Asn Ile Pro Leu Phe Asn Phe Lys Tyr Asn Ser Met Glu Leu Asn Phe Gln Phe Tyr Phe Leu Cys Phe Leu Leu Cys Phe Ile Pro Leu Leu Gln Ala Gln Asn Leu Gln Thr Tyr Ile Val Gln Leu His Pro Gln His Ala Ser 50 55 . 60 Thr Arg Thr Pro Phe Ser Ser Lys Phe Gln Trp His Leu Ser Phe Leu Glu Asn Phe Thr Asn Ile Pro Leu Phe Asn Phe Lys Tyr Ile Gln Trp Asn Ser Ile Pro Ile Leu Phe Leu Cys Phe Tyr Ser Va' Tyr Ser Pro 100 105 ~ 110 Ala Thr Ser Ile Ser Ser Gly Glu Asn Ser Ser Ser Arg Leu Leu Tyr Ser Tyr His Ser Ala Phe Glu Gly the Ala Ala Leu Leu Ser Glu Asn 130 135 , 140 Glu Leu Lys Ala Leu Lys Lys Ser Asn Asn Val Leu Ser Ile Tyr Pro Glu Arg Lys Leu Glu Val Gln Thr Thr Tyr Ser Tyr Lys Phe Leu Gly Leu Ser Pro Thr Lys Glu Gly Thr Trp Leu Lys Se. Gly Phe Gly Arg Gly Ala Ile Ile Gly Val Leu Asp Thr Gly Ile Trp Pro Glu Ser Pro Ser Phe Val Asp His Gly Met Ser Pro Ile Pro Lys Lys Trp Lys Gly Xaa Cys Gln Glu Gly Lys Asn Phe Asn Ser Ser Ser Cys Asn Arg Lys Leu Ile Gly Ala Arg Phe Phe Gln Ile Gly His Met Met Ala Ser Lys Thr Ser Lys Ser Ile Asp Phe Met Glu Asp Tyr Val Ser Pro Arg Asp Ser Gln Gly His G1y Thr His Thr Ala Ser Thr Ala Gly Gly Ala Pro Val Pro Met Ala Ser Val Leu Gly Asn Gly Ala Gly Glu Ala Arg Gly Met Ala Pro Gly Ala His Ile Ala Ile Tyr Lys Val Cys Trp Ser Ser Gly Cys Tyr Ser Ser Asp Ile Leu A1a Ala Met Asp Val Ala Ile Arg Asp Gly Val Asp Ile Leu Ser Leu Ser Ile Gly Gly Phe Pro Val Pro Leu Tyr Glu Asp Thr Ile Ala Ile Gly Ser Phe Arg Ala Met Glu Arg Gly Ile Ser Val Ile Cys Ala Ala Gly Asn Asn Gly Pro Ile Leu Ser Ser Val Ala Asn Glu Ala Pro Trp Ile Ala Thr Ile Gly Ala Ser Thr Leu Asp Arg Lys Phe Pro Ala Ile Ile Gln Leu Gly Asn Gly Lys Tyr Val Tyr Gly Glu Ser Leu Tyr Pro Gly Lys Gln Val His Asn Ser Gln Lys Val Leu Glu Ile Val Tyr Leu Asn Asp Gly Asp Asn Gly Ser Glu Phe Cys Leu Arg Gly Ser Leu Pro Arg Ala Lys Val His Gly Lys Ile Val Val Cys Asp Arg Gly Val Asn Gly Arg Ala Glu Lys Gly Gln Val Val Lys Glu Ser Gly Gly Val Ala Met Ile Leu A_a Asn Thr Ala Val Asn Met Glu Glu Asp Ser Val Asp VaI His Val Leu Pro Ala Thr Leu Ile Gly Phe Asp Glu Ser Ile Gln Leu Gln Se. Tyr Met Asn Ser Thr Arg Lys Pro Thr Ala Arg Ile Ile Phe Gly Gly Thr Val Ile Gly Lys Ser Ser Ala Pro Ala Val Ala Gln Phe Ser Se. Arg Gly Pro Ser Phe WO 00/22144 PCTlEP99/07633 Thr Asp Pro Ser Ile Leu Lys Pro Asp Val Ile Ala Pro Gly Val Asn Ile Ile Ala Ala Trp Pro Gln Asn Leu Gly Pro Ser Gly Leu Ala Glu Asp Ser Arg Arg Val Asn Phe Thr Val Leu Ser Gly Thr Ser Met Ala Cys Pro His Val Ser Gly Ile Ala Ala Leu Leu His Ser Ile His Pro Lys Trp Ser Pro Ala Ala Ile Lys Ser Ala Leu Met Thr Thr Ala Asp Thr Thr Asn His Gln Gly Lys Pro Ile Met Asp Gly Asp Thr Arg Ala 645 ~ 650 655 Gly Leu Phe Ala Ile Gly Ala Gly His Val Asn Pro Gly Arg Ser Asp Asp Pro G1y Leu Ile Tyr Asp Ile Asn Ala Asn Asp Tyr Ile Thr His Leu Cys Thr Ile Gly Tyr Lys Asn Ser Glu Ile Leu Ser Ile Thr His Lys Asn Val Ser Cys His Asp Val Leu Gln Lys Asn Arg Gly Phe Ser Leu Asn Tyr Pro Ser Ile Ser Val Ile Phe Lys Ala Gly Lys Thr Arg Lys Met Ile Thr Arg Arg Val Thr Asn Val Gly Ser Pro Asn Ser Ile Tyr Ser Val Giu Ile Val Ala Pro Glu Gly Val Lys Val Arg Val Lys Pro Arg Arg Leu Val Phe Lys His Val Asn Gln Ser Leu Ser Tyr Arg Val Trp Phe Ile Ser Arg Lys Arg Ile Gly Thr Gln Arg Arg Ser Phe Ala Glu Gly Gln Leu Met Trp Ile Asn Ser Arg Asp Lys Tyr Gln Lys 805 8i0 815 Val Arg Ser Pro Ile Ser Val Ala Trp Ala Ser Lys Lys <210> 9 <21i> 2492 <212> DNA
<213> Solanum tuberosum <220>
<221> CDS
<222> (3)..(2489) <400> 9 ,ZO

