EP0808370A1

EP0808370A1 - Stress-tolerant plants and methods of producing the same

Info

Publication number: EP0808370A1
Application number: EP96902974A
Authority: EP
Inventors: Fritz Eberhard-Karls-Universität Tübingen SCHÖFFL; Anja Eberhard-Karls-Universität Tübingen HÜBEL; Jeong Eberhard-Karls-Universität Tübingen HEE LEE
Original assignee: KWS Kleinwanzlebener Saatzucht AG
Current assignee: KWS SAAT SE and Co KGaA
Priority date: 1995-02-02
Filing date: 1996-02-01
Publication date: 1997-11-26
Also published as: WO1996023891A1; JPH10510998A; DE19503359C1

Abstract

The invention concerns plants with enhanced stress tolerance obtainable by introducing a nucleic acid which codes for a constitutively active transcription activator into a plant cell and by regenerating a transgenic plant. The plants concerned have higher levels of protective proteins under normal conditions than wild plant types.

Description

Stress-tolerant plants and processes for their production

The present invention relates to a plant with increased stress tolerance and a method for its production. In particular, the present invention relates to a plant with an increased level of protective proteins even under stress-free conditions.

Heat shock proteins (HSP) are synthesized in all organisms, including plants, in response to greatly increased temperatures or heat shock (HS). The biological significance of this reaction, known as the HS response, is the protection of cellular proteins and structures during a stress phase and / or in the subsequent recovery phase. The HS response can also be caused by other abiotic stress factors (stressors) such as Heavy metals, arsenite or dryness are triggered. The HS response is a general stress reaction which ensures the survival of the cell and thus of the organism under unfavorable environmental conditions. The protective effect of the HSPs is based predominantly on minimizing the damage caused by the denaturation of proteins due to the action of the stressors on the organism. Almost all HSPs are molecular chaperones (cf. Table 1), which form a reversible bond with partially denatured proteins and thereby enable or accelerate the correct renaturation (folding) of these molecules. HSPs are able to reduce the proportion of denatured protein due to the effects of stress, or to increase the proportion of correctly folded, biologically active proteins. Further details on the role of heat shock proteins in plants are described by Vierling, E. in Annu. Rev. Plant Physiology Plant Mol. Biol. 1991, volume 42, pages 579 to 620.

Although, as stated above, all organisms show an HS response when stressed, this response occurs in plants special meaning too. Plants are least able to avoid or avoid environmental stress due to their location, and are most exposed to the abiotic stressors. The small HSPs (HSP20 group with a molecular weight of approximately 17-20 kDa) are of particular importance for the response of the plants to stress. These proteins are highly expressed and there are several related genes (a family of genes). In contrast to other HSP groups (for example HSP70), there are no HSP20 or HSP20-like proteins which are already formed under normal conditions, for example at the normal ambient temperature. Without environmental stress, HSPs are only synthesized in certain stages of plant development, with this being observed especially in the late phases of pollen development and embryogenesis (the desiccation phase of seed maturation).

However, there are no plants known to have significant levels of HSPs under normal stress free conditions. In particular, no plants are known in which a whole group of HSPs or even all HSPs are present in the plant in biologically relevant concentrations under stress-free conditions.

The present invention was based on the technical problem of providing plants which have an increased stress tolerance and which can react in particular to a large number of different stressors with increased tolerance.

This problem is solved by a plant containing at least one constitutively expressed and active transcription activator for the constitutive expression of at least one protective protein. Another technical problem has been to provide a method for producing a plant with increased stress tolerance.

According to the invention, this technical problem is solved by a method comprising the steps:

(a) introducing a nucleic acid which codes for a constitutively active transcription activator into a plant cell, and

(b) Regeneration of a transgenic plant from the plant cell produced in step (a).

Another technical problem was the provision of means for producing a plant with increased stress tolerance.

This technical problem is solved by a nucleic acid which codes for a transcription activator which is constitutively active in the plant.

Figure 1: Alternative models of regulation of the HS response in higher eukaryotes.

Figure 2: Expression of the HSF-GUS fusion gene in transgenic Arabidopsis.

A) shows schematically the components of the gene fusion construct.

B) shows the constitutive GUS activities in transgenic Arajbidopsis plants. The GUS activities were determined for each construct in protein extracts from 30 individual transformants (F0) and their Fl progeny. GUS activity was measured in pMol NAD / mg protein / minute.

C) shows constitutive HSF and HSF-GUS (GH, HG) mRNAs by Northern blot hybridization

Figure 3: Constitutive expression of HSP18 in transgenic AraJidopsis that overexpresses HSF fusion proteins.

A) shows the detection of HSP18 in Western blots.

B) shows the detection of HSP18 mRNA by Northern blot hybridization.

WT = untransformed Arabidopsis; HG1, HG2, GH1, GH2 = single transformants, containing HSF-GUS or GUS-HSF.

