WO2003035856A2

WO2003035856A2 - Protein with kinase activity

Info

Publication number: WO2003035856A2
Application number: PCT/EP2002/011703
Authority: WO
Inventors: Irute Meskiene; Heribert Hirt; Claudia Jonak
Original assignee: Arc Seibersdorf Research Gmbh
Priority date: 2001-10-19
Filing date: 2002-10-18
Publication date: 2003-05-01
Also published as: WO2003035856A3; AU2002350570A1

Abstract

A protein with kinase activity is provided comprising (a) an amino acid sequence showing at least 90%, preferably at least 95%, identity to an amino acid sequence as given in Fig. 1, or (b) a specific fragment thereof comprising at least 400 amino acids.

Description

Protein with kinase activity

The present invention relates to a protein with kinase activity, as well as a polynucleotide molecule.

Unlike animals, plants are sessile organisms. To survive changes in the environment plants have evolved complex and sophisticated sensing and adaptation systems with allowance to response and adapt to many stress situations. Such stresses include abiotic factors, such as cold, drought, wounding, irradiation and osmotic stress, as well as biotic factors, such as pathogens. Stress responses are characterized by a complex spacial and temporal pattern of events encompassing immediate early responses which occur within seconds and minutes and include the opening of ion channels and the formation of reactive oxygen intermediates . These events are followed by the onset of a transcriptional activation of certain genes and the production of certain plant hormones, such as ethylene and jasmonic acid. Late responses occur' within hours and days and include the expression of genes that regulate various metabolic pathways or are involved directly in stress responses .

Environmental stresses, such as salinity or wounding have an enormous impact on crop productivity. Efforts to improve stress tolerance of plants focused on the overexpression of individual stress-induced genes. The beneficial outcome has, however, been rather small. Modulation of signalling regulators is a new way for improving the stress tolerance of plants .

Various strategies for making salt tolerable plants are described:

The US 5,639,950 relates to a DNA sequence including a bi-func- tional enzyme (termed P5CS) with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyses the first two steps in plant proline production. Uptake of this gene in plants was described to increase salt tolerance and drought resistance of the plants.

US 5,859,337 describes nucleic acids encoding polypeptides which confer salt tolerance in plants and other organisms . These polynucleotide sequences are termed "STZ" or "STO" .

In WO 99/54489 Al a method for obtaining plants, tolerant to abiotic stress conditions, in particular osmotic stress, is described, conferring to a plant the capacity to counteract the stress-induced phosphorylation of Cyclin Dependent Kinase (CDK) proteins .

US 5,965,705 relates to a gene, designated as CBFl, encoding a protein (CBFl) , which binds to a region regulating expression of genes which promote cold temperature and dehydration tolerance in plants. This gene is used to transform micro-organisms and can be used to transform plants.

US 4,594,323 relates to mutated proBA regions which are provided for conferring osmotic tolerance on osmotically sensitive hosts. The mutated DNA sequence is able to overproduce at least one enzyme in the biosynthetic pathway for an amino acid which imparts the desired osmotic tolerance.

It is an object of the present invention to provide further means and methods for improving stress tolerance in organisms and cells, especially salt tolerance in plant cells or plants.

This object is solved by a protein with kinase activity comprising

(a) an amino acid sequence showing at least 90%, preferably at least 95%, identity to an amino acid sequence as given in Fig. 1, or

(b) a specific fragment thereof comprising at least 400 amino acids .

Within the course of the present invention a protein with a kinase activity was isolated and characterized. The amino acid sequence of the protein with kinase activity is depicted in Fig.l and was named MsK4. This protein is a glycogen synthase kinase 3- like protein-kinase and shows 66% identity to GSK-3S and about 50% identity to different shaggy kinases from Drosophila mela- nogaster. While MsK4 shares 70% to 76% with other GSK-3 like pro- teins from alfalfa (s. C. Jonak et al . , "Inflorescence-Specific Expression of AtK-1, a Novel Arabidopsis Thaliana Homologue of Shaggy/Glycogen Synthase Kinase-3", Plant Molecular Biology, 27, 1995, (217-221)), it is highly related to AtKl/ASK_k (87% identity) from Arabidopsis (C. Jonak et al . , "Wound- nduced Expression and Activation of WIG, a Novel Glycogen Synthase Kinase 3", The Plant Cell, 12, 2000, (1467-1475)).

