WO2012021913A1

WO2012021913A1 - Methods for increasing resistance of plants to drought, salt and pathogens

Info

Publication number: WO2012021913A1
Application number: PCT/AT2011/000349
Authority: WO
Inventors: Thomas Roitsch; Francisco Perez Alfocea; Alfonso A. Albacete Moreno
Original assignee: University Of Graz; Spanish National Research Council
Priority date: 2010-08-18
Filing date: 2011-08-18
Publication date: 2012-02-23

Abstract

The present invention is related to a method of increasing the resistance of a plant to stress, preferably drought or salt stress, and/or a method of increasing the fruit yield of a plant or generating a plant displaying a higher fruit yield, comprising the step of administering to the plant or a progenitor thereof, a nucleic acid molecule comprising a promoter functional in at least one plant species and an invertase from an organism, wherein said promoter is a fruit- specific promoter and said promoter differs from the endogenous promoter which controls the expression of said invertase in the genome of said organism, and wherein the promoter and the invertase are functionally linked to allow for expression of the invertase.

Description

METHODS FOR INCREASING RESISTANCE OF PLANTS TO DROUGHT, SALT AND PATHOGENS

The present invention is related to methods for increasing the resistance of plants to drought, salt and pathogens.

The perspective of global warming coinciding with a further increase of the human population is projected to have a significant impact on the demands imposed on contemporary agricultural production, in particular the need to produce increasing amounts of foodstuffs in an environment less-than-optimal for plant growth. Therefore, there is an urgent need to generate plants that are not only optimised in terms of traditional criteria such as the yield of fruit production, resistance to plant pathogens, physical stress and the like, but also tolerate elevated temperatures at reduced amounts of water available to support growth and development.

It is likely that this challenge cannot be met using traditional methods of plant breeding only. However, the tools provided by modern plant molecular biology and genetics will turn out an asset, as they provide not only the means to identify target molecules linked to useful traits such as resistance to drought, pathogens and other biotic and abiotic stress conditions, but also ways to optimise plant metabolism in a manner favourable to achieve the above goals.

A plant's ability to grow and thrive is inevitably linked, in terms of metabolism, to the ability to produce and consume carbohydrates in a tissue-specific manner. More specifically, in higher plants, growth and metabolism of sink tissues is sustained by the carbohydrates synthesized in source leaves and transported, mainly in the form of sucrose, through the phloem into the sink tissues. Source-sink relationships have been shown to change with plant growth and development and in response to different biotic and abiotic stresses. The use of sucrose in the sink tissues requires cleavage of the glycosidic bond, catalysed both by sucrose synthase and invertases. Sucrose synthase cleaves sucrose into UDP-glucose and fructose, whereas invertases hydrolyse sucrose into the hexose monomers.

Three types of invertase isoenzymes are distinguished based solubility, sub-cellular localization, pH optima and isoelectric point (Roitsch and Gonzalez, 2004). Between them, cell-wall bound invertases have been shown to play a crucial function in carbohydrate partitioning and supply of photoassimilates to sink tissues (Tang et al., 1999; Goetz et al., 2001 ; Weschke et al., 2003). Cleavage of sucrose at the site of phloem unloading and transport of the generated hexoses into the sink cells, through the concerted action of cell-wall invertases and hexose transporters, generates differences in osmotic pressure that drive the transport of sucrose in the phloem. An apoplasmic unloading of sucrose is not only characteristic of symplasmically isolated tissues but also of actively growing tissues, like maize primary root tips, where the demand of photoassimilates cannot be satisfied solely by the symplasmic unloading (Bret-Harte and Silk, 1994). The high invertase activity reported in root tips and site of emergency of secondary roots supports a role of cell-wall invertase in active growth of this sink tissue (Eschrich, 1980). In Arabidopsis thaliana, the expression pattern of cell-wall and vacuolar invertases in the root during development and in response to different culture conditions suggests that cell wall invertase is involved in sucrose partitioning in conditions with a high assimilated demands in this tissue. In mature roots, however, cell wall invertase expression is not detected and vacuolar invertase expression would be responsible for sucrose incoming (Tymowska-Lalanne and Kreis, 1998). Phloem unloading in mature roots would then fit to the model proposed by Sturm et al. (1995), where the driving force for sucrose unload results from the cleavage of the sugar by sucrose synthase and vacuolar invertase in the cytosol. In carrot tap roots, the effect of antisense inhibition of vacuolar and cell wall invertases on plant phenotype suggests an important role in sucrose partitioning (Tang et al., 1999). In addition, vacuolar invertase may be a key regulator of cell expansion, due to the doubled osmotic potential generated by sucrose cleavage in the vacuole.

Therefore, one of the main roles of invertases is that of an enzyme that provides metabolites essential for suitable growth conditions. However, several documents of the prior art also discuss invertases in the context of stress conditions. Irrespective of this, it is generally understood in the art that for increasing the resistance of plants to stress conditions such as drought and salt stress constructs which are suitable to increase the content of osmotically active compounds or of ion transporters, are the means of choice to increase the resistance of plants.

Therefore, the problem underlying the present invention is to identify methods for increasing resistance of plants to drought, salt and pathogens.

The problem underlying the present invention is, among others, solved in a first aspect, which is also the first embodiment of the first aspect, by a method of increasing the resistance of a plant to stress, preferably drought or salt stress, and/or a method of increasing the fruit yield of a plant or generating a plant displaying a higher fruit yield, comprising the step of administering to the plant or a progenitor thereof, a nucleic acid molecule comprising a promoter functional in at least one plant species and an invertase from an organism, wherein said promoter is a fruit-specific promoter and said promoter differs from the endogenous promoter which controls the expression of said invertase in the genome of said organism, and wherein the promoter and the invertase are functionally linked to allow for expression of the invertase.

In a second embodiment which is also an embodiment of the first aspect the invertase is a plant-derived invertase, preferably an extracellular plant invertase.

In a third embodiment which is also an embodiment of the first and the second embodiment of the first aspect, the promoter is selected from a species of the family Solanaceae and is preferably selected from a tobacco species or a tomato species.

In a fourth embodiment which is also an embodiment of the first, second and third embodiment of the first aspect, the fruit-specific promoter is selected from the group comprising the fruit-specific promoter of vacuolar invertase InvLp6 from Lycopersocon piminellifolium, the E8 promoter, the 2A1 1 promoter, the mripl promoter, the TFP promoter, the TPRP-F1 promoter, and the asr promoter.

In a fifth embodiment which is also an embodiment of the first to fifth embodiment of the first aspect, the promoter is the fruit-specific promoter of vacuolar invertase InvLp6 from Lycopersocon piminellifolium and the invertase is the extracellular invertase CINl from Chenopdium rubrum.

In a sixth embodiment which is also an embodiment of the first to fifth embodiment of the first aspect, the nucleic acid molecule is one which hybridises, preferably under stringent conditions, to a nucleic acid molecule which is complementary to the nucleic acid molecule as defined in any one of said first to fifth embodiments of the first aspect.

In a seventh embodiment which is also an embodiment of the first to sixth embodiment of the first aspect, the plant is a species from the family of Solanaceae, the familiy of Brassicaceae or the family of Poaceae.

The problem underlying the present invention is, among others, solved in a second aspect, which is also the first embodiment of the second, by a nucleic acid molecule in accordance with the second aspect and its various embodiments. The problem underlying the present invention is, among others, solved in a third aspect, which is also the first embodiment of the third aspect by a vector comprising a nucleic acid molecule in accordance with the second aspect and its various embodiments.