Figure 4: Constructs for cloning the fusion products HSF1-GUS / GUS-HSF2.

FIG. 4a shows the construct pAthsfl, in which the Eco RV (41 base pairs before the start of translation) / Sacl fragment from Athsfl was cloned into the pBluescript vector;

FIG. 4b shows the base vector pBIN19-CaMV / Noster, which contains the 300 bp fragment of the Cauliflower Mosaic Virus 35S promoter as promoter, which still contains the TATA box and the transcription start of the native CaMV gene. To insert the CaMV promoter, the HindIII / Xba I fragment was cloned into the plant vector pBIN19, and then the transcription termination signal Noster was inserted as the SacI / EcoRI fragment into the pBIN19 vector with the CaMV promoter; FIG. 4c shows the HSF-GUS gene construct in the pBluescript vector (auxiliary vector). The coding region of the GUS gene lies in a DNA restriction fragment which had been subcloned into the plasmid via Hindlll / Sacl. For the insertion of Athsfl, the Athsfl gene from pAthsfl (FIG. 4a) was isolated as a HindII fragment. This resulted in the fragment in which the 5 'end with the EcoRV site (41 base pairs before the start of translation) is located. This fragment contains the associated transcription start for the native Athsfl gene. This gene fragment forms the starting point for the construction of the HG fusion with the constitutive plant promoter (FIG. 4d);

FIG. 4d shows the construct in which the HG fusion fragment between the EcoRV / SacI cleavage sites from FIG. 4c had been cloned into the basic vector pBIN19 cleaved with S al / SacI;

The abbreviations used have the following meaning:

Athsfl AS 1-520 with TATA-Box GUS AS 1-603 CaMV 300 bp with transcription start Nos-ter 300 bp r transcription start

FIG. 4e shows the gene construct in which the GH fusion fragment was cloned directly into the base vector. In the base vector pBIN19-CaMV-35S-Noster, GUS was cloned as the Xbal / Sacl fragment in the corresponding interfaces of the vector. In the vector generated in this way, containing GUS, there were more in the polylinker Restriction sites are available for the merger with Athsfl. Athsf was then inserted by cloning via the SacI site (24 base pairs after the translation start).

Athsfl: AS 6-625

CIS: AS 1-598

CaMV: 300 bp with start of transcription

Nos-ter: 300 bp: start of transcription

"Increased stress tolerance" as used herein means the ability of a plant according to the invention to show less impairment in performance under the influence of stress factors than the wild type plant, or even to maintain full performance. The increased stress tolerance is presumably based, without being bound by any theory, on the presence of one or more protective proteins under normal conditions in an increased concentration in comparison with the respective wild type plant. The performance of a plant can be seen, for example, in the growth rate, the stability of the characteristics, the resistance to pathogens, the quality and the yield of the harvested products, the nutrient efficiency, the efficiency of the water balance and the behavior towards crop protection agents.

"Transcription activator", as used herein, means a regulatory protein which stimulates the transcription of certain genes in the active state. For this purpose, the transcription activator must recognize (i) certain sequences (so-called responsive elements) in the promoters of the genes under its control and / or their flanking regions, (ii) bind to it and (iii) initiate or promote transcription of the genes.

A transcription activator can be present as a protein either in the active (derepressed) or in the inactive (repressed) state. The activator only stimulates transcription and thus the expression of the regulated genes in the active state.

Examples of transcription activators in plants are described in K.D. Scharf, S. Rose, W. Zott, F. Schöffl and L. Nover in: The EMBO Journal, Volume 9, pages 4495 to 4501 (1990).

"Constitutively expressed and active" in the context of the transcriptional activator means that the activator is constantly, i.e. is expressed under normal conditions such as under stress conditions and is constantly present in a form which allows its binding to the control elements of the DNA.

"Heat shock factor (HSF)" is to be equated with the term "heat shock transcription activator", which binds to the heat shock elements (HSE) in promoter sequences in the active state via its DNA binding domain and thereby influences the transcription of the corresponding genes. The transcription activator is activated to its DNA-binding form, for example by multimerization, such as trimerization, of individual activator proteins.

A naturally occurring HSF in wild-type plants needs one to be activated by stress, e.g. Heat stress, generated signal.