Proteins with kinase activity in the scope of the present application are enzymes which transfer a phosphate group from ATP to a substrate as acceptor, for example to an alcoholic hydroxy-, car- boxy-, amino-, phosphategroup, etc.. In order to determine the kinase activity of a protein any method known to the person skilled in the art can be applied. One example is to provide a protein to be tested and to carry out in vitro kinase assays with a specific substrate and to test phosphorylation of the substrate, for example with the help of radioactive phosphate.

Glycogen synthase kinase 3 (GSK-3) plays a key role in many physiological processes. In animals GSK-3 is an essential component for insulin signalling, cell fate specification and cell proliferation. While mammals have two GSK-3 isoforms, plants contain up to 10 different genes encoding distinct GSK-3s. Mammalian GSK-3 phosphorylates and inactivates glycogen synthase, thereby regulating glycogen metabolism.

The US 6 262 345 relates to a glycogen synthase 3, as well as cells which comprise this kinase.

The US 5 891 628 refers to the identification of polycystic kidney disease gene diagnostics and treatments whereby the level of PKDl polypeptide is measured in a biological sample. This document also describes an MsK4, however, this is a gene obtained from an adult kidney library and is not identical or even simai- lar to the inventive protein with kinase activity.

In Pay et al . ("The MsK family of Alfalfa Protein Kinase Genes Encodes Homologues of Shaggy/Glycogen Synthase Kinase-3 and Shows Differential Expression Patterns in Plant Organs and Development", The Plant Journal, 3(6), 1993 (847-856)) MsKl, MsK2 and MsK3 , protein kinases isolated from Medicago, show a 74% and 72% identity, respectively, to the sequence according to Fig. 1.

Davletova et al. ("Auxin and Heat Shock Activation of a Novel Member of the Calmodulin like Domain Protein Kinase Gene Family in Cultured Alfalfa Cells", Journal of Experimental Botany, 52 (355) , Seiten 215-221) relates to a Calmodulin like protein kinase (CPK) homologue identified in alfalfa and termed MsCPK3. This protein is neither structurally nor functionally similar to the inventive protein and shows only 15% identity to the sequence according to Fig. 1.

Computer analysis of the inventive MsK4 identified a potential chloroplast-targeting sequence motif. Immunochemical and biochemical analysis revealed that MsK4 kinase is localized in plas- tids in association with starch granules. In leaves, MsK4 protein was found to be located on starch granules of esophyll chloro- plasts. In roots, MsK4 was found in amyloplasts, starch storage forms of plastids, in epidermis and cortex as well as statolithes in the gravity-sensing columella cells of the root cap. In cultured cells, MsK4 protein was localized in amyloplasts which were entirely filled with starch grains. More detailed MsK4 localization studies with immunoelectron microscopy of root cells showed that MsK4 is localized directly on starch grains of amyloplasts.

Exposure of alfalfa roots to various abiotic stresses, revealed that MsK4 kinase is rapidly activated by high salt conditions. Immunoprecipitation of root protein extracts with the MsK4-spe- cific antibody and subsequent kinase assays revealed that untreated roots contained only very little MsK4 activity, but incubation of roots with more than 125mM NaCl activated the MsK4 kinase. These data show that plants contain a plastid-located MsK4 that associates with starch granules and is activated by hy- perosmotic conditions .

Therefore, the newly found MsK4 can be used to induce stress tolerance in cells and organisms, in particular plants and plant cells. This inventive MsK4 will further provide new ways to understand and improve salt tolerance of organisms and plants, which is becoming an ever more important problem in world-wide agriculture.

However, it has also surprisingly been found within the scope of the present invention that MsK4 is associated with starch granules .

Starch is, like glycogen in animals, a polymer of glucose, and of primary importance as an energy and carbon store. Moreover, starch metabolism in plants is highly sensitive to changes in the environment. Interestingly, before becoming famous as a key regulator of animal physiological and developmental processes, mammalian GSK-3 was identified as a serine/threonine protein kinase that regulates glycogen metabolism. Although the molecular mechanisms have not been determined yet in plants, it appears that one function of GSK-3s that has been conserved during plant and animal development is to control synthesis and/or breakdown of the sugar polymers starch/glycogen.