ga attctg aacccc tttaaatac catcaa att tca aca 47 ttc ccc ata IleLeu AsnPro PheLysTyr Gln IleIleSer Thr Phe Pro His aacattcca ttattcaac ttcaaatat aattcaatg gaactcaat ttc 95 AsnIlePro LeuPheAsn PheLysTyr AsnSerMet GluLeuAsn Phe caattctat tttctctgt tttctactc tgttttatt cccctgcta caa 143 GlnPheTyr PheLeuCys PheLeuLeu CysPheIle ProLeuLeu Gln getcaaaat ttgcaaact tatatagta caattacat ccacaacat gca 191 AlaGlnAsn LeuGlnThr TyrIleVal GlnLeuHis ProGlnHis Ala tcaacaaga acccctttt agttctaaa tttcagtgg cacctttca ttt 239 SerThrArg ThrProPhe SerSerLys PheGlnTrp HisLeuSer Phe cttgaaaat ttcacaaac attccatta ttcaacttc aaatatatt caa 287 LeuGluAsn PheThrAsn IleProLeu PheAsnPhe LysTyrIle Gln tggaactca attccaatt ctatttctc tgtttctac tctgtttat tcc 335 TrpAsnSer IleProIle LeuPheLeu CysPheTyr SerValTyr Ser cctgetaca agcatttcc tcaggtgaa aactcgagt tctcgcctt ttg 383 ProAlaThr SerIleSer SerGlyGlu AsnSerSer SerArgLeu Leu tactcttac cattctgca tttgaaggt tttgcagca cttctatct gaa 431 TyrSerTyr HisSerAla PheGluGly PheAlaAla LeuLeuSer Glu aatgagcta aaggcactg aagaaatcg aataatgtg ttatcaata tat 479 AsnGluLeu LysAlaLeu LysLysSer AsnAsnVal LeuSerIle Tyr ccggagagg aagcttgag gttcaaaca acttattct tacaagttc tta 527 ProGluArg LysLeuGlu ValGlnThr ThrTyrSer TyrLysPhe Leu ggacttagt cctacaaag gaaggtact tggttaaag tctggattt ggt 575 GlyLeuSer ProThrLys GluGlyThr TrpLeuLys SerGlyPhe Gly cgaggcgcg atcattgga gttcttgat actggaatt tggccagaa agt 623 ArgGlyAla IleIleGly ValLeuAsp ThrGlyIle TrpProGlu Ser ccaagtttt gttgatcat ggaatgtct cctattcca aagaaatgg aaa 67~

ProSerPhe ValAspHis GlyMetSer ProIlePro LysLysTrp Lys ggtntctgc caagaagga aaaaacttc aattcttca agttgcaat cgc 719 GlyXaaCys GlnGluGly LysAsnPhe AsnSerSer SerCysAsn Arg aagcttatt ggtgcaagg tttttccag ataggacac atgatggca tca 767 LysLeuIle G1yAlaArg PhePheGln IleGlyHis MetMetAla Ser aagacatcaaaa tcaatagat tttatggaggat tatgtatca cctcga 815 LysThrSerLys SerIleAsp PheMetGluAsp TyrValSer ProArg gattctcaaggc catggtaca catacagcatct actgcaggg ggaget 863 AspSerGlnGly HisGlyThr HisThrAlaSer ThrAlaGly GlyAla cccgttccaatg gcgagtgtg cttggaaatgga gcaggagag getcga 911 ProValProMet AlaSerVal LeuGlyAsnGly AlaGlyGlu AlaArg gggatggcccct ggtgetcat atcgcgatatac aaagtttgt tggtct 959 GlyMetAlaPro GlyAlaHis IleAlaIleTyr LysValCys TrpSer agtggttgttat agttctgat atacttgcagca atggatgta getatt 1007 SerGlyCysTyr SerSerAsp IleLeuAlaAla MetAspVal AlaIle agagatggagta gacatattg tctctttcaatt ggtggtttc cctgtt 1055 ArgAspGlyVal AspIleLeu SerLeuSerIle GlyGlyPhe ProVal ccactttatgag gatactatt getattggcagt tttcgaget atggaa 1103 ProLeuTyrGlu AspThrIle AlaIleGlySer PheArgAla MetGlu cgtggaatttca gttatatgt getgcaggaaat aatggtcca attcta 1151 ArgGlyIleSer ValIleCys AlaAlaGlyAsn AsnGlyPro IleLeu agttcagtagca aatgagget ccttggattgcc actattggt getagc 1199 SerSerValAla AsnGluAla ProTrpIleAla ThrIleGly A1aSer acacttgacagg aaatttcca gcaataattcag ctaggtaat ggcaag 1247 ThrLe~sAspArg LysPhePro AlaIleIleGln LeuGlyAsn G1yLys tatgtgtatgga gaatccttg tacccgggcaaa caagttcat aattct'1295 TyrValTyrGly GluSerLeu TyrProGlyLys GlnValHis AsnSer cagaaagttctt gagattgtt tatctcaatgac ggtgataat ggaagt 1343 GlnLysValLeu GluIleVal TyrLeuAsnAsp GlyAspAsn GlySer gaattttgctta agagggtct ctgccaagaget aaagtccat ggaaaa 1391 GluPheCysLeu ArgGlySer LeuProArgAla LysValHis GlyLys atcgttgtatgt gatcgtgga gttaatggaaga gcagagaaa ggtcaa 1439 IleValValCys AspArgGly ValAsnGlyArg AlaGluLys GlyGln gttgttaaagaa tcaggtggt gttgccatgatc ctagcaaat acagca 1487 ValValLysGlu SerGlyGly ValAlaMetIle LeuAlaAsn ThrAla gtaaatatggag gaagattct gtggacgtacat gtcctacct gcaaca 1535 ValAsnMetGlu GluAspSer ValAspValHis ValLeuPro AlaThr $~