Genes that are regulated by HSF are, for example, the genes for protective proteins, such as the heat shock proteins. "Protective proteins" as used herein are gene products of genes whose expression is regulated by HSF. The best known protective proteins include the heat shock proteins, which act as molecular chaperones in the cells and thereby protect other proteins or biochemical and physiological processes in the cell when exposed to stress. Protective proteins can have enzymatic activities that catalyze biochemical reactions and thus protect cells and organisms from stress-related damage (e.g. due to oxidative stress, for an overview see (CH Foyer, P. Descourvieres and KJ Kunert in: Plant, Cell and Environment, Volume 17 , Pages 507 to 523 (1994)), anaerobiose (Y. Yang, H.-B. Kwon, H.-P. Peng, M.- C. Shin in: Plant Physiology, volume 101, pages 209 to 216 (1993 )) regarding heavy metal toxicity (D. Neumann, O. Lichtenberger, D. Günther, K. Tschiersch and L. Nover in: Planta, Volume 194, pages 360 to 367 (1994)) Photoinhibition (G. Schuster, D. Even, K. Kloppstech and J. Ohad in: The EMBO Journal, Volume 7, pages 1 to 6 (1988)) and oxidative stress (P. Mehlen, J. Briolag, L. Smith, C. Diaz-Latoud, M. Fabre, D. Pauli, A.-P. Arrigo in: Eur. J. Biochemistry, Volume 215, pp. 277-284 (1993)), including the proteins of the HSP20 family.

"Stress factors (stressors)" are biotic or abiotic factors that can reduce the performance of a plant.

Such factors can be: pathogen infestation (viruses, viroids, fungi, bacteria, insects, nematodes), wounding, heat (especially during flower formation and during seed ripening), cold (especially during germination), UV radiation, high light intensity , high concentration of heavy metal, salt and / or ozone, acidity, dryness and 0 ₂ deficiency. "Wild type plant" herein means a plant which shows a normal stress tolerance and which provides the starting plant or plant cell for the process according to the invention.

"Fusion protein", as used herein, means a fusion protein consisting of a transcription activator and at least one further protein component which usually does not occur in the corresponding transcription activator. The fusion protein has the DNA-binding property of the transcription activator and, if appropriate, others The activator component of the fusion protein has the ability to bind DNA and regulate the transcription of the genes under its control, and it can carry the foreign protein component at its N-terminus, C-terminus and / or within the protein chain The foreign protein portion preferably comprises approximately 500-600 amino acids.

"Increased level of protective protein" means that the plant according to the invention with increased stress tolerance contains one or more protective proteins in a concentration which is higher than in the comparable wild type with normal stress tolerance. An elevated level is already present, for example, when the plant with increased stress tolerance under stress-free conditions reaches a concentration of protective proteins which corresponds to 5%, preferably approximately 15-20% or more, of the concentration of protective proteins under stress conditions.

"Normal conditions" as used herein means that no "stressors" as discussed above affect the plant. A transition from normal conditions to stress conditions is caused, for example, by an increase in temperature from approximately 20-25 ° C. (normal) to approximately 37 ° C. (stress). Stress reactions can also be triggered at temperatures lower and / or higher than 37 ° C. An important switching point for the regulation of the HS response, ie the HSP synthesis, is the transcription of the HSPs. The coordinated expression of the HSPs is brought about by the central regulator, ie the HS transcription factor (HSF). The HSF thus plays a key role in initiating the HS response. This function consists in recognizing heat shock promoters, binding them and stimulating the transcription of the HS genes (which code for HSP). This principle of regulation is preserved in all higher eukaryotes, including the plants (cf. FIG. 1). The central regulator protein HSF is constitutive (ie permanent), but it is inactive under normal or optimal growth conditions with regard to its ability to bind to DNA and to activate the transcription of the HS genes. The effect of stress on the plant is that the HSF is activated, ie it gains its ability to bind the DNA and transcription activation of the HS genes, and this in turn leads to the increased production of HSPs. The activation of HSF often correlates with a trimerization or multimerization of HSF monomers. The HSF trimers bind to conserved HS promoter elements (referred to as HSE which contain the consensus motif [nGAAnnTTCn] _n ). The transcripts of the HS genes generated in this way are then preferably translated during a heat shock and enriched in relatively large amounts, with the result that a high level of HSPs and other protective proteins is present in the plant under stress.

One possibility of influencing the HS response seemed to be the ectopic overexpression of HSF. An HSF gene under the control of a strong constitutive promoter should lead to overexpression of the HSF and consequently to an increased transcription of the HS genes with the result of an increased HSP level. As invented by the inventors Found surprisingly, this approach did not lead to overexpression of the HSPs.

In contrast, it was found within the scope of the present invention that an elevated level of protective proteins in a plant is obtained under normal conditions if the plant contains a transcription activator, preferably an HSF, which is constitutively expressed and constitutively in the active state, so that it is present Plant constitutively expressed at least one protective protein. Preferably, however, several protective proteins, such as the members of the HSP20 family, as well as HSP70 and superoxide dismutase, are constitutively expressed simultaneously in such a plant under the influence of the transcription activator. The constitutive expression of the protective proteins can even affect all protective proteins of the plant.