Therefore, another aspect of the present invention relates to the influence of MsK4 on the starch metabolism in plants. By regulating the synthesis and breakdown as well as the structure of starch the carbohydrate metabolism of the plant or cells can be regulated. For example an increased carbon and energy demand can be satisfied by regulating the expression of the inventive MsK4 in the plant/cells. Due to this surprising fact that the inventive MsK4 plays a major role in the starch metabolism, cells and plants can be designed, for example by genetic modifications, which have an increased or decreased expression of MsK4 or which expression can be up- or downregulated according to the specific needs .

A further aspect of the present application is therefore the use of the inventive protein with kinase activity or its nucleic acid sequence for initiating the synthesis or for increasing the synthesis of starch in cells and plants or for inhibiting or down- regulating the breakdown of starch in cells and plants. Furthermore, cells and plants with - compared to wild-type cells and plants - increased quantities of starch are provided.

A further aspect of the present application is furthermore the use of the inventive protein with kinase activity or its nucleic acid sequence for inhibiting or downregulating the synthesis or for increasing or initiating the breakdown of starch in cells or plants. Furthermore, cells and plants with - compared to wild- type cells or plants - less or no starch are provided.

A still further aspect of the present application is the use of the inventive protein with kinase activity or its nucleic acid sequence for the production of cells and plants with a modified structure of starch. Furthermore, cells and plants with - compared to wild-type cells or plants - a modified structure of starch are provided.

In the scope of the present application, identity in amino acid sequence is preferably higher than 98%, especially preferred higher than 99%.

The percentage of identity between two amino acid sequences can be determined preferably electronically with the help of any algorithm allowing alignment of sequences, e.g. FAST/A algorithm and others. Of course, also proteins with additional amino acids at one or both ends of said sequence as given in Fig.l are comprised by the present application.

In the scope of the present application, the term "fragment" relates to a protein comprising less than 432 amino acids, which has kinase activity which can be measured as mentioned above. Such a fragment may comprise 431 or less amino acids.

Preferably the protein comprises an amino acid sequence as given in Fig.l. This precise sequence was found in Medicago sativa, isolated and purified. The kinase activity of this protein, MsK4, was shown to be rapidly induced in roots by exposure of the plant to high salinity conditions.

A further aspect of the present invention relates to a polynucleotide molecule which comprises a sequence encoding a protein with kinase activity according to the present invention as mentioned above. The sequence of this polynucleotide molecule can be derived electronically with the help of any known algorithm. With this polynucleotide molecule any organism can be genetically manipulated and thereby comprise or express the inventive protein with kinase activity or also show inhibited or reduced expression of MsK4. Such methods are well known in the state of the art and comprise for example knock-out methods. Such an inventive polynucleotide molecule which is comprised by the present application is deposited at GenBank under the accession number Bank IT 429023 AF 432225.

According to a preferred aspect of the present invention a polynucleotide molecule is provided which comprises

(a) a sequence with at least 88%, preferably at least 90%, still preferred at least 95%, most preferred at least 98%, identity to a polynucleotide sequence as given in Fig. 2 or

(b) a sequence which hybridizes under stringent conditions with said sequence (a) .

Here again, the percent identity can be easily determined electronically with any known algorithm, for example FAST/A. Preferably, the sequence of the inventive polynucleotide molecule has at least 99%, preferably at least 99.5% identity to a polynucleotide sequence as given in Fig.2. The polynucleotide sequence according to Fig.2 was isolated from Medicago sativa and was shown to encode a glycogen synthase kinase 3-like protein kinase. It was shown that the activity of this encoded protein kinase is rapidly induced by exposure of the plants to high salinity condition.

An example for highly stringent hybridisation conditions is 6x SSC and 65°C. However, stringent hybridisation conditions for a polynucleotide molecule with the given length will usually involve hybridisation at a temperature of 15 to 25 degrees below melting temperature (Tm) of the expected duplex (for molecules with over 200 nucleotides) (Sambrook et al . , 1989). The Tm of a nucleic acid duplex can be calculated using a formula based on the percentage G+C contained in the nucleic ' acids and by taking the length into account. Such a formula is for example Tm=81.5- 16.6 (log [Natrium⁺] ) -0.41 (%G+C- (600/N) ) , where N is the chain length. Therefore the person skilled in the art will be able to determine the condition for the hybridisation reaction which will be a function of factors such as concentration of salt or forma- ide in the hybridisation buffer, the temperature of the hybridi- sation and the post hybridisation wash condition.