In a preferred embodiment, the vector is a plant expression vector or a plant-specific virus.

The problem underlying the present invention is, among others, solved in a fourth aspect, which is also the first embodiment of the fourth aspect, by a cell comprising a nucleic acid molecule in accordance with the second aspect and its various embodiments and/or a vector in accordance with the third and its various embodiments.

In a preferred embodiment, the cell is a plant cell.

The problem underlying the present invention is, among others, solved in a fifth aspect, which is also the first embodiment of the fifth aspect, by a plant, comprising a nucleic acid molecule in accordance with the second aspect and its various embodiments and/or a vector in accordance with the third aspect and its various embodiments and/or a cell in accordance with the fourth aspect and its various embodiments.

In a second embodiment of the fifth aspect which is also an embodiment of the first embodiment of the fifth aspect the organism is a plant species selected from the family of Solanaceae, the family of Brassicaceae or the family of Poaceae.

The problem underlying the present invention is, among others, solved in a sixth aspect, which is also the first embodiment of the sixth aspect, by the use of a nucleic acid molecule in accordance wit the second embodiment and its various embodiments and/or a vector in accordance with the fourth aspect and its various embodiments and/or a cell in accordance with the fifth aspect and its various embodiments for the generation of a transgenic plant.

In a second embodiment of the sixth aspect which is also an embodiment of the first embodiment of the sixth aspect the transgenic plant is resistant to drought and salt stress.

Additionally, the present inventors have found that a nucleic acid molecule comprising a promoter functional in at least one plant species and an invertase from an organism, wherein said promoter differs from the endogenous promoter which controls the expression of said invertase in the genome of said organism, and wherein the promoter and the invertase are functionally or operably linked to allow for expression of the invertase, is useful in a method of increasing the fruit yield of a plant or generating a plant displaying a higher fruit yield. In a preferred embodiment such method is a method according to the present invention. Insofar, also this nucleic acid molecule represents a further aspect of the instant application.

In an embodiment of the nucleic acid molecules in its diverse forms as subject to the instant application, the promoter is a fruit-specific promoter.

In an embodiment of the nucleic acid molecules in its diverse forms as subject to the instant application, the promoter and the invertase are from the same plant species. Alternative, in a further embodiment, the promoter and the invertase are from different species.

In an embodiment of the nucleic acid molecules in its diverse forms as subject to the instant application, the invertase is a one of an organism, whereby the organism is selected from the group comprising plants, animals, bacteria and fungi. In a preferred embodiment the invertase is a plant-derived invertase, preferably an extracellular plant invertase.

In an embodiment of the nucleic acid molecules in its diverse forms as subject to the instant application, the promoter is selected from a species of the family Solanaceae, Brassicacea or Poaceae. In a preferred embodiment, the family is Solanaceae and the species is selected from a tobacco species or a tomato species.

In an embodiment of the nucleic acid molecules in its diverse forms as subject to the instant application, the promoter is a fruit-specific promoter. Preferably such fruit-specific promoter is one selected from the group comprising the fruit-specific promoter of vacuolar invertase InvLp6 from Lycopersocon piminellifolium, the E8 promoter, the 2A1 1 promoter, the mripl promoter, the TFP promoter, the TPRP-F1 promoter, and the asr promoter. In a preferred embodiment, the promoter is the fruit-specific promoter of vacuolar invertase InvLp6 from Lycopersocon piminellifolium and the invertase is the extracellular invertase CINl from Chenopdium rubrum.

A further aspect of the present invention is related to a vector which comprises a nucleic acid molecule as described herein and in particular a nucleic acid molecule according to the present invention. Preferably, such vector is a plant expression vector or a plant-specific virus comprising said nucleic acid molecule.

A further aspect of the present invention is related to a cell which comprises a nucleic acid molecule as described herein and in particular a nucleic acid molecule according to the present invention, or a vector according to the present invention.. Preferably, such cell is a plant cell comprising said nucleic acid molecule.

A further aspect of the present invention is related to a plant which comprises a nucleic acid molecule as described herein and in particular a nucleic acid molecule according to the present invention. Preferably, such plant is a a plant species selected from the family of Solanaceae, the family of Bransicaceae or the family of Poaceae. More preferably such plant is a transgenic plant.

The present inventors have surprisingly found that the activity of invertase is a suitable target for solving the problems underlying the invention. Moreover, the present inventors have surprisingly found that increasing the activity of invertase by way of expression of a fusion construct comprising a species-specific promoter operably linked to an invertase leads to an increase of the plant's resistance to stress conditions such as drought and increased salt concentrations. Even more surprisingly, the inventors have found that this approach leads to satisfactory results if said plant is a species other than the species from which the species- specific promoter has been taken. A particular surprising effect can be achieved in case the promoter is a fruit-specific promoter.

Without wishing to be bound by any theory, the present inventors assume that elevated levels of invertase are taken by plant cells as an indication that stress conditions are to be expected, allowing for an early adaptation in anticipation of such stress conditions. Insofar, the technical teachings of the present invention is to increase the activity of an invertase in a plant, preferably by expressing in said plant a construct comprising an invertase under the control of a suitable promoter. However, there are many other ways that can be used to increase the invertase activity in a plant, for example the application to the plant of an activator molecule or the introduction of more than one copy of the regular invertase under the regular invertase promoter of the plant. Analyses of the drought and salt stress transgenic plants revealed that they are characterized by elevated levels of plant hormones that belong to the class of cytokinins. This finding indicates that the increase in invertase activity results in atemporally and spatially specific increase in cytokinins that are involved in mediating the stress resistance caused by the increased invertase activity.

A preferred approach when practicing the teachings of the present invention involves the design and use of suitable nucleic acid sequences that relate to invertase in order to influence the levels of invertase in a plant of interest. This may be achieved in various ways which all require the person practicing the invention to carefully design and provide specific nucleic acid constructs.

In a preferred embodiment, a nucleic acid molecule comprising a promoter functional in at least one plant species, as used herein, refers to a nucleic acid sequence coding for an invertase of an organism and also comprising, preferably on the 5' side or upstream of the coding sequence, a promoter functional in a plant species. In a preferred embodiment, the term "promoter functional in at least one plant species", as used herein, is a promoter that, if present in a cell of said plant species or any environment that comprises a functional transcription and/or translation machinery of said plant species, controls the expression of a nucleic acid, i.e. directs the components of the transcription/translation machinery to the nucleic acid such that the latter is transcribed to produced mRNA, which, in turn, is adequately processed and finally translated by a ribosome to produce a protein. The person skilled in the art is able to identify and obtain suitable promoters and invertase sequences and to construct nucleic acid sequences comprising promoters and invertase sequences using standard molecular biology techniques and/or methods for the chemical synthesis of nucleic acid sequences.

In a preferred embodiment, the term "invertase", as used herein, refers to an enzyme that has invertase activity. In a preferred embodiment, as used herein, this term refers both to the nucleic acid sequence encoding the enzyme, the translated protein or its activity, respectively. In an embodiment, the activity of an invertase is defined as the ability to catalyse the hydrolysis of sucrose into the hexose monomers. Respective assay systems for measuring the activity of invertases are known to the ones skilled in the art, and, for example, described in Roitsch et al. (1995) (Roitsch T., Bittner M., Godt D.E. ( 1995).