The increased stress tolerance of a plant manipulated in this way is based, without being bound to any theory, presumably on an increased concentration of one or more protective proteins even under normal conditions, so that protective proteins are already present in large quantities when a stress factor occurs. to immediately minimize the damage caused, for example, by denaturing proteins due to the stressful situation. Such a plant with increased stress tolerance already has a level of at least one protective protein under normal conditions, ie stress-free conditions, which makes up approximately 5% of the level of protective protein which is built up in a plant which is fully under stress. The plant according to the invention preferably already contains about 15 to 20% or more of the stress protein level of a plant under stress conditions under normal conditions, with very particular preference the members of the HSP20 family already reaching 20% of the maximum level under stress conditions under normal conditions. A so-called heat shock factor is preferably considered as the activator of the protective proteins. These transcription activators (transcription factors) usually bind to conserved elements within the HS promoters, which often have the consensus motif [nGAAnnTTCn] _n . An example of such a heat shock factor from plants is the AraJidopsis heat shock factor (cf. Hübel, A. and Schöffl, F. in Plant Molecular Biology, volume 26, pages 353 to 362 (1994). Further heat shock factors are known to the person skilled in the art well-known in the art, as described for example in KD Scharf, T. Materna, E. Treuter and L. Nover in: L. Nover (ed.) Plant Promotors and Transcription Factors, pages 121 to 158, Springer Verlag, Berlin-Heidelberg ( 1994).

As stated above, the transcription activator in wild type plants is either not at all present under normal conditions, or else in a form which does not allow DNA binding, in particular binding to HSE. In the context of the present invention, it could be shown that such an inactive transcription activator can be converted into a constitutively active transcription activator by manipulating its native, inactive structure, preferably by changing the protein sequence. The techniques for changing a given protein sequence are well known to those skilled in the art, as described, for example, in Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989). The modification of the inactive transcription activator takes place with the aim of giving the activator a constitutive DNA binding ability.

A preferred modification of an HSF to activate it is the fusion with another protein (foreign protein), the foreign protein preferably being fused to the N-terminus or C-terminus of the HSF. Suitable fusion partner for an HSF are, for example, β-glucuronidase, and further so-called reporter proteins, for example luciferase, structural or enzyme proteins, and proteins which are simultaneously suitable for selecting cells, tissues and plants with a modified HSF. Neomycin phosphotransferase II, phosphinothricin acetyltransferase and hygmycin phosphotransferase are particularly suitable for this. Furthermore, the HSF can also be modified by attaching any other, including synthetic, proteins.

Whether a modification of the inactive HSF has converted it into a constitutively active HSF can be determined simply by examining the modified HSF to determine whether it has acquired the ability to bind DNA under normal conditions through the modification. This ability to bind DNA can be determined using conventional methods known from the prior art, such as, for example, the gel retardation assays familiar to the person skilled in the art, as described in D.D. Mosser, N.G. Theodorakis and R.J. Morimoto in: Molecular & Cellular Biology, Volume 8, pages 4736 to 4744 (1988).

Plants from the Brassicaceae family, the Magnoliatae class and the Lilietae class are preferred as plants with increased stress tolerance, members of the genera Brassica, Beta, Solanum, Lycopersicum, Helianthus, Glycine, Zea, Hordeum, Triticum being particularly preferred , Seeale and Oryza.

The increased stress tolerance of the plants according to the invention is evident in their ability to maintain their full performance under the action of stress factors, but at least under given stress conditions they show less impairment of their full performance than the wild type plant from which the plant according to the invention originates has gone. The performance of a plant can be seen, for example, in its growth rate, the stability of the characteristics, seeds and fruit substance, the resistance to pathogens, the quality and the yield of the harvested products, the nutrient efficiency, the efficiency of the water balance and the tolerance towards it Pesticides.

The plants according to the invention preferably show an improved stress tolerance to at least one of the biotic stress factors, such as pathogen attack (for example viruses, viroids, fungi, bacteria, insects, nematodes), and / or abiotic stress factors, wounding, heat (in particular during flower formation and during fruit and seed ripeness), cold (especially during germination), UV radiation, high light intensities, high concentrations of heavy metals, salt and ozone, acidity, dryness, and 0 ₂ deficiency. Plants which have an increased tolerance to several of the stress factors mentioned are particularly preferred; they are very particularly preferably more tolerant to all stress factors.

Preferred plants according to the invention have an elevated level of at least one protective protein, but preferably several protective proteins, even under stress-free conditions, ie under conditions in which the plants are not exposed to one of the above-mentioned stress factors (normal conditions). The presence of an elevated level of protective protein can be determined simply by determining the content of protective proteins in a plant according to the invention and comparing this content with the protective protein content of the wild type plant. The protective proteins can be isolated and quantified from the plants using conventional methods known from the prior art. A nucleic acid which codes for the desired transcription activator is used as the vehicle for introducing a constitutive active transcription activator into the plant.

The nucleic acid is a DNA, for example in the form of a plasmid, which can be introduced into plants by known methods. For example, the suitable plasmid for this purpose is the Ti plasmid from Agrobacterium tumefaciens, or one of the known techniques for direct DNA transfer is used.

The nucleic acid introduced into the plant preferably codes for a constitutively active HSF, with a fusion protein consisting of an HSF and another protein or protein fragment being particularly preferred as the active form.