In the scope of the present application the term "polynucleotide molecule" relates to a DNA as well as an RNA molecule.

Preferably the polynucleotide molecule comprises a sequence as given in Fig.2. A sequence identical to the one given in Fig.2 has shown to code for MsK4 with kinase activity inducible by stress factors.

A still further embodiment of the present invention relates to a polynucleotide molecule comprising a specific fragment of the above mentioned polynucleotide molecule according to the present invention of at least 20, preferably at least 50, still preferred at least 100 nucleic acids.

Hereby, a "specific" fragment relates to any fragment of the above mentioned polynucleotide molecule which is not identical to any known nucleic acid sequence but comprised only by a fragment of the above sequence and therefore specific only for the above sequence. Such a fragment can be determined by a person skilled in the art without undue burden by comparing a given fragment with known published sequences. This will preferably be carried out electronically in a database, e.g. Derwent. If a given fragment is not identical to any already known published nucleic acid sequence and is however identical to a fragment of one of the above defined polynucleotide molecules this fragment will be comprised by the present application. Here, of course, fragments which are larger e.g. which comprise at least 500 or at least 1000 nucleic acids are also comprised by the present application. However, the small fragments are more stable and therefore easier to manipulate and to be used in assays than longer fragments.

Preferably a polynucleotide molecule comprising a sequence which is complementary to the above mentioned polynucleotide molecule according to the present invention is provided by the present invention. With this complementary sequence it is possible to provide molecules which will hybridise to a part of the polynucleotide molecule according to the present invention and therefore can be used for the detection of a polynucleotide molecule ac- cording to the present invention or for the amplification of a given region of the inventive polynucleotide molecule or even for knock-out methods. Therefore, proteins according to the present invention of any given organism can be detected and characterised. Here again the complementary molecule may comprise the full length of the inventive polynucleotide molecule or a specific fragment thereof.

Preferably the polynucleotide molecule comprises a detectable marker. This marker may be any known marker e.g. fluorescent, radioactive, a marker for enzymatic or chemical detection. Here the term "detectable marker" refers to any marker which is attached to or integrated into the polynucleotide molecule and which thereby renders the molecule detectable. Hereby, the polynucleotide molecule may comprise the full length of the inventive molecule or only a fragment thereof .

According to an advantageous embodiment of the present invention a biologically functional vector comprising an inventive polynucleotide molecule as mentioned above is provided. Of course, the vector will comprise any necessary elements which are usually present in a vector, in particular, regulatory elements. These regulatory elements may be for example promoters which may be induced by drought, cold, salinity, wound, anoxia etc.. However, this regulatory element may also be a promoter for expression in particular tissues or which induces high expression of a specific protein. Such vectors are well known in the state of the art and the person skilled in the art will select the optimal vector without undue burden.

To obtain high constitutive expression of a transgene in all tissues, so far the 35S cauliflower mosaic virus promoter has been used extensively (Holmberg N. and Bulow L., 1998, Trends Pi. Sci. 3:61-66). This promoter has several advantages. It has a broad host range and can be used in both dicotyledonous and monocotyle- donous plants and varies little between different organs and developmental stages (Benfey P. and Chua N., 1990, Science 250:959- 966) .

Promoters for expression in particular tissues have been used and include the Arabidopsis promoter from the Rubisco small subunit gene AtSlA, which only drives expression in green tissues (Krebbers et al., 1988, Plant Mol. Biol . 11:745-759), or the patatin type I promoter, which mediates exclusive expression in potato tubers (Rocha-Sosa M. et al . , 1989, EMBO J. 8.23-29).

Preferably the vector further comprises a selection marker. Thereby any cell transfected by this vector can be easily detected according to well known methods in the state of the art.

Advantageously the vector further comprises an inducible promoter, said promoter preferably being regulated by salt concentration. Thereby, a vector is provided which will induce strong resistance against salinity to any cell into which it is transfected since the promoter will be regulated by the salt concentration e.g. with mounting salt concentration the promoter will up-regulate expression of the inventive protein with kinase activity or fragment thereof and vice versa.