ttgatt ggttttgac gaatcaatt cagttgcaaagc tatatgaac tca 1583 LeuIle GlyPheAsp GluSerIle GlnLeuGlnSer TyrMetAsn Ser acgcga aaaccaaca getcgaatc atatttggagga acagttata gga 1631 ThrArg LysProThr AlaArgIle IlePheGlyG1y ThrValIle Gly aaatct agtgcacct getgtagca caattttcttca aggggtcca agt 1679 LysSer SerA1aPro AlaValAla GlnPheSerSer ArgGlyPro Ser tttact gatccttca attctcaaa cctgatgtgatt getccaggt gtc 1727 PheThr AspProSer IleLeuLys .ProAspValIle AlaProGly Val aacata attgetget tggccgcaa aatctaggtcct agtggcctg get 1775 AsnIle IleAlaAla TrpProGln AsnLeuGlyPro SerGlyLeu Ala gaggat tcaagaaga gtaaacttc actgtcttatca ggaacttca atg 1823 GluAsp SerArgArg ValAsnPhe ThrValLeuSer GlyThrSer Met gettgt cctcatgtt agtggcatt getgcactactc cattcaatt cat 1871 AlaCys ProHisVa1 SerGlyIle AlaAlaLeuLeu HisSerIle His cctaaa tggtcacca getgcaatc aaatccgcg ctaatgaca actgca 1919 ProLys TrpSerPro AlaAlaIle LysSerAla LeuMetThr ThrAla gacaca acaaaccac caaggaaaa ccaatcatg gatggtgac acacga 1967 AspThr ThrAsnHis GlnGlyLys ProIleMet AspGlyAsp ThrArg getgga cttttcgcc ataggaget ggacatgta aatcctgga agatcc 2015 AlaGly LeuPheAla IleGlyAla GlyHisVal AsnProGly ArgSer gatgat cccggattg atatatgac attaatgca aatgactat atcact 2063 AspAsp Pro.GlyLeu IleTyrAsp IleAsnAla AsnAspTyr IleThr cacctt tgcactatt ggttacaaa aactctgaa atcctcagc attact 2111 HisLeu CysThrIle GlyTyrLys AsnSerGlu IleLeuSer IleThr cacaag aatgttagc tgccacgac gttttacag aaaaacagg ggtttt 2159 HisLys AsnValSer CysHisAsp ValLeuGln LysAsnArg GlyPhe agtctc aattacccc tctatttcc gtaatcttt aaggcagga aaaacg 2207 SerLeu AsnTyrPro SerIleSer ValIlePhe LysAlaGly LysThr agaaaa atgatcaca aggagagtg acaaatgtg gggagtcct aattca 2255 ArgLys MetIleThr ArgArgVal ThrAsnVal GlySerPro AsnSer atc tac tca gtt gaa att gtg gca cca gaa gga gtt aaa gtg aga gtt 2303 IleTyr SerValGlu IleValAla ProGluGly ValLysVal ArgVal aaaccg cgacgtctg gtatttaaa catgttaat caaagttta agttac 2351 LysPro ArgArgLeu ValPheLys HisValAsn GlnSerLeu SerTyr agagtt tggtttata tcaaggaag agaattggg actcaaagg agaagc 2399 ArgVal TrpPheIle SerArgLys ArgIleGly ThrGlnArg ArgSer tttgca gaaggacaa ttgatgtgg atcaactcc agagataaa taccag 2447 PheAla GluGlyGln LeuMetTrp IleAsnSer ArgAspLys TyrGln aaagtt agaagtcct atttcagtt gcatgggca tcaaagaag tga 2492 LysVal ArgSerPro IleSerVal AlaTrpAla SerLysLys <210> 10 <211> 829 <212> PRT
<213> Solanum tuberosum <400> 10 Ile Leu Phe Asn Pro Phe Lys Tyr Pro His Gln Ile Ile Ser Thr Asn Ile Pro Leu Phe Asn Phe Lys Tyr Asn Ser Met Glu Leu Asn Phe Gln Phe Tyr Phe Leu Cys Phe Leu Leu Cys Phe Ile Pro Leu Leu Gln Ala Gln Asn Leu Gln Thr Tyr Ile Val Gln Leu His Pro Gln His Ala Ser Thr Arg Thr Pro Phe Ser Ser Lys Phe Gln Trp His Leu Ser Phe Leu Glu Asn Phe Thr Asn Ile Pro Leu Phe Asn Phe Lys Tyr Ile Gln Trp Asn Ser Ile Pro Ile Leu Phe Leu Cys Phe Tyr Ser Val Tyr Ser Pro Ala Thr Ser Ile Ser Ser Gly Glu Asn Ser Ser Ser Arg Leu Leu Tyr Ser Tyr His Ser Ala Phe