The introduced nucleic acid itself preferably contains the regulatory elements required for the expression of the desired transcription activator, the elements required for the expression in plants being known and described in the prior art. Alternatively, the introduced nucleic acid can also be brought under the control of regulatory elements already present in the plant cell. F. Schöffl, M. Rieping and G. Baumann in: Developmental Genetics, Volume 8, pages 365 to 374 (1987) describes the constitutive expression of an HS gene under the control of the CaMV-35S promoter in tobacco.

The methods for introducing a nucleic acid into a plant cell are well known to those skilled in the art. The regeneration of a complete plant from a plant cell, transformed with a nucleic acid, is also familiar to the person skilled in the art and can be carried out using conventional methods from the prior art. The plants according to the invention are produced by introducing a nucleic acid which codes for a constitutively active transcription activator into a plant cell and regenerating the transgenic plant from the plant cell transformed with the nucleic acid.

All measures for producing a transgenic plant are known from the prior art. Usually after a nucleic acid has been introduced into plant cells, selection is made for those plant cells which have taken up the nucleic acid and stably express it. A complete plant is then regenerated from these transformants using conventional methods. It is particularly advantageous if the selection marker required for the selection of the appropriately transformed plants is provided by the foreign protein which serves as a fusion partner of the HSF.

The plants of the invention are of great use in the field of agriculture, particularly where under difficult growing conditions, i.e. Plants must be grown under the influence of stress factors.

The following examples illustrate the invention. Most of the experiments were carried out with Arabidopsis thaliana, which is known to be a popular model system for crops. This model embodies all the important genetic, physiological, biochemical and molecular properties of higher plants. In particular, this plant represents a model system for the regulation of the HS response in crop plants. The heat shock factor from Arabidopsis, ATHSFl:

The heat shock factor is expressed constitutively, but its activation for DNA binding is strictly linked to heat induction (at 37 ° C), and activation requires the transition from monomers to trimers. ATHSFL is described in detail in Hübel, A. and Schöffl, F. in Plant Molecular Biology, Volume 26, pages 353 to 362 (1994).

Manufacture of constitutively active ATHSFl:

Two types of gene fusion products, HSF-GUS (HG) and GUS-HSF (GH), were made as shown in Figure 2A. The fusions were constitutively expressed using the CaMV 3SS promoter in transgenic Arabidopsis plants. It does not matter for the activity of the fusion product whether ATHSF1 was fused with the foreign protein at the N- or at the C-terminus. The construct used for the transformation was produced by subcloning ATHSF1 cDNA in the plant vector BIN19, which contains the glucoronidase (GUS) reporter gene under the control of the constitutive CAMV35S promoter, and the termination sequence of the nopaline synthase (NOS-ter). The ATHSF1 portion was 15 amino acids missing at the C-terminus in the HSF-GUS fusion and 5 amino acids at the N-terminus in the GUS-HSF fusion. In the GUS-HSF fusion, the GUS content is reduced by 5 amino acids at the C-terroinus.

Expression of the fusions in Arabidopsis

The GUS activities were determined for each construct in protein extracts from 30 different transformants (F0) and the Fl subsequent generation. GUS activity was measured in pMol NAD / mg protein / minute. Arabidopsis thaliana (ecotype Columbia) was treated with Agrobacterium tumefaciens, containing the vector plasmids derived from BIN19, with the help of Valvekens, M. van Montagu and M. van Lijsebettens in: Proceedings of the National Academy of Science, USA, Volume 85, Pages 5536 to 5540 (1988) described method transformed. F1 seeds were generated from self-transformed transformants and the GUS activities were determined in the leaf tissue protein extracts using the fluorescence method as described by RA Jefferson, TA Kavnagh and MW Bevan in: The EMBO Journal, Vol. 6, pages 3901 to 3907 (1987). The CIS activities found are shown in FIG. 1B.

Furthermore, the mRNA levels of HSF and the HSF fusions in the wild type as well as in the transgenic Arajbidopsis were determined. For this purpose, the total RNA was isolated from the leaf tissue of individual transformants and from wild type plants following or without heat stress (HS) at 37 ° C. for 3 hours. The RNAs were analyzed by Northern blot hybridization using 30 μg

Total RNA / lane and a 32P-labeled ATHSFl cDNA sample as described by Severin and Schöffl in K. Severin and F. Schöffl in: Plant Molecular Biology, volume 15, pages 827 to 833 (1990). The mRNA levels of the chimeric genes were significantly increased in comparison to the levels of the authentic ATHSF1 transcript, as can be seen from FIG. IC. These data show that the chimeric HSFs are stably expressed in the transgenic plants in addition to the endogenous wild-type ATHSF1.

Constitutive HSP synthesis

So far, 5 genes are known for the small HSP in Arabidopsis, three for class I (which also includes the HSP18 tested below), two for class II (see Vierling, E. see above). mRNAs are so well preserved within a family that they cross-react in hybridizations.