Different plant promoters induced by drought, cold, salinity or anoxia have also been used in various studies (Homberg N. and Bϋlow L., 1998, Trends Pi. Sci. 3:61-66). For example the osmotin promoter responds to salinity, drought, and the hormone abscisic acid (Raghotha a et al . , 1993, Plant Mol. Biol. 23: 1117-1128), and the glyceraldehyde-3-phosphate dehydrogenase 4 promoter responds to anoxia (Kδhler et al . , 1996, Plant J. 10:175-183).

According to a further embodiment of the present invention a plant cell is provided which comprises an inventive vector as mentioned above. Due to the expression of the inventive protein with the kinase activity or an active fragment thereof the inventive plant cell will show higher tolerance to stress factors, for example salinity stress. The vector will preferably be selected according to a given plant cell or the plant cell will be selected according to a given vector. Of course, the cell can be only transiently transfected by the vector. It is, however, preferable that the cell is stably transfected by the vector. Thereby a plant cell is given which stably expresses the inventive protein over a number of generations . Since plant cells are in general sensitive towards stress factors the inventive plant cell will be particularly tolerant towards a given stress factor, in particular high salinity, dryness, etc.

Still preferred the plant cell comprises said vector integrated into its genome. Thereby the plant cell is stably transfected and will give stress tolerance on to a number of generations.

Advantageously the plant cell exhibits a salt tolerance of at least 150% of a wild type cell. Since salt stress is an important factor which causes great economic losses, plant cells which exhibit a salt tolerance of at least 150% of the wild type cell will be of great economic importance. Furthermore, such cells will be most suitable for plants which are to be cultivated on grounds with high salt concentrations. Furthermore, such plant cells are useful in vitro as in vivo to test various plants, cultivation conditions and various media and salts.

According to another aspect of the present invention a plant is provided comprising at least a subpopulation of inventive plant cells as mentioned above. Of course, said subpopulation should be large enough to confer the stress tolerance to the plant as a whole. Therefore, a plant is provided which shows a higher stress tolerance than the wild type plant and therefore will be particularly useful for the cultivation on high salinity grounds.

A still further aspect of the present invention relates to a method for producing an inventive protein as mentioned above comprising the steps

(a) providing an inventive vector,

(b) transfecting a suitable host selected from the group consisting of a plant cell, plant and plant tissue with said vector,

(c) selecting and cultivating stably transfected hosts,

(d) expressing said protein and

(e) isolating and purifying said protein. Here again the same definitions and preferred embodiments as mentioned above apply. This method will allow the production of high quantities of the inventive protein in a short period of time and with minimal costs. The transfection step will be carried out according to any known method, e.g. by electroporation, salt transfection, lipo- fectamin, etc. The selection will preferably be carried out due to the selection marker in the vector, which may be for example used to select on media containing antibiotics. However, the selection step may also be carried out by cultivating said transfected cells on a medium with high salt concentration, whereby only stably transfected host cells will be able to grow whereby at the same time the expression of said protein will be enhanced.

The isolation and purification step may also be carried out according to any known method, e.g. chromatography, immunological methods, electrophoresis, etc.

In case said host is a plant cell various in vitro methods which are highly reliable and can be carried out within a relative short period of time are provided.

The production of plant or plant tissues may be carried out in vitro by the production of a callus. The cultivation of plants on a small scale, e.g. as a trial, or on a higher scale, e.g. in fields are possible. Whether a plant cell, a plant tissue or a whole plant will be cultivated will depend on the respective aims, e.g. whether the method is carried out for analytical reasons or as a method for the production of the inventive protein on an industrial scale.

According to a still further embodiment of the present invention a method is provided for identifying an inventive polynucleotide molecule as mentioned above comprising the steps

(a) providing a labelled inventive polynucleotide molecule or a conserved fragment thereof,

(b) providing a library of polynucleotide molecules potentially containing polynucleotide molecules encoding a protein with kinase activity or parts thereof,

(c) identifying said protein encoding polynucleotide molecule with said labelled polynucleotide molecule and optionally

(d) isolating said protein encoding polynucleotide molecule. For this the labelled polynucleotide molecule is preferably a conserved fragment of the inventive molecule, e.g. a fragment which is selected in conserved regions and which is specific for the inventive polynucleotide molecule. With this method respective polynucleotide molecules of any given organism can be detected and in a further step also the encoded proteins . For this inventive method, a DNA library or genomic library of a given organism may be provided.