Glu Gly Phe Ala Ala Leu Leu Ser Glu Asn Glu Leu Lys Ala Leu Lys Lys Ser Asn Asn Val Leu Ser Ile Tyr Pro Glu Arg Lys Leu Glu Val Gln Thr Thr Tyr Ser Tyr Lys Phe Leu Gly Leu Ser Pro Thr Lys Glu Gly Thr Trp Leu Lys Ser Gly Phe G1y Arg Gly Ala Ile Ile Gly Val Leu Asp Thr Gly Ile Trp Pro Glu Ser Pro Ser Phe Val Asp His Gly Met Ser Pro Ile Pro Lys Lys Trp Lys Gly Xaa Cys Gln Glu Gly Lys Asn Phe Asn Ser Ser Ser Cys Asn Arg Lys Leu Ile Gly Ala Arg Phe Phe Gln Ile Gly His Met Met Ala Ser Lys Thr Ser Lys Ser Ile Asp Phe Met Glu Asp Tyr Val Ser Pro Arg Asp Ser Gln Gly His Gly Thr His Thr Ala Ser Thr Ala G'_y Gly Ala Pro Val Pro Met Ala Ser Va1 Leu Gly Asn Gly Ala Gly Glu Ala Arg Gly Met Ala Pro Gly Ala His Ile Ala Ile Tyr Lys Val Cys Trp Ser Ser Gly Cys Tyr Ser Ser Asp Ile Leu Ala Ala Met Asp Va1 Ala Iie Arg Asp Gly Val Asp Ile Leu Ser Leu Ser Ile Gly Gly Phe Pro Val Pro Leu Tyr Glu Asp Thr Ile Ala Ile Gly Ser Phe Arg Ala Met Glu Arg Gly Ile Ser Val Ile Cys Ala Ala Gly Asn Asn Gly Pro Ile Leu Ser Ser Val Ala Asn Glu Ala Pro Trp Ile Ala Thr Ile Gly Ala Ser Thr Leu Asw Arg Lys Phe Pro Ala Ile Ile Gln Leu Gly Asn Gly Lys Tyr Val Tyr Gly Glu Ser Leu Tyr Pro Gly Lys Gln Val His Asn Ser Gln Lys Val Leu Glu Ile Val Tyr Leu Asn Asp Gly Asp Asn Gly Ser Glu Phe Cys Leu Arg Gly Ser Leu Pro Arg Ala Lys Val His Gly Lys Ile Val Val Cys Asp Arg Gly Val Asn Gly Arg Ala Glu Lys Gly Gln Val Val Lys Giu Ser Gly G'_y Val Ala Met Ile Leu Ala Asn Thr A~a Val Asn Met Glu Glu Asp Ser Val Asp Val His Val Leu Pro Ala Thr Leu Ile Gly Phe Asp Glu Ser Ile Gln Leu Gln Ser Tyr Met Asn Ser Thr 5i5 520 525 Arg Lys Pro Thr Ala Arg Ile Ile Phe Gly Gly Thr Val Ile Gly Lys $5 Ser Ser Ala Pro Ala Val Ala Gln Phe Ser Ser Arg Gly Pro Ser Phe Thr Asp Pro Ser Ile Leu Lys Pro Asp Val Ile Ala Pro Gly Val Asn Ile Ile Ala Ala Trp Pro Gln Asn Leu Gly Pro Ser Gly Leu Ala Glu Asp Ser Arg Arg Val Asn Phe Thr Val Leu Ser Gly Thr Ser Met A1a Cys Pro His Val Ser Gly Ile Ala Ala Leu Leu His See Ile His Pro Lys Trp Ser Pro Ala Ala Ile Lys Ser Ala Leu Met Thr Thr Ala Asp Thr Thr Asn H-s Gln Gly Lys Pro Ile Met Asp Gly Asp Thr Arg Ala Gly Leu Phe Ala Ile Gly Ala Gly His Val Asn Pro Gly Arg Ser Asp Asp Pro Gly Leu Ile Tyr Asp Ile Asn Ala Asn Asp Ty= Ile Thr His Leu Cys Thr Ile Gly Tyr Lys Asn Ser Glu Ile Leu Ser Ile Thr His Lys Asn Val Ser Cys His Asp Val Leu Gln Lys Asn Arg Gly Phe Ser Leu Asn Tyr Pro Ser Ile Ser Val Ile Phe Lys Ala Giy Lys Thr Arg Lys Met Ile Thr Arg Arg Val Thr Asn Val Gly Ser Pro Asn Ser Ile Tyr Ser Val Glu Ile Val Ala Pro Glu Gly Val Lys Val Arg Val Lys Pro Arg Arg Leu Val Phe Lys His Vas. Asn Gln Ser Leu Ser Tyr Arg Val Trp Phe Ile Ser Arg Lys Arg Ile Gly Thr Gln Arg Arg Ser Phe Ala Glu Gly Gln Leu Met Trp Ile Asn Ser Arg Asp Lys Tyr Gln Lys Val Arg Ser Pro Ile Ser Val Ala Trp Ala Ser Lys Lys <210> 11 <211> 3140 <212> DNA
<213> Solanum tuberosum .~6 <220>