Basically, the invertase assay is performed in an embodiment as follows. A soluble protein extract is obtained by homogenisation of the tissue in a homogenisation buffer. An insoluble protein fraction is obtained by shaking the insoluble pellet in high salt buffer overnight. After dialysis of these fractions, vacuolar, neutral and extracellular invertase activity in the corresponding fractions are measured by determining the amount of glucose released in a reaction with sucrose as a substrate and at the corresponding pH by use of a buffer. Glucose released is measured by use of a coupled assay with glucose oxidase and peroxidase enzymatic activities. The concentration of glucose released in the reaction is calculated from the OD value by use of a calibration curve. In all cases, control reactions using the same volume of water instead of sucrose in the reaction mixture are performed. Invertase activity for each sample is preferably determined in triplicate and normalised to the concentration of protein in the assay determined by the Bradford method (1976) with the Bio Rad kit.

In a preferred embodiment, the term "endogenous promoter", which controls the expression of an invertase in an organism, as used herein, refers to the promoter that controls expression of the invertase in the genetically unmodified organism from which the invertase has been taken, for example the wild type species. In other words, the endogenous promoter can be found by isolating genomic nucleic acid of the organism and sequencing, starting in 5' or upstream direction from the coding invertase. In line, in a preferred embodiment, a promoter that "differs from the endogenous promoter which controls the expression of said invertase in the organism", as used herein, refers, by contrast, to a heterologous promoter, which controls the expression of an invertase if the nucleic acid sequences of the heterologous promoter is fused to the 5' end of the nucleic acid sequence of the invertase in a suitable manner. In other words, the promoter that differs from the endogenous promoter cannot be found by isolating genomic nucleic acid of the organism and sequencing, starting in 5' or upstream direction from the coding invertase, but will be present in association and controlling the invertase- coding sequence only in artificial nucleic acid constructs.

The organism from which the invertase has been taken is of minor importance, as long as the enzyme is translated to yield an active protein. In a preferred embodiment, the organism is the plant species; in other words, the source species of the promoter is also the source of the invertase. This is a particularly useful approach if promoters of the species, from which the invertase has been taken, are available that allow for a stronger and/or inducible expression as the existence and - possibly - availability of all suitable elements for transcription and translation is ensured. Moreover, the use of an inducible promoter allows the conduction of pilot experiments using various amounts of inductor in order to titrate the expression and to identify optimal expression levels. Therefore, in a preferred embodiment, the promoter is from the organism from which the invertase has been taken and allows for the altered or inducible expression of invertase, preferably at levels higher than those allowed for by the endogenous promoter of the invertase. In another preferred embodiment, the organism from which the invertase is taken is a second plant species, preferably Chenopodium rubrum or any other dicot or dicotyledonous plant, or any other plant.

In a preferred embodiment, the invertase is selected from the group comprising extracellular invertase CINl from Chenopdium rubrum or any other dicot plant, or any other plant. In a preferred embodiment, the invertase is the extracellular invertase CEN1 from Chenopdium rub rum.

However, invertases are wide spread and an invertase to be used for a nucleic acid sequence within the invention may in principle be taken from a variety of organisms, not only plants. In a preferred embodiment, the invertase is from an organism selected from the group comprising bacteria and fungi.

The invertases in accordance with the present invention are defined by their nucleic acid sequences as disclosed herein. The invertases in general have the following amino acid sequence in their catalytic center: cystein (C) - proline (P) - asparagine (D) which, at the nucleic acid level, corresponds to TGT/C - CCT/C/A/G - GAT/C in case of cell wall invertases, and cysteine (C) - valine (v) - asparagines (D), or at the nucleic acid level, TGT/C, GTT/C/A/G - GAT/C for vacuolar invertases.

In principle, the invertase within the invention need not be an enzyme specifically designated in the prior art as an invertase. Many enzymes are promiscuous, i.e. one enzyme shows more than one activity, for example the hydrolysis of peptide and ester bonds. In a preferred embodiment, the invertase is an enzyme having more than one activities, one of them, not necessarily the most prominent one, being invertase activity. Also, in principle, artificial invertases may be created by grafting active residues of an invertase catalytic centre on the scaffold of a protein that is not an invertase or not even an enzyme. Preferably, a protein structurally homologous to an invertase, i.e. having a similar three dimensional fold, is used as the scaffold. This way, an enzymatically active invertase may be generated that has virtually no detectable nucleic acid and or amino acid sequence homology to any known invertase except for the motif comprising the catalytically active residues. The person skilled in the art knows how to acquire and compare structural information, for example using X-ray crystallography, Nuclear Magnetic Resonance spectroscopy, structural modelling and suitable data bases, for example the DALI server (Holm, L., and Sander, C. Mapping the protein universe. Science 273, 595-603 (1996). Therefore, in a preferred embodiment, the invertase is a protein and or nucleic acid of synthetic origin, preferably generated by modifying a protein scaffold such that an invertase catalytic centre is present.

The person skilled in the art is aware that many different promoters exist that can be used, depending on the individual application envisioned. In particular, promoters may be constitutional promoters, i.e. they are able to effect a constant level of expression of the coding nucleic acids controlled by them, as in the case of "house-keeping genes", i.e. essential genes that are constantly expressed. Alternatively, promoters may regulate expression differentially, meaning that expression is controlled depending on the conditions, for example the presence of transcriptional activators or repressors. In a preferred embodiment of the invention, the promoter is a constitutional promoter. If, for example, a plant is to be grown in an environment where the plant would be constantly exposed to a condition detrimentally affecting growth, development and/or well-being of the plant such as constant shortage of water, and expression of the nucleic acid under the control of the promoter strengthens the plant, it makes sense to choose a constitutional promoter as there is no need for the plant to adjust expression. However, if the plant is only temporarily exposed to such a condition, for example, the shortage of water is only temporary and possibly predictable, it makes sense to choose a promoter that is able to regulate expression differentially. This way, the promoter may allow for expression only under such a detrimental condition, meaning that under different conditions the plant is able to save the energy and resources required for the expression of the coding nucleic acid sequence under the control of the promoter. Therefore, in another preferred embodiment, the promoter may be a promoter that regulates expression differentially.

Moreover, promoters may or may not be functional in a group of species only or may or may not be species-specific, and the person skilled in the art is able to test, using routine experimentation, the specificity of a promoter. A species-specific promoter may be chosen if the expression of the invertase nucleic acid sequence is to be restricted to a species. This situation may arise, for example, if one species is more susceptible to less-than-optimal growth conditions to be overcome than other species that are present. Alternatively, it may be useful to restrict the expression of the invertase to the species of interest, for example a crop of agricultural value, whereas the expression in unwanted species, for example weeds, is not supported in order to select for the growth and thriving of the species of interest and to discourage the growth of weeds. Therefore, in a preferred embodiment, the promoter may be species-specific. In another preferred embodiment, the promoter may be specific for a plant species. Other promoters are widely recognised and functional in a broad range of species, and it could make sense to use such a promoter, for example if a given invertase is to be expressed, as part of large trial, in a variety of plant species, in order to dispense the need to design and clone a construct for each and every species to be tested. Therefore, in another preferred embodiment, the promoter may be species-unspecific. Besides, the specificity of a promoter may also relate to the location. A promoter may be tissue-specific, i.e. allows for different levels of expression in different tissues, for example fruit, leaves, roots and the like. For example, it could make sense to use a promoter that is fruit-specific in order to specifically increase the fruit yield of the plant of interest. Alternatively, it may make sense to use a promoter specific for those tissues that are particularly susceptible to a specific detrimental growth condition, for example a sink tissue that needs to be provided with energy-rich compounds by other tissues, such as, for example, fruit, leaves, roots. In a preferred embodiment, the promoter is a fruit-specific promoter. In another preferred embodiment, the promoter is specific for a sink tissue.