Whether or not said polynucleotide molecule found in said library encodes a protein with kinase activity or parts thereof can be determined according to any known method. For example the found polynucleotide molecules will be inserted into a given vector. Said vector will be transfected into a suitable host, stably transfected hosts will then be selected and cultivated. The protein expressed, isolated and purified as described above will be tested for kinase activity as mentioned above, e.g. with an acceptor substrate and ATP.

The present invention is described in the following examples and in the figures in more detail. Of course, the present invention is not restricted to these examples and figures.

Fig.l shows the MsK4 amino acid sequence isolated from Medicago sativa,

Fig.2 shows a nucleotide sequence of MsK4 isolated from Medicago sativa,

Fig.3 shows the specificity of anti-MsK4 antibody,

Fig.4 shows the activity of MsK4 induced by NaCl, KCl, and sorbi- tol,

Fig.5 shows the kinetics of the MsK4 activation,

Fig.6 shows the localization of MsK4 in amyloplasts and chloro- plasts,

Fig.7 shows the localization of MsK4 on starch grains,

Fig.8 shows the immunoblots of extract, untreated and treated starch Examples :

Example 1

Function of MsK4

In search of^' GSK-3-like genes from Medicago sativa a full cDNA encoding a new member of this family has been isolated (s. Fig. 2) . .

To study the function of MsK4 protein kinase (s. Fig. 1), a pep- tide antibody against the C terminus of MsK4 was produced. In crude extracts from cultured alfalfa cells the affinity purified antibody recognized a single 50kD-protein, in good agreement with the calculated molecular mass of MsK4. In Fig.3A a protein gel blot analysis of cultured alfalfa cells with the anti-MsK4' antibody without (lane 1) or with MsK4 peptide competition (lane 2) is shown. This immunoreaction was specific because binding of the antibody could be competed by preincubation of the antibody with the C-terminal MsK4 peptide (Fig.3A) .

To test whether the antibody could specifically immunoprecipitate the MsK4 kinase, the in vitro translated-proteins of alfalfa GSK- 3s MsKl, MsK4 and WIG were incubated with MsK4 antibody. In Fig.3B an autoradiogramm of 35S-methionine labelled in vitro- translated proteins of MsKl, MsK4 and WIG (lanes 1 to 3, respectively) and immunoprecipitation of in vitro-translated proteins of MsKl, MsK4 and WIG with anti-MsK4 antibody (lanes 4 to 6, respectively) is shown. As shown in Fig.3B the MsK4 antibody exclusively immunoprecipitated MsK4 but did not immunoprecipitate the other alfalfa kinases .

It could therefore be shown that the_. given antibody specifically immunoprecipitates the MsK4 kinase.

Example 2

Testing of MsK4 activity

Alfalfa seedlings were exposed to increasing concentrations of NaCl . Roots were harvested 10 min after addition of NaCl. Immuno- precipitation of protein extracts using the MsK4 specific antibody and subsequent kinase assays revealed that untreated roots ^' contained only very little MsK4 activity, but incubation of roots with more than 125 mM NaCl activated the MsK4 kinase (Fig.4, upper lane) . Maximal MsK4 activity in roots was obtained by exposure to 750mM NaCl. The same samples were also immunoprecipitated with an antibody specific for the alfalfa wound-induced GSK3 WIG. No activation of WIG kinase was obtained by any NaCl concentration (Fig.4, second lane).

To test whether induction of MsK4 activity is specific for high NaCl concentration or is a general response to hyper-osmotic stress roots were exposed to different amounts KCl or sorbitol. Immunokinase assays revealed that MsK4 activity is induced in a similar way by high concentrations of KCl and sorbitol indicating that MsK4 could be involved in mediating a general hyper-osmotic stress response (Fig.4 lower lanes).

Exposure of alfalfa roots to various abiotic stresses revealed that MsK4 kinase is rapidly activated by high salt concentrations but not by drought, cold, wounding, or water.

Example 3

Time course of MsK4 activation

To analyse the time course of MsK4 activation, roots were exposed to water (Fig. 5, top lane) or 250mM NaCl (Fig. 5, bottom lane) and harvested at different timepoints. A slight increase in the immunopurified MsK4 kinase activity was already detected two minutes after addition of NaCl. Maximal activation was obtained after 10 minutes (Fig.5). No induction of MsK4 activity was detected when roots were treated with water.