<221>
CDS

<222> (2298) (1)..

<400>

actcatttattc tcctttcta tgtctttta ctatgtttt gtttgcata 48 ThrHisLeuPhe SerPheLeu CysLeuLeu LeuCysPhe ValCysIle caagetcaagat ttgcaaact tacatagtt cagttacat ccacatgga 96 GlnAlaGlnAsp LeuGlnThr TyrIleVal GlnLeuHis ProHisGly gcaacaagaccc ccttttagc tctaaacta caatggcac ctttctttc 144 AlaThrArgPro ProPheSer SerLysLeu GlnTrpHis LeuSerPhe cttgcaaaagca gtttcctct ggagaacaa gactcgtct tctcgtctt 192 LeuAIaLysAla yalSerSer GlyGluGln AspSerSer SerArgLeu ttgtactcttac cattctgcg atggaaggt tttgcaget cgactcact 240 LeuTyrSerTyr HisSerAla MetGluGly PheAlaAla ArgLeuThr gaagatgaggtt gagttgtta agggaatct aatgatgtg ttgtcgata 288 G1uAspGluVal GluLeuLeu ArgGluSer AsnAspVal LeuSerIle cgtgetgagagg aggcttgaa attcagact acttattct tacaagttc 336 ArgAlaGluArg ArgLeuGlu IleGlnThr ThrTyrSer TyrLysPhe 100 . 105 110 ttgggattaagt ccaacgaga gaaggaget tggttgaag tctggattt 384 LeuGlyLeuSer ProThrArg GluGlyAla TrpLeuLys SerGlyPhe ggtcgaggggcg atcattgga gtgttggat actggagtt tggccagaa 432 GlyArgGlyAla IleIleGly ValLeuAsp Th=GlyVal TrpProGlu agtccaagtttt gatgatcat gggatgcca cctgetcca cagaagtgg 480 SerProSerPhe AspAspHis GlyMetPro ProAlaPro GlnLysTrp aggggtgtctgc caaggagga caggatttt aattcttct agttgtaat 528 ArgGlyValCys GlnGly.Gly GlnAspPhe As~:SerSer SerCysAsn cgcaagcttatt ggtgcaagg tttttcaga aaaggacat cgtgtgget 576 ArgLysLeuIle GlyAlaArg PhePheArg LysGlyHis ArgValAla tcaatgacatca tcaccagat gcagtggag gaatatgtg tcgccacgg 624 SerMetThrSer SerProAsp AlaValGlu GluTyrVal SerProArg gattcccatggc catggtaca catacagca tccactget ggaggaget 672 AspSerHisGly HisGlyThr HisThrA1a SerThrAla GlyGlyAla gcagttccattg getggtgtg ctcggaaat ggagcaggg gaggetcga 720 AlaValProLeu A1aGlyVal LeuGlyAsn GlyAlaGly GluAlaArg gggatggcc ccgggtgcc cacattgca atatat aaagtatgctgg ttc 768 GlyMetAla ProGlyAla HisIleAla IleTyr LysValCysTrp Phe agtggttgt tacagctct gatatactt gcagca atggatgtggcc atc 816 SerGlyCys TyrSerSer AspIleLeu AlaAla MetAspValAla Ile agagatgga gtagacata ttgtcactc tcactt ggtggcttccct att 864 ArgAspGly ValAspIle LeuSerLeu SerLeu GlyGlyPhePro Ile ccactttat gatgatact attgccatt ggaagt ttccgagccatg gag 912 ProLeuTyr AspAspThr IleAlaIle GlySer PheArgAlaMet Glu 290 295 ' 300 catggaatt tcagttata tgtgetgca gggaat aatggaccaatc caa 960 H=sGlyIle SerValIle CysAlaAla GlyAsn AsnGlyProIle Gln agttcagta gccaacggt getccttgg attgcc actattggtget agc 1008 SerSerVal AlaAsnGly AlaProTrp IleAla ThrT_leGlyAla Ser acacttgac aggagattt ccagcgtca gttcag ttaggcaacgga aag 1056 ThrLeuAsp ArgArgPhe ProAlaSer ValGln LeuGlyAsnGly Lys ttcctgtac ggagaatcc ttgtaccct gggaag aaagttcctagc tct 1104 PheLeuTyr GlyGluSer LeuTyrPro GlyLys LysValProSer Ser cagaagaat cttgagatc gtttatgta aaggat aaggacaaggga agt 1152 GlnLysAsn LeuGluIle ValTyrVal LysAsp LysAspLysGly Ser gaattttgc ttgagagga tcgctatca aaagca caagtccgaggg aaa 1200 GluPheCys LeuArgGly SerLeuSer LysAla GlnValArgGly Lys atggttgtg tgtgatagg ggagtcaat ggaagg gcagaaaaaggc cag 1248 MetValVal CysAspArg GlyValAsn GlyArg AlaGluLysGly Gln gttgtgaag gaggcaggt ggtgetgcc atgatc ttagcaaataca gca 1296 ValValLys GluAlaGly GlyAlaAla MetIle LeuAlaAsnThr Ala ataaatatg gaggaagat tccattgat gtccat gtcctcccagca acg 1344 IleAsnMet GluGluAsp SerIleAsp ValHis ValLeuProAla Thr ttgattggc ttcgatgaa tcaattcaa ttacaa aactacctgaac