As preferably used herein a fruit-specific promoter is a promoter the activity of which may be detected in fruits. Such detection may be accomplished by methods well known to a person skilled in the art such as, but not limited to, Northern blots, RT-PCR, DNA micro-analysis of total RNA from fruit tissue. It is to be acknowledged that in an embodiment, the activity of the fruit-specific promoter is not limited to fruit tissue, but may, in principle, also be detected in (an) other tissue(s), preferably other plan tissue(s). In connection with the term fruit- specific promoter it will be acknowledged by the person skilled in the art that when used in a heterologous system, i.e. a system where such promoter does not naturally occur but preferably upon having been inserted into such system by means of genetic engineering, or a different plant species than the one where it is naturally occurring, may show different activity. Such different activity may be indicated by a different expression level, differences in the tissues and cells, respectively, where said promoter is expressed, the time and developmental stage when the promoter is active or not active, and the like.

The promoter to be used to control the invertase coding sequence may be taken from a variety of sources as long as it is functional in a plant species. In an embodiment, the promoter is taken from a member of the family Solanaceae. In another embodiment, the promoter is taken from a tobacco species. In another embodiment, the promoter is taken from a tomato species. In an embodiment, the promoter is the fruit-specific tomato promoter of the vacuolar invertase InvLp6 from Lycopersocon piminellifolium. In another embodiment, the promoter is a promoter that is taken from a species that differs from the members of the family Solanaceae. In another embodiment, the promoter is taken from a member of the family Solanaceae that differs from the tomato species of said family. In another embodiment, the promoter is taken from a member of the family Solanaceae that differs from the tobacco species of said family. In an embodiment, the promoter is selected from the list comprising the fruit-specific tomato promoter of the vacuolar invertase InvLp6 (=PFBRU), the promoters E8 (He et al., 2008), 2A1 1 (Amemiya et al (2006), mripl (Burger et al (2006), TFP Shen et al (2004), TPRP-F1 (Carmi et al. (2003), asr (Hong et al. 2002). Other fruit-specific promoters are known to the one skilled in the art.

In another embodiment, the promoter is from a plant-specific virus. In another embodiment, the promoter is from a plant-specific bacterium.

In terms of the design and use of suitable nucleic acid sequences that relate to invertase to be used in order to influence the levels of invertase in a plant of interest, numerous combinations of invertases and promoters may be used. In fact all possible combinations of promoters mentioned herein and all invertases mentioned herein may be used. In a preferred embodiment, the promoter is PBFRU and the invertase is Cinl . In another preferred embodiment, the promoter is InvLp6 and the invertase is Cinl. In another preferred embodiment, the promoter is InLp6 and the invertase is from a plant that is a member of the family Solanaceae but differs from a tomato species. In another preferred embodiment, the promoter is InLp6 and the invertase is from a plant that is a member of the family Solanaceae but differs from a tobacco species. In another preferred embodiment, the promoter is InLp6 and the invertase is from a bacterium. In another preferred embodiment, the promoter is InLp6 and the invertase is from a fungus.

Care should be taken to ensure that a functional construct is used when practicing the present invention. In a preferred embodiment, the term "the promoter and the invertase are operably linked to allow for expression", as used herein, means that the promoter and the invertase are not only associated as part of a single nucleic acid molecule, but are also arranged such that the cellular transcription/translation machinery recognises the combination of them. In particular, the promoter needs to direct an RNA polymerase to the coding nucleic acid sequence such that the correct transcription product is synthesised which, in turn and following adequate processing, is translated by a ribosome to yield the functional protein. The person skilled in the art knows about the design of functional expression of a coding nucleic acid sequence to be controlled by a heterologous promoter.

Within the invention is a nucleic acid molecule which hybridises, preferably under stringent conditions, to the nucleic acid sequence of any of the constructs mentioned herein is within the invention. Such stringend conditions are, for example, described in Sambrook J., Fritsch E.F. and Maniatis T. ( 1989). Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor New York.

Furthermore, it is within the present invention that the invention is related to a nucleic molecule which, but for the degeneracy of the genetic code, would hybridise, preferably under stringent conditions, to the nucleic acid molecules disclosed herein, each preferably coding for an invertase and/or a promoter.

Furthermore, it is within the present invention that the invention is related to a polypeptide or peptide encoded by any nucleic acid molecule or parts thereof that is within the invention.

It is also within the present invention that the respective nucleic acid molecules comprising a promoter and an invertase sequence according to the present invention, are cloned into a vector. Preferably such vector is a plant vector or a plant expression vector. For the purpose of producing any polypeptide encoded by the nucleic acids according to the present invention or those described herein an expression vector is used, whereby such expression vector is a viral, microbial, plant or animal vector, preferably a plant vector. It is also within the present invention that such vector is inserted into a cell, whereby such cell is preferably a plant cell. It is also within the present invention that the plant cell is grown into a mature plant. In a further embodiment the cell and the mature plant generate a seed containing such vector or a cell containing such vector. Preferably the seed and/or the plant is a hybrid plant which is preferably not capable of being propagated by common biological means, i. e. crossing and propagation. It is also within the present invention that the nucleic acid or a vector or cell comprising the nucleic acid is in a tissue or organ or organism. In a preferred embodiment, the organism is a plant. In another preferred embodiment, the organism is a bacterium. In another preferred embodiment, the organism is a plant-specific virus.

Methods for the introduction of a nucleic acid construct and vector, respectively, into a plant cell, which is preferably an embryonic plant cell, are known to the one skilled in the art and, for example, described in Clough S.J. and Bent A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743. Preferred transformations methods are Agro, particle bombardment, and floral dip. More preferably, Arabidopsis plants are transformed by floral dip according to the method of Clough and Bent (1998). Plants are grown under long days until flowering and clipped to encourage proliferation of secondary bolts. An Agrobacterium strain, carrying the construct in a binary vector, is grown in a large culture in YEB at 28°C. Subsequently, the Agrobacterium is centrifuged and resuspended to a

in 5% sucrose solution. Silwett is added to a concentration of 0,05% and flowers are dipped by inmersion in the solution for 2-3 seconds. Dipped plants are covered with Saran wrap during 24 hours, and in darkness, to maintain high humidity. Subsequently, they are uncovered and grow normally. Watering is stopped as seeds become mature and seeds are selected in antibiotic-containing plates.

Also, seeds derived from such a transgenic plant or plant cell are within the present invention.

In a further aspect of the present invention, the nucleic acids, vectors, cells and plants as well as other organisms containing such vectors encoding for any of the invertases as disclosed herein, are used for the production of the respective invertase. For such purpose, the respective cells expressing the nucleic acid coding for the invertase is cultivated in an appropriate reaction vessel containing an appropriate medium and subsequently the invertase is isolated and/or purified. Such cultivation and isolation/purification methods are known to the one skilled in the art.

In a still further aspect the present invention is related to the use of any of the invertases as disclosed herein, as a target molecule. A target molecule as used herein is a molecule which is either targeted in vivo or in vitro or in silico. Such targeting can, in a preferred embodiment, mean that the invertase is subjected to a screening process, preferably an in vitro screening process or an in silico screening process. In a preferred embodiment, the goal of such a screening process is to identify activators of the invertase activity.

In an in silico screening, the target molecule, i. e. any of the invertases as described herein, is used as a molecule against which the fit of other molecules is tested or other molecules are designed by means of computational analysis and design so as to fit to the target molecule preferably such as to inhibit or promote the activity of the target molecule. Preferably such fitting is related to the active center of the invertase. The thus identified compound, either identified by in vitro and/or by in silico screening can ultimately be used in connection with the methods disclosed herein. The thus identified compound can thus be a plant growth promoter or a plant protective agent, particularly in case such compound is actually decreasing the activity of an invertase, more preferably a root invertase and its activity respectively, in accordance with the present invention.