The constitutive expression of HSP18 in transgenic AraJbidopsiε that overexpress HSF fusion proteins is shown in FIG. 3.

For Western blot analysis, the proteins were isolated without or following an HS (cf. also FIG. 1) by rapidly homogenizing leaf tissue in buffers containing 8 M urea. Analysis samples (30 μg / lane) were subjected to SDS-PAGE and transferred to nitrocellulose using the electroblot method. HSP18 was detected by an antibody directed against a recombinant Arabidopsis HSP17.6. The gene sequence for HSP17.6 is described in K.W. Helm and E. Vierling in: Nucleic Acids Research, Volume 17, page 7995 (1989). The bands were visualized by incubation with mouse anti-chicken IgG to which alkaline phosphatase was coupled and staining with the nitrotetrazolium blue / 5-bromo-4-chloro-3-indolyl-phosphate system as described in E. Engvall and PJ Perlmann in: Journal of Immunology, Volume 109, page 129 (1972).

The Northern blot hybridizations were carried out as described in K. Severin and F. Schöffl in: Plant Molecular Biology, Volume 15, pages 827 to 833 (1990).

The result of the HSP expression studies shows that in the wild type (WT) at room temperature (RT) no HSPlδmRNA is detectable. Antisera directed against recombinant HSP18 from Arabidopsis can be used to detect constitutively expressed small HSPs in the transgenic plants. The antisera, although directed against an individual HSP, recognize all members of an HSP family because of the strong conservation of the HSP structure. There is a in the Western blot clear difference between WT (no expression) and transgenic plants at RT. The level of constitutively expressed HSP found in the transgenic plants at room temperature is approximately 15 to 20% of the amount of HSPs induced by HS (determined with the aid of immunodiffusion). This corresponds to approximately 1 to 2 ng HSP18 / μg total protein. After a heat shock, the amount of small HSP in the transgenic plants increases to the value that is also achieved in the heat-induced wild type (cf. FIG. 3).

The mRNA levels for HSP70, the most conserved of all protective proteins, are also strongly constitutively increased in HSF-GUS (GUS-HSF) transgenic plants compared to the uninduced wild type. This shows that the constitutive stress response is not only limited to the small HSP, but also includes HSP70 and other HSPs. In other words, the constitutively active HSF induces a range of HSPs.

Constitutive activity of the HSF fusion proteins and HSF interactions.

Depression of the HSF activity of the fusion proteins expressed in Arabidopsis (HSF-GUS and GUS-HSF) could be demonstrated by three experiments:

1. The HSF fusion proteins are constituted by trimerization.

2. The HSF fusion proteins bind constitutively to HSE sequences.

3. The heat shock proteins are expressed constitutively.

It was shown that at normal temperature (20 to 25 ° C), ie under stress-free conditions, the HSF fusion proteins are active are, but the naturally occurring HSF is inactive. Only after an HS can the activity of the naturally occurring ATHSFL be observed.

Transferability of the HSF activation from Arabidopsis to other plants

The HSF-GUS or GUS-HSF gene constructs described above allow heterologous expression in plants other than Arabidopsis. The fusion products from Arabidopsis lead to the constitutive expression of HSPs in heterologous plants.

Furthermore, in plants other than Arabidopsis, the corresponding HSFs can also be activated in a manner analogous to the activation in Arabidopsis described in detail above, so that the HSPs are constitutively expressed by the homologously constitutively active HSFs.

Constitutive expression of heat shock proteins in Hairv root crops and sugar beet plants

Media:

bacteria

Media are used to grow the bacteria, as described by Maniatis T. et al. in "Molecular cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press (1989). plants

The media used are derived from that of Murashige T. et al. in "A revised medium for rapid growth and bioassays with tobacco tissue cultures", Physiol. Plans. 15: 473-497 (1962) specified MS medium and by that of Gamborg O.L. et al. in the nutrient requirements of suspension cultures of soybean root cells ", Exp. Cell Res. 50: 151-158 (1968).

Tribes and vectors:

Aαrobacterium strains

LB 4404 and Ril5834-Wildstamm (both commercially available; for example Clontech Laboratories, Palo Alto, California, or the American Type Culture Collection under the deposit number ATCC 15834).

Plasmid

HSF1 / GUS and GUS / HSF2 (BIN19 derivatives; Bevan M., (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721).

Methods used:

All standard molecular biological methods - such as restriction analysis, plasmid isolation, mini-preparation of plasmid DNA, transformation of bacteria, etc. - were, unless otherwise stated, as described by Maniatis et al., "Molecular Cloning: A Laboratory Manual ", (1989).

Plant material

7-10 day old sugar beet plants of different genotypes were grown for transformation in vitro.