tca 1392 LeuIleGly PheAspGlu SerIleGln LeuGln AsnTyrLeuAsn Ser acaaaaaga ccaacaget cgattcata tttgga ggaacggtaa~a gga 1440 ThrLysArg ProThrAla ArgPheIle PheGly GlyThrValIle Gly aagtctaga gcacctgca gtagetcag ttttcg tcaagggggcca agc 1488 LysSerArg AlaProAlaVal AlaGlnPhe SerSerArg GlyProSer tatactgat ccttcaattctc aaacctgat ttgattget ccaggggta 1536 TyrThrAsp ProSerIleLeu LysProAsp LeuIleAla ProGlyVal aacataatt gccgettggcca caaaactta ggccccagt ggtcttccc 1584 AsnIleIle AlaAlaTrpPro GlnAsnLeu GlyProSer GlyLeuPro gaagattca cgaagagtaaat ttcactgtt atgtcaggg acctcaatg 1632 GluAspSer ArgArgValAsn PheThrVal MetSerGly ThrSerMet gcatgtcct catgtaagtgga attgccgca ttgctccat tcagetcat 1680 AlaCysPro HisValSerGly IleAlaAla LeuLeuHis SerAlaHis cctaaatgg actccagcagca ataagatcc gcattaatg accactgca 1728 ProLysTrp ThrProAlaAla IleArgSer AlaLeuMet ThrTarAla gatacaget gatcatatggga aaaccaatc atggatgga gatgcacca 1776 AspThrAla AspHisMetGly LysProIle MetAspGly AspAlaPro getaaactt tttgcagetgga getggacac gtgaaccct ggaagagcc 1824 AlaLysLeu PheAlaAlaGly AlaGlyHis ValAsnPro GlyArgAla atcgatcct ggattgatatat gadatccag gttgatgaa tatatcact 1872 IleAspPro GlyLeuIleTyr AspIleGln ValAspGlu TyrIleThr catctttgc actatcggatac agaaattct gaagtcttc agcattact 1920 HisLeuCys ThrIleGlyTyr ArgAsnSer GluValPhe SerIleThr cataggaat gtcagctgccat gacatttta cagaacaac aggggtttc 1968 HisArgAsn ValSerCysHis AspIleLeu GlnAsnAsn ArgGlyPhe agcctaaat tacccctcaatt tcaataact ttcagagca ggaatgact 2016 SerLeuAsn TyrProSerIle SerIleThr PheArgAla GlyMetThr agaaagata.atcaagaggaga gtaacaaat gtggggaac cctaactct 2064 ArgLysIle IleLysArgArg ValThrAsn ValGlyAsn ProAsnSer atttactca gttgacattgag gcacctgag ggagtcaaa gtgagagtg 2112 IleTyrSer ValAspIleGlu AlaProGlu GlyValLys ValArgVal aagccacgt cgtctgatattt aaacatgtg aaccaaagc ttaagctai 2160 LysProArg ArgLeuIlePhe LysHisVal AsnGlnSer LeuSerTyr agagtttgg tttatatcacga aagawaata gagtctaaa aggatgagc 2208 ArgValTrp PheIleSerArg LysXaaI1e GluSerLys ArgMetSer ttt gca gag ggg caa ttg aca tgg ttc aat gta gga aac aaa gcc acg 2256 Phe Ala Glu Gly Gln Leu Thr Trp Phe Asn Val Gly Asn Lys Ala Thr aaa gtt aaa agt cct att tcc gtc aca tgg gca tca atg aag 2298 Lys Val Lys Ser Pro Ile Ser Val Thr Trp Ala Ser Met Lys tgatcactat caccactatc acaagcacca tatatttcat tgtcttagtt caaaatttcc 2358 aattaggaat ttcacatcac attataaatt gatgttagag cagatacact ttatctttcc 2418 acaaagaaga aatgatcgat aatcattgaa atgatttgtg ttttactaag tagatgtgtc 2478 tccacaatgt taagaagtat taatatgtat aaatagatta gacaaagcac gagattgcgc 2538 ctgagtgagg nattttctca agtttacacc ttttgaacta aattactcat aaaccagtat 2598 gacagacaaa aaattcaaga aattggcgag gcaaaagaaa acatacaata taatctcaac 2658 ttttaacaaa ttgcaagcca tttgaattag cataccgctc cataaatctc atgaacctgt 2718 cccagtctcg tggagtccgc ataatatact tagcttcaat tcctgcaggc tttccattaa 2778 caaacttagc attgacatca actgacgtta gaaccccttc ttcgtcaatc atgtagaatc 2838 cagtgatatc ccctacttca ccagatgaat caaatacgga gggttgatca aacctgaata 2898 tagccatacc atttgtccat cccttgactt tgttaatttc acatctggta ttgtttgctc 2958 atcagttcct tgtatgaact gaatttttgg ttgaaccatc attatacata gtctggacat 3018 tttctggttt ttgatattgg tactgaaacg cgaacgggat aggcacaatc gttggccaat 3078 tgaatgaaga acctgcactt tgatgaacta tccttgatgc tattcctaca gtacatgaca 3138 Ca <210> 12 <211> 766 <212> PRT