In connection with the in vitro screening process, the target molecule as such is provided and one or several compounds, preferably taken from a library, are tested typically by contacting the compound with the target molecule, whether or not any of the compounds have an impact on the target, preferably whether or not there is an increase or decrease in the activity of the target, i. e. the invertase, activity. As the target molecule is an invertase, assays are known to the one skilled in the art to evaluate whether a compound, also referred to herein as a candidate compound, has an activating effect on the invertase. Such molecules can further be used as a candidate, lead or compound for the manufacture of an agrochemical product. Preferably such agrochemical product is an agrochemical suitable to increase the drought and/or salt resistance or to increase the fruit yield.

The reagents and organism mentioned herein, in particular the nucleic acid constructs comprising invertase and promoter, may be used to engineer plants with useful properties, for example resistance to detrimental factors, to be cultivated on a large scale basis for the purpose of agricultural production. In a preferred embodiment, the term "resistance" to a detrimental factor, for example the presence of a pathogen, as used herein, refers to the ability of a plant to tolerate the harmful effects imposed by said factor better than a plant lacking said ability would. For example, a plant showing a resistance to a pathogen may be able to fight, tolerate and/or survive infection by a pathogen, at least better than a plant that does not display said resistance. In a preferred embodiment, the term "tolerance", as used herein, is used synonymously with the term resistance with respect to a detrimental. For example, the terms "drought tolerance" or "resistance to drought" refer to the ability of a plant to tolerate a shortage of water better than a plant that lacks said ability. In a preferred embodiment, the term„drought", as used herein, refers to a situation wherein the amount of water available to support the growth, development and/or well-being of a plant is reduced compared to the amount of water previously available. In a preferred embodiment, the amount of water is reduced such that it is a limiting factor with respect to the growth, development and/or well- being of a plant. In a preferred embodiment, the term "drought" may comprise repeated cycles of reduced and standard amounts of water available to support the growth, development and/or well-being of a plant. In another preferred embodiment of the invention, the term "drought" as used herein, means that the plant has at its disposal less water than the amount that would be optimal to support growth and development of said plant. This tolerance may be reflected by the ability to remain viable or alive under prolonged periods in the presence of the detrimental factor. For example, a plant showing drought tolerance may remain viable with no watering for a period longer than a plant lacking this trait would. In a preferred embodiment, a plant showing drought resistance or tolerance is able to tolerate a lack of watering water for 1 , 2, 4, 7 or 11 days. In a preferred embodiment, the term "hydric stress" as used herein, is synonymous with "drought".

In a preferred embodiment, the term "salt", as used herein, refers to any inorganic or organic salt the plant may be exposed to as a result of the presence of said salt in the substrate used to grow said plant or in the water used to water said plant. In a preferred embodiment, the salt is an inorganic salt. In a more preferred embodiment, the salt is sodium chloride.

In a preferred embodiment, the term "salt tolerance", as used herein, refers to the ability of a plant to tolerate salt concentrations higher than those optimal in terms of growth, development and/or well-being of said plant better than a plant lacking said ability would. Salt tolerance refers to the ability to grow or remain viable under conditions comprising watering using water comprising, in an embodiment, 1 to 1000 mM sodium chloride, in a preferred embodiment, 25 to 500 mM sodium chloride, in a more preferred embodiment, 50 to 300 mM sodium chloride and, in an even more preferred embodiment, 60 to 200 mM sodium chloride and, in a most preferred embodiment, 75 mM sodium chloride.

In a preferred embodiment, an increase in the fruit yield is reflected by the fresh weight of selected fruits in g or the average fresh fruit weight per plant in g orthe fruit yield per plant in g, each in comparison to a standard plant, preferably a wild type plant.

Furthermore, the use of any of the nucleic acid sequences, cells, organs, organisms and tissues mentioned herein for the modification of a plant genome, preferably to increase the invertase activity, is within the invention.

Moreover, the activity of invertase may be increased in a plant by multiplying the copies of the endogenous combination of promoter and construct, for example by introducing additional copies and effecting their transient expression or even insertion into the genome for permanent expression. The underlying rationale is that multiple copies increase the frequency of expression using the endogenous transcription and translation machinery without having to resort to heterologous invertases and/or promoters. Therefore, in a preferred embodiment of the invention, a nucleic acid molecule comprising an endogenous invertase of a plant and its respective endogenous promoter is used for the modification of the genome of said plant. In another preferred embodiment, such nucleic acid is inserted into the plant genome.

It is also within the present invention that any nucleic acid molecule mentioned herein or cell, organ, organism or vector comprising said molecule is used for the manufacture of a medicament for the treatment or prevention of a disease of a plant. In a preferred embodiment, as used herein, the terms "nucleic acid molecule", "nucleic acid sequence", "nucleic acid construct" or the like are used synonymously and interchangeably.

It is within the invention that the methods, uses and reagents of the invention relate to all kinds of plant species. In principle, plant species that can be grown in a large scale for the industrial production of fruits, crops, seeds, oils, starch and the like may be used. In a preferred embodiment, the plant is a higher plant. In another preferred embodiment, the plant is a member of the family Solanacea. In another embodiment, the plant is a tomato species. In another embodiment, the plant is a tobacco species. In another embodiment, the plant is a plant that can be grown to obtain an industrially useful product, preferably a foodstuff. However, in principle, the methods and uses may also be applied to lower plants, more specifically unicellular plants or lower plants such as algae. This could be envisioned to increase, say, the yield of a plant product produced by algae in a bioreactor. Moreover, it may be possible to adapt such lower or unicellular plants such that they grow in culture medium and/or soil that would otherwise have been unsuitable for cultivation, say, due to high salt content. Again, this way, it may be possible to select in favour of engineered organisms and discourage the growth of contaminants. In a preferred embodiment, the plant is a plant grown in a liquid culture medium. In another preferred embodiment, the plant is a unicellular plant. In another embodiment, the plant is an alga.

In a preferred embodiment, all infections with all kinds of diseases and/or the presence of conditions or factors detrimentally affecting growth, development and well-being of a plant are considered as stress or, in another embodiment, indications of the inability to cope with stress of various kinds.

In a preferred embodiment, the term "development", as used herein, refers to any event associated with the formation and/or differentiation of any part of the plant, including any event associated with the multiplication of said plant. In a preferred embodiment, the term "development" comprises the formation of leaves, the formation of fruits, the ripening of fruits and the formation of seeds.

In another preferred embodiment, the disease is or is related to stress. In a preferred embodiment, the term "stress", as used herein, refers to a situation wherein the plant is exposed to conditions that differ from the optimal growth conditions. In a preferred embodiment, the plant is exposed to drought, salt, heat, cold, higher-than-optimal salt conditions, a lack of nutrients, lack of light, physical harm and/or pathogens. In a preferred embodiment, the term "hydric stress" as used herein, is synonymous with "drought".

In another preferred embodiment, the plant disease is related to a pathogen. In a preferred embodiment, the term "pathogen", as used herein, refers to any organism that interacts with the plant in a manner detrimental to the plant but advantageous to the pathogen. For example, the pathogen could be a fungus or bacterium that colonises wounded parts of the plant, consuming nutrients and water produced by the plant. Bacterial pathogens include, but are not limited to, pathogenic strains from the genus Arthrobacter, Pseudomonas, Xanthomonas, , Agrobacterium, Burkholderia, Proteobacteria, Bacillus, Clavibacterium, Clavibacter, Corynebacterium, Erwinia. Fungal pathogens include but are not limited to pathogenic strains from the genus Alternaria, Botrytis, Verticillium, Plasmodiophora, Hyaloperonospora, Fusarium, Thielaviopsis, Rhizoctonia, Phakospora, Pvthium, Phvtophthora, Puccinia.