DNA transfer in aqua bacteria

a) Transformation

The plas id DNA was prepared using the method of P.J.J. Hooykaas and T. Mozo, "Agrobacterium molecular genetics" in Plant Molecular Biology, Manual B3: 1-9, (1994).

b) DNA analysis

The DNA transfer into the agrobacterium was checked by isolating bacterial DNA according to the method described by P.J.J. Hooykaas and T. Mozo, "Agrobacterium molecular genetics" in Plant Molecular Biology, Manual B3: 1-9, (1994). The restriction cleavage of the DNA, the transfer to nitrocellulose and the hybridization against the corresponding radioactive probe gave information about a successful DNA transfer into the agrobacteria.

Production of "Hairy Root" Cultures of Sugar Beet by Co-Transfer of T-DNAs from Agrobacterium tumefaciens and A. rhizogenic plasmids: Cultivation of the bacteria

Both strains of Agrobacteria required for infection were grown overnight in selective antibiotic medium (LB medium) at 27 ° C. After centrifuging and again taking up the sediment in 1/10 MS medium, the bacteria could be used for the infection.

Hvpokotyl explant infection

Sterile sugar beet seedlings of different genotypes, 7 to 10 days old, were used for the hypocotyl infection. The injured 1 cm long pieces of hypocotyl (only shoot pole) were briefly placed in an agrobacterial suspension (A. Tumefaciens and A.

₉ rhizogenes were mixed in a 1: 1 ratio, about 10 cells / ml) were immersed. The infected explants were kept on MSB medium (MS with 0.5 mg / 1 BAP and 30 mg / 1 kanamycin) for 4 weeks at 24 ° C. and steady light. The root tips of the induced roots were **. B5 medium transferred with 500 mg / 1 cefotaxime and 300 mg / 1 kanamycin. After about 8 weeks (subculture every 4 weeks) the roots could be subcultured in B5 liquid medium for further propagation.

Biochemical detection of the expression of the fusion protein

The X-Gluc assay was performed according to Jefferson RA, (1987) "Assaying Chimeric Genes in Plants: The GUS-Gene Fusion System", Plant Molecular Biology Reporter 5: 387-405. Detection of heat stress proteins (Western blot / ELISA.

The antibodies used were produced in rabbits (immunization with recombinant Arabidopsis thaliana heat shock factor ATHSP17.6). The heat stress proteins were extracted from plant tissue using a buffer containing 6 M urea. The extracts were separated using a 15% SDS-PAGE. In addition to the recombinant ATHSP17.6, heat-stressed plant tissue was used as a positive control. The heat stress was carried out in SIB buffer (1 mM NaP, 1% sucrose, pH 6.0) at 40 ° C. for 2 hours with shaking.

Transformation of sugar beet with AgrrojacteriuΛi tumefaciens:

Cultivation of the bacteria

The LB4404 agrobacterial strain required for infection was grown overnight in selective antibiotic medium (AB minimal medium) at 27 ° C. After centrifuging and again taking up the sediment in 1/10 MS medium, the bacteria could be used for infection.

Leaf explant infection

For the cotyledon infection 7 to 10 day old sterile sugar beet seedlings of different genotypes were used. The cotyledons injured at the basal end were incubated for approx. 4 min in an agrobacterial suspension (approx. 10 ⁹ cells / ml). The infected explants were kept on 1/10 MS medium for 3 to 4 days at 24 ° C. and a 16 hour day and then on MS medium with 0.5 mg / 1 BAP, 0.05 mg / 1 NAA, 500 mg / 1 carbenicillin and 300 mg / 1 kanamycin transferred. Until the end of the selection, subculturing was carried out every 2 to 3 weeks on the same medium. The transgenic shoots selected after a few weeks were propagated on MS with 0.5 mg / 1 BAP and 500 mg / 1 carbenicillin and then rooted on 4 MS with 5 mg / 1 IBA and 500 mg / 1 carbenicillin (16 hour day, 24 ° C).

The biochemical detection of the expression of the fusion protein and the detection of the heat stress proteins were carried out as indicated for the "Hairy Root" cultures.

The above experiments showed that the transgenic "hairy roots" of the sugar beet and the transgenic sugar beet plants constitutively expressed the heat stress proteins.

Constitutive expression of heat shock proteins in tobacco plants

The heterologous HSF-GUS expression effects a constitutive HSP synthesis:

The effect of constitutive heat shock protein synthesis, which is observed in Arabidopsis and is based on the transgenic Arabidopsis HSF-GUS, can also be demonstrated in transgenic tobacco plants. As a result, the HSF fusion protein from Arabidopsis is also effective in heterologous plants.

The constitutive expression of heat shock proteins in the tobacco plants increases the resistance of the plants to various stress conditions. Drought tolerance

Transgenic HSF-GUS tobacco plants show an increased drought tolerance. The drying out of cut leaves is significantly slowed down compared to WT, the differences in water loss are up to 20, sometimes even 30%, depending on the drying time and the temperature. Transgenic plants that were dried out to a water loss of 29.5% could be revitalized again; WT plants that lost 33.25% water in the same period (33 hours) could not be revitalized.