<213> Solanum tuberosum <400> 12 Thr His PheSer PheLeuCys LeuLeuLeu CysPheVal CysIle Leu Gln Ala AspLeu GlnThrTyr IleValGln LeuHisPro HisGly Gln Ala Thr ProPro PheSerSer LysLeuGln TrpHisLeu SerPhe Arg Leu Ala AlaVal SerSerGly GluGlnAsp SerSerSer ArgLeu Lys Leu Tyr TyrHis SerAlaMet GluGlyPhe AlaAlaArg LeuThr Ser Glu Asp ValGlu LeuLeuArg GluSerAsn AspVa~_Leu SerIle Glu Arg Ala Glu Arg Arg Leu Glu Ile Gln Thr Thr Tyr Ser Tyr Lys Phe Leu Gly Leu Ser Pro Thr Arg Glu Gly Ala Trp Leu Lys Ser Gly Phe Gly Arg Gly Ala Ile Ile Gly Val Leu Asp Tr~r Gly Val Trp Pro Glu Ser Pro Ser Phe Asp Asp His Gly Met Pro Pro Ala Pro Gln Lys Trp Arg Gly Val Cys Gln Gly Gly Gln Asp Phe Asn Ser Ser Ser Cys Asn Arg Lys Leu Ile Gly Ala Arg Phe Phe Arg Lys Gly His Arg Val Ala Ser Met Thr Ser Ser Pro Asp Ala Val Glu Glu Tyr Val Se. Pro Arg Asp Ser His Gly His Gly Thr His Thr Ala Ser Thr Ala Gly Gly Ala Ala Val P=o Leu Ala Gly Val Leu Gly Asn Gly Ala Giy Glu Ala Arg Gly Met Ala Pro Gly Ala His Ile Ala Ile Tyr Lys Val Cys Trp Phe Ser Gly Cys Tyr Ser Ser Asp Ile Leu Ala Ala Met Asp Val Ala Ile Arg Asp Gly Val Asp Ile Leu Ser Leu Ser Leu Gly Gly Phe Pro Ile Pro Leu Tyr Asp Asp Thr Ile Ala Ile Gly Ser Phe Arg Ala Met Glu His Gly Ile Ser Val Ile Cys Ala Ala Gly Asn Asn Gly Pro Ile Gln Ser Ser Val Ala Asn Gly Ala Pro Trp Ile Ala Thr Ile Gly Ala Ser Thr Leu Asp Arg Arg Phe Pro Ala Ser Val Gln Leu Gly Asn Gly Lys Phe Leu Tyr Gly Glu Ser Leu Tyr Pro Gly Lys Lys Val Pro Ser Ser Gln Lys Asn Leu Giu Ile Val Tyr Val Lys Asp Lys Asp Lys Gly Ser Glu Phe Cys Leu Arg Gly Ser Leu Ser Lys Ala Gln Val Arg Gly Lys Met Val Val Cys Asp Arg Gly Val Asn Gly Arg Ala Glu Lys Gly Gln Val Val Lys Glu Ala Gly Gly Ala Ala Met Ile Leu Ala Asn Thr Ala Ile Asn Met Glu Glu Asp Ser Ile Asp Val His Val Leu Pro Ala Thr Leu Ile Gly Phe Asp Glu Ser Ile Gln Leu Gln Asn Tyr Leu Asn Ser Thr Lys Arg Pro Thr Ala Arg Phe Ile Phe Gly Gly Thr Val Ile Gly Lys Ser Arg Ala Pro Ala Val Ala Gln Phe Ser Ser Arg G1y Pro Ser Tyr Thr Asp Pro Ser Ile Leu Lys Pro Asp Leu Ile Ala Pro Gly Val Asn Ile Ile Ala Ala Trp Pro Gln Asn Leu Gly Pro Ser Gly Leu Pro Glu Asp Ser Arg &rg Val Asn Phe Thr Val Met Ser Gly Thr Ser Met Ala Cys Pro His Val Ser Gly Ile Ala Ala Leu Leu His Ser Ala His Pro Lys Trp Thr Pro Ala Ala Ile Arg Ser Ala Leu Met Thr Thr Ala Asp Thr Ala Asp His Met Gly Lys Pro Ile Met Asp Gly Asp Ala Pro Ala Lys Leu Phe Ala Ala Gly Ala Gly His Val Asn Pro Gly Arg Ala 595 60~ 605 Ile Asp Pro Gly Leu Ile Tyr Asp Ile Gln Val Asp Glu Tyr Ile Thr His Leu Cys Thr Ile Gly Tyr Arg Asn Ser Glu Val Phe Ser Ile Thr His Arg Asn Val Ser Cys His Asp Ile Leu Gln Asn Asn Arg Gly Phe Ser Leu Asr_ Tyr Pro Ser Ile Ser Ile Thr Phe Arg Ala Gly Met Thr Arg Lys Ile Ile Lys Arg Arg Val Thr Asn Val Gly Asn Pro Asn Ser Ile Tyr Ser Val Asp Ile Glu Ala Pro Glu Gly Val Lys Val Arg Val Lys Pro Arg Arg Leu Ile Phe Lys His Val Asn Gln Ser Leu Ser Tyr Arg Val Trp Phe Ile Ser Arg Lys Xaa Ile Glu Ser Lys Arg Met Ser Phe Ala Glu Gly Gln Leu Thr Trp Phe Asn Val Gly Asn Lys Ala Thr Lys Val Lys Ser Pro Ile Ser Val Thr Trp Ala Ser Met Lys ~2 <210> 13 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 13 Gln Thr Tyr Ile Val <210> 14 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 14 Ile Val Gln Leu His <210> 15 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 15 Ser Ser Arg Leu Leu <210> 16 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artif=cial sequence <400> 16 Gln Thr Thr Tyr Ser <210> 17 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 17 Ser Ser Ser Cys Asn <210> 18 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 18 Val Leu Gly Asn Gly <210> 19 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sec_uence: artificial sequence <400> 19 Gly Ala His Ile Ala <210> 20 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sea_uence: artif'_cial sequence <400> 20 Phe Arg Ala Met Glu <210> 21 <212> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 21 Val Ile Cys Ala Ala <210> 22 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 22 Ala Ala Gly Asn Asn <210> 23 <211> 5 <212> PRT
<213> Artificial Sequence <220> , <223> Description of Artificial Sequence: artificial secuence <400> 23 Ser Ser Val Ala Asn <2i0> 24 <211> 5 <212> PRT
<213> A=tificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 24 Tyr Gly Glu Ser Leu <210> 25 <211> 5 <212> PRT
<213> Ar~ificial Sequer_ce <220>
<223> Description of Artificial Sequence: artificial sequence <400> 25 Gly Ser Glu Phe Cys <210> 26 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 26 Cys Leu Arg Gly $er <210> 27 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sea_uence: artificial sequence <400> 27 Arg Gly Val Asn Gly <210> 28 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 28 Pro Ala Thr Leu Ile Gly <210> 29 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 29 Ile Phe Gly Gly Thr <210> 30 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 30 Pro Gln Asn Leu Gly <210> 31 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 31 Val Asn Phe Thr Val <210> 32 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sea_uence: artificial sequence <400> 32 His Val Ser Gly Ile <210> 33 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 33 Gly Phe Ser Leu Asn <210> 34 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 34 Arg Arg Val Thr Asn <210> 35 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 35 Pro Asn Ser Ile Tyr <210> 36 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 36 Leu Ser Tyr Arg Val <210> 37 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 37 Ser Pro Ile Ser Val <210> 38 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 38 Val Ile Cys Ala Ala <210> 39 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial s ecrlenc a <400> 39 Cys Ala Ala Gly Asn <210> 40 <211> 5 <212> PRT
<223> Artificial Secruence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 40 Ala Ala Gly Asn Asn <210> 41 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 41 Val Ile Cys Ala Ala G_y Asn Asn Gly <210> 42 <211> 5 <212> PRT
<213> Artificial Sequence WO 00/22144 PCT/EP99/0~633 <220>
<223> Description of Artificial Sequence: artificial sequence <400> 42 Ile Ile Gly Val Leu <210> 43 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 43 Gly Val Leu Asp Thr <210> 44 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 44 Thr His Thr Ala Ser Thr <210> 45 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 45 Ser Arg Asp Ser <210> 46 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 46 Arg Asp Ser Gly <210> 47 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 47 His Val Ser Gly Ile <210> 48 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 48 Phe Thr Val Ser Gly Thr <210> 49 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 49 Ser Tyr His Ser Ala <210> 50 <211> 5 <212 > PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 50 4~
Gly Leu Ser Pro Thr <210> 51 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 51 Trp Leu Lys Ser Gly <210> 52 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 52 Phe Asn Ser Ser Ser <210> 53 <211> 5 <212 > PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 53 Ala Ser Thr Ala Gly <210> 54 <211> 5 <212 > PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 54 Ala Ala Met Asp Val 4~
<210> 55 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 55 Trp Ile Ala Thr Ile <210> 56 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 56 Gly Pro Ser Gly Leu <210> 57 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 57 Ile Ala Ala Leu Leu His <210> 58 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 58 Lys Pro Ile Met Asp <210> 59 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 59 Val Ser Cys His Asp <210> 60 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 60 Tyr Pro Ser Ile Ser <210> 61 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: artificial sequence <400> 61 Ser Leu Ser Tyr Arg