A multitude of uses involving any of the nucleic acids, vectors, organs, tissues and organisms of the present invention are within the present invention. In an embodiment, a use within the invention comprises increasing the overall invertase activity present in a cell or tissues or plant.

In another preferred embodiment, a use within the present invention relates to a plant that is a member of the family Solanaceae. In a preferred embodiment, the plant is a tomato species. In another preferred embodiment, the plant is a tobacco species. In a preferred embodiment, the plant is a member of the family Solanaceae that differs from the tobacco or tomato species of said family.

Within the invention are multiple methods and uses that relate to the increase of invertase activity. Two main strategies exist that can be used to effect an increase in invertase activity. On the on hand, the overall invertase activity may be increased by expressing, under the control of a suitable promoter, an invertase, preferably a copy in addition to the endogenous promoter-invertase combination. On the other hand, the invertase activity of a plant may be in creased by an activator. In a preferred embodiment, the term "activator" of invertase activity, as used herein, refers to a molecule that interacts with the invertase such that its activity is increased. For example, the activator may be an allosteric activator, i.e. a molecule that binds to the enzyme at a site other than the catalytic center in a manner that promotes the catalysis of the reaction, for example by lowering the energy required to reach the transition state of the catalytic reaction. Such an activator may be administered to the plant. In an embodiment, the activator is injected into plant tissue. In another embodiment, the activator is added and taken up by the plant via the watering water. In another embodiment, the activator is a biomolecule, preferably a protein or a nucleic acid, preferably a RNA molecule, and a nucleic acid molecule coding for this biomolecule is used for expression in the plant. In a preferred embodiment, the activator is a small molecule. In a preferred embodiment, the term "small molecule", as used herein, is a molecule that obeys the Lipinski's rules of five, i.e. comprises less than 5 hydrogen bond donors, less than 10 hydrogen bond acceptors, has a molecular weight of 500 g/mol or less and a partition coefficient of less than 5.

In a preferred embodiment, the activator of an/the invertase is a polypeptide. As preferably used herein, a polypeptide is a polymer comprising at least two amino acids which are linked to each other by a peptide bond. More preferably, the polypeptide comprises 6, 10, 25 or more amino acids, whereby the upper range is preferably 50, 100, 200 and 500 amino acids. In connection with the present invention, the term polypeptide and protein are used in a synonymous manner.

In another preferred embodiment, the activator of an/the invertase is a nucleic acid, preferably a ribonucleic acid. In another preferred embodiment, the activator is an aptamer. Aptamers are D-nucleic acids which are either single-stranded or double-stranded and which specifically interact with a target molecule. The manufacture or selection of aptamers is, e. g., described in European patent EP 0 533 838. In contrast to RNAi, siRNA, siNA, antisense-nucleotides and ribozymes, aptamers do not degrade any target mRNA but interact specifically with the secondary and tertiary structure of a target compound such as a protein. Upon interaction with the target, the target typically shows a change in its biological activity. The length of aptamers typically ranges from as little as 15 to as much as 80 nucleotides, and preferably ranges from about 20 to about 50 nucleotides.

In any event, regardless of the chemical identity of the activator, the person skilled in the art knows how to identify such an activator, for example by screening chemical libraries comprising a plurality of potential activator molecules for molecules that increase the activity of invertase. Various screening methods and processes are discussed elsewhere herein. The invention is now further illustrated by the attached figures, examples and the sequence listing from which further features, embodiments and advantages may be taken.

Fig. 1 is a photograph showing the physical appearance of transgenic tomato plants (L93) comprising the nucleic acid construct (InvLp6:Cinl) including the fruit- specific promoter of tomato and the cDNA of extracellular Invertase from Chenopodium rubrumas well as the appearance of non-transgenic wild type plants (P-73) grown in a green house following drought stress.

Fig. 2 is a diagram indicating the water potential of the substrate, expressed as cbar, where wild type (P73) and transgenic plants (L93 and L10-L91) are grown. As may be taken therefrom, the water potential of the substrate of pots with transgenic plants is consistently lower that the potential of the substrate of the pot with a wild type plant. This indicates that the transgenic plants use less water than the wild type plants.

Fig. 3 is a diagram indicating the water content of plant tissues of plants subjected to three cycles of subsequent drought and rehydration indicated as RWC (% water on total fresh weight). As may be taken thereform, although the relative water content (RWC) declines both in the wild type plants and the different transgenic lines after watering has been stopped, the RWC of wild type plant is consistently lower than the RWC of the different transgenic lines. This result indicates that the transgenic plants are able to maintain a higher RWC during stress conditions important for physiological and growth process.

Fig. 4 is a diagram indicating the efficiency of photosynthesis measure by PAM chlorophyll fluorescence (Fvm) in a part of a leave subject to adaptation to darkness fro 15 minutes.

Fig. 5 is a diagram indicating the efficiency of photosynthesis (Fv/Fm)in leaves without adaptation to darkness.

Fig. 6 is a photograph showing the phenotype of transgenic and wild type plants following five days of drought stress.

Fig. 7 is a photograph showing the phenotype of transgenic and wild type plants following five days of drought stress.

Fig. 8 is a photograph showing the phenotype of transgenic and wild type plants following five days of drought stress.

Fig. 9 is a diagram indicating the soil water potential during a second cycle of drought stress. WT dried the substrate faster than the transgenic lines evaluated. Significant differences were found from the day 5 to day 7 of drought stress. This is indicating that transgenic plants use less water than the WT.

is a diagram indicating the relative water content (RWC) during a second cycle of drought stress. Initially all transgenic plants showed increased RWC than the WT but, later on, no significant differences were observed.

is a diagram indicating the evolution of maximum quantum efficiency of Photosystem II (Fv/Fm) during the drought period in plants adapted to darkness. Initially, when the drought stress started, no differences were found in the Fv/Fm between the WT and transgenic plants but, later on, chlorophyll fluorescence in the WT plants was lower than in the CIN 1 plants throughout the drought period.

is a diagram indicating evolution of maximum quantum efficiency of Photosystem II (Fv/Fm) during the drought period in plants without previous adaptation to darkness. Initially, when the drought stress started, no differences were found in the Fv/Fm between the WT and transgenic plants but, later on, chlorophyll fluorescence in the WT was lower than in the CINl plants throughout the drought period.

is a photograph showing the physical appearance of the transgenic line 22-8-10 expressing the LPBFRUCG::CIN1 construct and the WT the first day after a second rehydration cycle.

is a photograph showing the physical appearance of the transgenic line 22-8-8 expressing the LPBFRUCG::CIN1 construct and the WT the first day after a second rehydration cycle.

is a photograph showing the physical appearance of the transgenic line 22-8- 91expressing the LPBFRUCG::CIN1 construct and the WT the first day after a second rehydration cycle.

is a diagram indicating the relative water content (RWC) at the end of a third cycle of drought stress (10 days). The transgenic lines 22-8-91 and 22-8-93 showed an increased RWC than the other two transgenic lines evaluated and the WT.

is a diagram indicating the soil water potential during at the end of a third cycle of drought stress (10 days). Both the WT and one of the transgenic lines (22-8-8) dried the substrate faster than the transgenic lines evaluated. This is indicating that transgenic plants are using less water than the WT.

is a diagram indicating the fresh weight of selected fruits in g, comparing a wild type plant (P-73) and various transgenic plants comprising the InvLp6:Cinl construct, whereby HO stands for homozygous (+/+), HE stands for heterozygous (+/-), and A stands for azygous (-/-).