Example 4

Localization of MsK4

Sequence analysis of the MsK4 protein revealed a putative N-ter- minal chloroplast targeting sequence. In order to verify this predictions immunofluorescence with MsK4-specific antibody (Fig. 6, A, C, E, G) and corresponding bright field (Fig. 6,^' B, D, F, H, respectively) was performed in root epidermis and cortex (A, B) , in statolithes of colluella cells (C, D) , in suspension cultures cells (E, F) and in leaf mesophyll cells (G, H) . In roots MsK4-specific antibody decorated amyloplasts in epidermis and cortex (Fig.6A) as well as statolithes in the gravity-sensing columella cells of the root cap (Fig.6C). In cultured cells MsK4 protein localized to the amyloplasts which were entirely filled with starch grains (Fig.6E) . In leaves MsK4 protein was detected in chloroplasts of mesophyll cells associated with starch granules (Fig.βG) .

In Fig. 6 localization of MsK4 in chloroplasts and amyloplasts associated with starch grains is shown.

Immunoelectron microscopy of lateral root cap without (Fig. 7 A) and after (Fig. 7 B) preincubation of the MsK4-specific antibody with MsK4 peptide was performed, whereby a is amyloplast; cw is cell wall; n is nucleus; v is vacuole.

To corroborate the data obtained by immunolocalization and TEM, starch granules were isolated from suspension cultured cells. Protein gel blot analysis of the starch associated proteins using the MsK4 specific antibody showed that MsK4 protein copurified with the starch granules (Fig.8, lane 2). Washing of starch with 1% SDS or treatment with thermolysin abolished the 50kD-signal on the immunoblots (Fig.8, lane 3 and 4, respectively) indicating that MsK4 is loosely associated with starch grains . In lane 1 of Fig.8 the protein cell blot analysis of total protein extract is shown.

Claims

1. A protein with kinase activity comprising

(b) a specific fragment thereof comprising at least 400 amino acids .

2. The protein according to claim 1, characterized in that it comprises an amino acid sequence as given in Fig. 1.

3. A polynucleotide molecule, characterized in that it comprises a sequence encoding a protein according to claims 1 or 2.

4. The polynucleotide molecule according to claim 3 , characterized in that it comprises

5. The polynucleotide molecule according to claim 3 or 4, characterized in that it comprises a sequence as given in Fig. 2.

6. A polynucleotide molecule comprising a specific .fragment of the polynucleotide molecule according to any one of claims 3 to 5 of at least 20, preferably at least 50, still preferred at least 100, nucleic acids.

7. A polynucleotide molecule comprising a sequence which is complementary to the polynucleotide molecule according to any one of claims 3 to 6.

8. The polynucleotide molecule according to any one of claims 3 to 7, characterized in that it comprises a detectable marker.

9. A biologically functional vector comprising a polynucleotide molecule according to any one of claims 3 to 8.

10. The vector according to claim 9, characterized in that it further comprises a selection marker.

11. The vector according to claim 9 or 10, characterized in that it further comprises an inducible promoter, said promoter preferably being regulated by salt concentration.

12. A plant cell, characterized in that it comprises a vector according to any one of claims 9 to 11.

13. The plant cell according to claim 12, characterized in that it comprises said vector integrated into its genome.

14. The plant cell according to claim 12 or 13, characterized in that it exhibits a salt tolerance of at least 150% of the wild type plant cell.

15. A plant comprising at least a sub-population of plant cells according to any one of claims 12 to 14.

16. A plant tissue comprising at least a sub-population of plant cells according to any one of claims 12 to 14.

17. A method for producing a protein according to claim 1 or 2 comprising the steps

(a) providing a vector according to any one of claims 9 to 11,

(c) selecting and cultivating stably transfected hosts,

(d) expressing said protein and

(e) isolating and purifying said protein.

18. A method for identifying a polynucleotide molecule according to claim 3 comprising the steps

(a) providing a labelled polynucleotide molecule according to any one of claims 4 to 8 or a conserved fragment thereof,

(b) providing a library of polynucleotide molecules potentially containing polynucleotide molecules encoding a protein with kinase activity or parts thereof, (c) identifying said protein encoding polynucleotide molecule with said labelled polynucleotide molecule and optionally

(d) isolating said protein encoding polynucleotide molecule.