Claims (21)

Claims

1. A recombinant DNA molecule comprising:
(i) a nucleic acid molecule encoding a subtilisin-like serine protease or encoding a biologically active fragment of such a protein, selected from the group consisting of (a) nucleic acid molecules comprising a nucleotide sequence encoding a protein comprising the amino acid sequence as given in SEQ ID NO: 2, 8, 10 or 12;
(b) nucleic acid molecules comprising a nucleotide sequence as given in SEQ ID NO: 1, 7, 9 or 11;
(c) nucleic acid molecules encoding a protein comprising at least the D region, H region, substrate binding site and/or S region of the subtilisin-like serine protease encoded by a nucleic acid molecule of (a) or (b); or (d) nucleic acid molecules hybridizing with the complementary strand of a nucleic acid molecule as defined in any one of (a) to (c);
(e) nucleic acid molecules encoding a protein the amino acid sequence of which is at least 65% identical to the amino acid sequence encoded by a nucleic acid molecule of any one of (a) to (c);
(f) nucleic acid molecules, the nucleotide sequence of which is degenerate as a result of the genetic code to a nucleotide sequence of a nucleic acid molecule as defined in any one of (a) to (e); or (ii) a nucleic acid molecule encoding a mutant non-active or a hyper-active form of or an antibody against the subtilisin-like serine protease encoded by a nucleic acid molecule of (i); or (iii) a nucleic acid molecule which specifically hybridizes with a nucleic acid molecule of (i) or the complementary strand thereof.

2. The recombinant DNA molecule of claim 1 wherein the nucleic acid molecule is DNA, cDNA, genomic DNA or synthetically synthesized DNA.

3. The recombinant DNA molecule of claim 1 wherein the nucleic acid molecule is derived from a plant, preferably Arabidopsis or potato.

4. The recombinant DNA molecule of any one of claims 1 to 3 wherein said nucleic acid molecule is operably linked to regulatory elements allowing the expression of the nucleic acid molecule in plants.

5. A vector comprising a recombinant DNA molecule of any one of claims 1 to 4.

6. A host cell containing a vector of claim 5 or a recombinant DNA molecule of any one of claims 1 to 4.

7. A method for the production of transgenic plants with altered stomata characteristica compared to wild type plants comprising the introduction of a recombinant DNA molecule of any one of claims 1 to 4 or the vector of claim 5 into the genome of a plant, plant cell or plant tissue.

8. A transgenic plant cell comprising stably integrated into the genome a recombinant DNA molecule of any one of claims 1 to 4 or a vector of claim 5 or obtainable according to the method of claim 7, wherein the expression of the nucleic acid molecule results in an increased expression or activity of subtilisin-like serine proteases in transgenic plants compared to wild type plants.

9. A transgenic plant or a plant tissue comprising plant cells of claim 8.

10. The transgenic plant of claim 9 which displays a decreased stomata density, lower conductance of stomata and/or wherein the water consumption is lowered compared to wild type plants.

11. A transgenic plant cell which contains stably integrated into the genome a recombinant DNA molecule of any one of claims 1 to 4 or part thereof, a vector of claim 5 or obtainable according to the method of claim 7, wherein the presence, transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis or the activity of subtilisin-like serine proteases in transgenic plants compared to wild type plants.

12. The plant cell of claim 11, wherein the reduction is achieved by an antisense, sense, ribozyme, co-suppression and/or dominant mutant effect.

13. A transgenic plant or plant tissue comprising the plant cells of claim 11 or 12.

14. The transgenic plant of claim 13 which displays increased stomatal density and/or higher conductance of stomata and/or increased content of sugars and/or protein in plant leaves compared to wild type plants.

15. The transgenic plant of any one of claims 9, 10, 13 or 14, the plant cell of any one of claims 8, 11 or 12, or the plant tissue of claim 9 or 13, wherein said plant, plant cell or plant tissue is derived from a monocotyledonous or dicotyledonous plant.

16. The transgenic plant, plant cell or plant tissue of claim 15, wherein said plant is derived from maize, rice, barley, wheat, rye, oats, tomato, melon, banana, chicoree, salad, cabbage, potato, tobacco, alfalfa, clover, oilseed rape, sunflower, peanut, soybean, cotton, sugar beet, linseed, flax, millet, hemp, sugar cane, bean, pea or tree.

17. Harvestable parts or propagation material of plants of any one of claims 9, 10, 13 or 14 to 16 comprising plant cells of claim 8, 11, 12, 15 or 16.

18. A kit comprising a recombinant DNA molecule of any one of claims 1 to 4 or a vector of claim 5.

19. A method for the production of a transgenic plant comprising an increased yield and/or increased stomatal density compared to wild type plants, wherein (a) a plant cell is genetically modified by the introduction of a foreign nucleic acid molecule the presence of which or the expression of which results in a decreased activity of a subtilase;
(b) a plant is regenerated from the cell prepared according to step (a); and (c) further plants, if any, are generated from the plant prepared according to step (b).

20. A method for the production of a transgenic plant having a decreased water consumption and/or decreased stomatal density compared to wild type plants wherein (a) a plant cell is genetically modified by the introduction of a foreign nucleic acid molecule the presence of which or the expression of which results in an increased activity of a subtilase;
(b) a plant is regenerated from the cell prepared according to step (a); and (c) further plants, if any, are generated from the plant prepared according to step (b).

21. Use of a nucleic acid molecule encoding or regulating the expression of a subtilisin-like serine protease or a nucleic acid molecule hybridizing with such a nucleic acid molecule, a nucleic acid molecule as defined in any one of claims 1 to 4, a recombinant DNA molecule of any one of claims 1 to 4, or a vector of claim 5 for the production of giants with improved fresh and dry weight, for enhancing the content of sugars and/or protein in plant leaves for the production of plants with reduced leaf temperatures or with reduced water loss and lower water consumption, for the modulation (enhancement) of CO2 uptake into and H2O release from leaves, for sustained photosynthesis under high intensity conditions or for the improvement of disease resistance of plants.