Fig. 19 is a diagram indicating the average fresh fruit weight per plant in g, comparing a wild type plant (P-73) and various transgenic plants comprising the InvLp6:Cinl construct.

Fig. 20 is a diagram indicating the total fruit yield per plant in g, comparing a wild type plant (P-73) and various transgenic plants comprising the InvLp6:Cinl construct.

Fig. 21 is a diagram indicating the leaf content with respect to hexoses for the above wild type and transgenic plants.

Fig. 22 is a diagram indicating the leaf content with respect to sucrose for the above wild type and transgenic plants.

Fig. 23 is a diagram where the data obtained for hexoses and as inidicated in Fig. 21 were plotted against the data obtained for sucrose and as indicated in Fig. 22 for the determination of a correlation between these two paramters with sucrose being the substrate for the transgenic invertase and hexose being the product of said invertase. As may be taken from said figure there is no obvious correlation between these two parameters. (DST: days of salt treatment; DAA: Days after anthesis)

Fig. 24 is a diagram indicating the sucrolytic activity of sucrose synthase in leaves of wild type and transgenic plants.

Fig. 25 is a diagram indicating the sucrolytic activity of cytoplasmatic invertase in leaves of wild type and transgenic plants (NI: neutral invertase or invertase activity in the cytoplasma.

Fig. 26 is a diagram indicating the sucrolytic activity of vacuolar invertase in leaves of wild type and transgenic plants (VAI = vacuolar acid invertase).

Fig. 27 is a diagram indicating the sucrolytic activity of extracellular invertase in leaves of wild type and transgenic plants.

Fig. 28 is a diagram indicating the concentrations of hexoses in fruits of the above wild type and transgenic plants.

Fig. 29 is a diagram indicating the concentrations of sucrose in fruits of the above wild type and transgenic plants.

Fig. 30 is a diagram where the data obtained for hexoses and as inidicated in Fig. 21 were plotted against the data obtained for sucrose and as indicated in Fig. 22 for the determination of a correlation between these two paramters with sucrose being the substrate for the transgenic invertase and hexose being the product of said invertase. As may be taken from said figure there is no obvious correlation between these two parameters.

Fig. 31 is a diagram indicating the sucrolytic activity of sucrose synthase in fruits 20DAA of wild type and transgenic plants.

Fig. 32 is a diagram indicating the sucrolytic activity of cytoplasmatic invertase in fruits

20DAA of wild type and transgenic plants.

Fig. 33 is a diagram indicating the sucrolytic activity of vacuolar invertase in fruits

20DAA.

Fig. 34 is a diagram indicating the sucrolytic activity of extracellular invertase in fruits

20DAA of wild type and transgenic plants.

Fig. 35 is a diagram indicating the concentrations of hexoses in mature fruits of the above wild type and transgenic plants.

Fig. 36 is a diagram indicating the sucrolytic activity of sucrose synthase in mature fruits of wild type and transgenic plants.

Fig. 37 is a diagram indicating the sucrolytic activity of cytoplasmatic invertase in mature fruits of wild type and transgenic plants.

Fig. 38 is a diagram indicating the sucrolytic activity of vacuolar invertase in mature fruits of wild type and transgenic plants.

Fig. 39 is a diagram indicating the sucrolytic activity of extracellular invertase in mature fruits of wild type and transgenic plants.

Fig. 40 represents photographs of wild type and transgenic tobacco plants expressing a

InvLp6g:Cin construct seven days after cessation of watering.

Fig. 41 represents photographs of wild type and transgenic tobacco plants expressing a

InvLp6:Cin construct eleven days after cessation of watering.

Fig. 42 represents photographs showing the effect of watering wild type and three independent lines of transgenic tobacco plants for three weeks using 100 mM sodium chloride, the transgenic plants expressing a PBFRU:Cinl construct containing the InvLp6 promoter.

Examples

Example 1: Cloning of promoter-invertase constructs

In a first step the invertase promoter of vacuolar invertase LPBFRUCG (clone InvLp6g, acc. No. Z 12028) was subcloned as Pstl-Ncol fragment from plasmid pWC19 into the Pstl-Ncol cut vector pGEM-T to generate plasmid pEBLl.

In a second step the cDNA for invertase CINl was isolated as Ncol-Apal fragment from the plasmid pREl 171-20 and fused to the LPBFRUCG promoter in the Ncol-Apal cut plasmid pEBLl to generate plasmid pEBL2.

In a third step the complete promoter-invertase fusion (expression cassette) was isolated as Sall-Sacl fragment from plasmid pEBL2 and ligated into the Sail and Sacl cut plant transformation vector pBHOl to generate plasmid pEBLMCGl.

Example 2: Generation of transgenic plant cells

Generation of transgenic tomato plants:

Transformation of Lycopersicon esculentum with construct pEBLMCGlwas done following essentially the protocol of Fillatti et al. (1987).

Generation of transgenic tobacco plants:

Construct pEBLMCGlwas transformed in tobacco (Nicotiana tabacum cv SRI) using standard Agrobacterium (LBA4404) transformation procedures (Horsch et al. 1985).

Example 3: Exposure of transgenic tomato plants expressing an InvLp6:Cinl construct to drought (Fig 1-17)

Seeds were germinated under selective conditions on Agar plates and seedlings were transferred to soil. One or several cycles of drought stress was applied by stopping watering for the indicated time periods following by reassuming the normal watering scheme. The result of this experiment is shown in Figs. 1 to 17.

Example 4: Exposure of transgenic tomato plants expressing an InvLp6:Cinl construct to elevated concentrations of salt (Fig 18-39)

Seeds were germinated under selective conditions on Agar plates and seedlings were transferred to soil. Salt stress was applied watering with a solution containing sodium chloride (NaCl) at the indicated concentration. The result of this experiment is shown in Figs. 18 to 39. Example 5: Exposure of transgenic tobacco plants expressing an InvLp6:Cinl construct to drought

Seeds were germinated under selective conditions on Agar plates and seedlings were transferred to soil. Drought stress was applied by stopping watering.

Example 6: Exposure of transgenic tobacco plants expressing an PBFRU.Cinl construct to elevated concentrations of salt

Seeds were germinated under selective conditions on Agar plates and seedlings were transferred to soil. Salt stress was applied watering with a solution containing sodium chloride (NaCl) at the indicated concentration.

References:

Throughout the present specification the following references were recited the disclosure of which is herein incorporated by reference in their entirety.

Amemiya Tsuyoshi, Kanayama Yoshinori, Yamaki Shohei, Yamada Kunio, Shiratake Katsuhiro (2006) Fruit-specific V-ATPase suppression in antisense-transgenic tomato reduces fruit growth and seed formation. Planta-223, 1272-1280 Balibrea Lara M.E., Gonzalez Garcia M.-C, Fatima T., Ehness R., Lee T.K., Proles R., Tanner W. and Roitsch T. (2004). Extracellular invertase is an essential component of cytokinin-mediated delay of senescence. Plant Cell 16, 1276-1287.

Bret-Harte M. and Silk W.K. (1994). Nonvascular, symplasmic diffusion of sucrose cannot satisfy the carbon demands of growth in the primary root tip of Zea mays L. Plant Physiol. 105, 19-33.