Tolerance to photo-oxidative stress

Photo-oxidative stress, generated by high temperature (37 ° C) and high light intensities (increased from 50 to 650 PFD), produced a significant difference (up to 30%) in the fluorescence, measured in, in transgenic tobacco plants compared to WT Fv / Fm; this difference is due to an increased Fo of the dark-adapted WT plants, which already occurs at time 0 (before the photo-oxidation stress). These results indicate that the photosynthetic reaction center is evidently stabilizing in transgenic plants and that non-photochemical quenching (disposal of radicals via enzymatic routes) may be enhanced.

Heavy metal tolerance

Transgenic plants in which part of the root has been cut off show a significantly weakened reaction to iron in Fe-EDTA solutions (200 to 400 μM). With identical treatment, WT plants show severe symptoms in the form of "oozing" and necrosis. Table 1

Biochemical and functional properties of HS proteins

HSP family ¹ properties

HSP 100 HSP 104 is important for thermotolerance in yeast associated with regulatory proteins in the cytoplasm

HSP90 ^b (inactive forms) in animal cells (hormone receptors, protein kinases etc.) the most conserved HSP, ATPase, reversible interaction with the nucleolus, the dissociation of

HSP70 ^b requires other (denatured) proteins ATP, possibly a negative regulator of the HS response, may possibly interact with HSF molecular "chaperone" for the correct arrangement of

HSP60 ^b multimeric protein complexes in mitochondria, chloroplasts, possibly cytoplasm

HS-dependent aggregation of this HSP in the cytoplasm,

HSP20 ^C Formation of granular bodies in plants, function as chaperone according to the mammalian HSP26 in vitro

8 kDa protein that is involved in the recognition of proteins for the

Ubiquitin is involved in proteolytic degradation

HSF ^b HS factor, positive regulator of the transcription of HS genes

a) Classified based on the molecular weight in kDa; there are different sizes in different organisms, but the structure and function is conserved within the families b) multiple isoforms; frequently heat-inducible proteins c) Multiple HSPs; HSP families in plants (the members are transported to the chloroplasts or to the ER); there is only one HSP in humans

Claims

claims

1. Plant with increased stress tolerance, containing at least one constitutively expressed and active transcription activator for the constitutive expression of at least one protective protein which is usually only induced by stress factors.

2. Plant according to claim 1, characterized in that the transcription activator is a heat shock factor (HSF).

3. Plant according to claim 2, characterized in that the transcription activator is a fusion protein from an HSF and another protein.

4. Plant according to one of claims 1 to 3, characterized gekenn¬ characterized in that the plant is selected from the family of the Brassicaceae, the class of Magnoliatae and the class of Liliatae.

5. Plant according to claim 4, characterized in that the plant is selected from the genera Brassica, Beta, Solanum, Lycopersicum, Helianthus, Glycine, Zea, Hordeum, Triticum, Seeale and Oryza.

6. Plant according to one of claims 1 to 5, characterized gekenn¬ characterized in that the plant shows increased stress tolerance compared to the stress factors pathogen attack, wounding, heat, cold, UV radiation, high light intensity, high concentration of heavy metal, salt and / or ozone , Acidity, dryness and / or 0 ₂ deficiency.

7. Plant according to one of claims 1 to 6, characterized gekenn¬ characterized in that the plant even under stress-free conditions gene has an elevated level of at least one protective protein.

8. Plant according to claim 7, characterized in that the level of several protective proteins is increased.

9. Nucleic acid which codes for a transcription activator constitutively active in a plant.

10. Nucleic acid according to claim 9, characterized in that the nucleic acid is a DNA.

11. Nucleic acid according to one of claims 9 or 10, characterized in that the encoded transcription activator is an HSF.

12. Nucleic acid according to one of claims 9 to 11, characterized in that the encoded transcription activator is a fusion protein of an HSF and another protein.

13. Nucleic acid according to one of claims 9 to 12, characterized in that it further comprises one or more regulatory elements for the expression of the coding sequence in a plant.

14. Plant cell containing a nucleic acid according to one of claims 9 to 13.

15. Plant which is obtainable from a plant cell according to claim 14.

16. A method for producing a plant with increased stress tolerance, comprising the steps: a) introducing a nucleic acid, which codes for a constitutively active transcription activator, into a plant cell, and

b) Regeneration of a transgenic plant from the plant cell produced in step a).

17. The method according to claim 16, characterized in that the nucleic acid is a DNA.

18. The method according to claim 16, characterized in that the nucleic acid is an RNA.

19. The method according to any one of claims 16 to 18, characterized in that the transcription activator is an HSF.

20. The method according to any one of claims 16 to 19, characterized in that the transcription activator is a fusion protein from an HSF and another protein.

21. The method according to claim 20, characterized in that the further protein simultaneously serves as a selection marker.