Burger Anita-L, Watts Leonora, Botha Frederik-C (2006) Grapevine promoter directs gene expression in the nectaries of transgenic tobacco . Physiologia Plantarum 126, 418-434 Carmi Nir, Salts Yehiam, Dedicova Beata, Shabtai Sara, Barg Rivka (2003) Induction of parthenocarpy in tomato via specific expression of the rolB gene in the ovary. Planta 217, 726-735

Elliott K. J., Bulter, W. O., Dickinson, C. D., Konno, Y., Vedvick, T. S., Fitzmaurice, L., Mirkov, T. E. (1993) Isolation and characterisation of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening. Plant. Mol. Biol. 21, 515-524 Eschrich W. (1980). Free space invertase, its possible role in phloem unloading. Ber. Deutsch. Bot. Ges. Bd. 93, S363-378.

Farrar J.F. and Jones D.L. (2000). The control of carbon acquisition by roots. New Phytol. 147, 43-53.

Goetz M., Godt D.E., Guivarc'h A., Kahmann U., Chriqui D. and Roitsch T. (2001). Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. Proc. Natl. Acad. Sci. U.S.A. 98, 6522-6527.

Greiner S., Krausgrill S. and Rausch T. (1998). Cloning of a tobacco apoplasmic invertase inhibitor: Proof of function of the recombinant protein and expression analysis during plant development. Plant Physiol. 1 16, 733-742.

Greiner S., Rausch T., Sonnewald U. and Herbers K. (1999). Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers. Nature Biotech. 17, 708-71 1.

He Zhu-Mei , Jiang Xiao-Ling, Qi Yu, Luo Di-Qing (2008) Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression. Genetical33, 207-214.

Heyer A.G., Raap M., Schroeer B., Marty B. and Willmitzer L. (2004). Cell wall invertase expression at the apical meristem alters floral, architectural, and reproductive traits in Arabidopsis thaliana. Plant J. 39, 161-169.

Hong Se-Ho, Kim In-Jung, Yang Deok-Chun, Chung Won-Il (2002) Characterization of an abscisic acid responsive gene homologue from Cucumis melo. Journal of Experimental Botany 53, 2271-2272

Link M., Rausch T. and Greiner S. (2004). In Arabidopsis thaliana, the invertase inhibitors AtC/VIFl and 2 exhibit distinct traget enzyme specificities and expression profiles. FEBS Lett. 573, 105-109.

Miller E.M. and Chourey P.S. (1992). The maize invertase-deficient miniature-l seed mutation is associated with aberrant pedicel and endosperm development. Plant Cell 2, 1 107- 1 1 19.

Rausch T. and Greiner S. (2004). Plant protein inhibitors of invertases. Biochim. Biophys. Acta 1696, 253-261.

Roitsch T., Balibrea, M. E., Hofmann, M., Proels, R., Sinha, A. K. (2003) Extracellular intertase: key metabolic enzyme and PR protein. J. Exp. Bot. 54, 513-524 Roitsch T., Bittner M. and Godt D.E. (1995). Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analogue and tissue-specific expression suggest a role in sink-source regulation. Plant Physiol. 108, 285-294.

Roitsch T. and Gonzalez M.-C. (2004). Function and regulation of plant invertases: sweet sensations. Trends Plant Sci. 9, 606-613.

Shen-Wen-Tao, Zhou-Peng, Guo-An-ping, Wang-Jian-wei (2004) Specific expression of rotavirus outer capsid protein VP7 in transgenic tomato fruits. Acta Botanica Yunnanica 26, 207-212 Sturm A., Sebkova V., Lorenz K., Hardegger M., Lienhard S. and Unger C.

(1995). Development- and organ-specific expression of the genes for sucrose synthase and three isoenzymes of acid β-fructosidase in carrot. Planta 195, 601-610.

Tang G.-Q., Luscher M. and Sturm A. (1999). Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning. Plant Cell 1 1, 177-189.

Tymowska-Lalanne Z. and Kreis M. (1998). Expression of the Arabidopsis thaliana invertase gene family. Planta 207, 259-265.

Von Schweinichen and Buttner M. (2005). Expression of a plant cell wall invertase in roots of Arabidopsis leads to early flowering and an increase in whole plant biomass. Plant Biol. 7, 469-475.

Weber H., Borisjuk L., Heim U., Sauer N. and Wobus U. (1997). A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. Plant Cell 9, 895-908.

Weil M., Krausgrill S., Schuster A. and Rausch T. (1994). A 17-kDa Nicotiana tabacum cell-wall peptide acts as in-vitro inhibitor of the cell-wall isoform of acid invertase. Planta 193, 438-445.

Weschke W., Panitz R., Gubatz S., Wang Q., Radchuk R., Weber H. and Wobus U. (2003). The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues in barley caryopses during early development. Plant J. 33, 395-41 1.

Wobus U. and Weber H. (1999). Sugars as signal molecules in plant seed development. Biol. Chem. 380, 937-944.

The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

1. A method of increasing the resistance of a plant to stress, preferably drought or salt stress, and/or a method of increasing the fruit yield of a plant or generating a plant displaying a higher fruit yield, comprising the step of administering to the plant or a progenitor thereof, a nucleic acid molecule comprising a promoter functional in at least one plant species and an invertase from an organism, wherein said promoter is a fruit-specific promoter and said promoter differs from the endogenous promoter which controls the expression of said invertase in the genome of said organism, and wherein the promoter and the invertase are functionally linked to allow for expression of the invertase.

2. The method according to claim 1, wherein the invertase is a plant-derived invertase, preferably an extracellular plant invertase.

3. The method according to any one of claims 1 to 2, wherein the promoter is selected from a species of the family Solanaceae and is preferably selected from a tobacco species or a tomato species.

4. The method according to any of claims 1 to 3, wherein the fruit-specific promoter is selected from the group comprising the fruit-specific promoter of vacuolar invertase InvLp6 from Lycopersocon piminellifolium, the E8 promoter, the 2A1 1 promoter, the mripl promoter, the TFP promoter, the TPRP-F1 promoter, and the asr promoter.

5. The method according to any one of claims 1 to 4, wherein the promoter is the fruit- specific promoter of vacuolar invertase InvLp6 from Lycopersocon piminellifolium and the invertase is the extracellular invertase CIN 1 from Chenopdium rubrum.

6. The method according to any one of claims 1 to 5 wherein the nucleic acid molecule is one which hybridises, preferably under stringent conditions, to a nucleic acid molecule which is complementary to the nucleic acid molecule as defined in any one of said claims 1 to 5.

7. The method according to any of claims 1 to 6, wherein the plant is a species from the family of Solanaceae, the familiy of Brassicaceae or the family of Poaceae.

8. A nucleic acid molecule as defined in any of claims 1 to 7.

9. A vector, preferably a plant expression vector or a plant-specific virus, comprising a nucleic acid molecule as defined in any one of claims 1 to 7.

10. A cell, preferably a plant cell, comprising a nucleic acid molecule as defined in any one of claims 1 to 7 and/or a vector according to claim 9.

1 1. A plant, comprising a nucleic acid molecule as defined in any one of claims 1 to 7 and/or a vector according to claim 9 and/or a cell according to claim 10.

12. The plant according to claim 1 1, wherein the organism is a plant species selected from the family of Solanaceae, the family of Brassicaceae or the family of Poaceae.

13. Use of a nucleic acid molecule as defined in any one of claims 1 to 7 and/or a vector according to claim 9 and/or a cell according to claim 10 for the generation of a transgenic plant.

14. The use according to claim 13, wherein the transgenic plant is resistant to drought and salt stress.