ES2370864B1 - Procedure to increase the accumulation of starch and alter the structure of starch in plants. - Google Patents

Abstract

Procedure to increase the accumulation of starch and alter the structure of starch in plants. The procedure consists in growing plants in an atmosphere in which volatiles emitted by a microorganism are present, without any physical contact between the microorganisms and the plant, except that the plant comes into contact with the volatiles emitted by the microorganism. It is based on the discovery that volatiles emitted by Gram positive or negative bacteria, yeasts and microscopic fungi promote an increase in accumulated starch and the structural alteration of this biopolymer. The effect is also observed in separate leaves of whole plants.

Description

Procedure to increase the accumulation of starch and alter the structure of starch in plants.

Technical field

The invention relates to a process for increasing the amount of starch accumulated with respect to plants grown under normal conditions. This procedure also allows to increase the size of the starch granule, alter the amylose / amylopectin balance and degree of starch branching. Additionally, the invention also refers to a procedure to increase the amount of starch accumulated in separate leaves of the plants.

Background of the invention

Plants perceive biotic stimuli recognizing a multitude of different signaling compounds that originate in the organisms with which they interact. Some of these substances represent molecular patterns associated with pathogens, which generally act as triggers for defense reactions. They are perceived at low concentrations and comprise various structures, including those of carbohydrates, proteins, glycoproteins, peptides, lipid and sterols (Hahlbrock et al. 2003: Nonself recognition, transcriptional reprogramming, and secondary metabolite accumulation during plant / pathogen interactions, Proc Natl. Acad. Sci USA 100 (supl 2), 1456914576).

Microorganisms also synthesize many volatile compounds with molecular masses less than 300 Da, low polarity, and high vapor pressure (Schöller et al. 2002: Volatile metabolites from actinomycetes, J. Agric.FoodChem.50,2615-2621; Schultzand Dickschat2007: Bacterialvolatiles: thesmellofsmall organisms, Nat. Prod. Rep. 24, 814-842; Splivallo et al. 2007a: Discrimination of truf fl e fruiting body versus mycelial aromas by stir bar sorptive extraction, Phytochemistry 68, 2584-2598). Contact with microorganisms or agents that trigger plant defense reactions not only affects these defense reactions, but, very often, leads to a decrease in photosynthesis, and a transition from the source state (in which assimilable organic compounds are produced) to the sump (in which they are imported such assimilable compounds of tissues in which they are stored) (for review, see Berger et al. 2007: Plant physiology meets phytopathology: plant primary metabolism and plant-pathogen interactions, J. Exp. Bot. 58, 4019-4026). An indication of the sump status in infected leaves is the upward regulation of cell wall invertase, which results in the reduction of sucrose export from the infected leaf to other parts of the plant. In some cases, the sacchaolytic enzyme sucrose synthase (SuSy) is regulated upwards after contact with microorganisms, which can be used to distribute sucrose to the callus deposition and promote biosynthesis of cell wall polysaccharides at the sites of infection (Essmann et al. 2008: Leaf carbohydrate metabolism during defense, Plant Signaling & Behvior 3, 885887). Contact with pathogens can also result in downregulation of genes involved in starch metabolism (Cartieaux et al. 2008: Simultaneous interaction of Arabidopsis thaliana with Bradyrhizobium sp. Strain ORS278and Pseudomonas syringae pv. TomatoDC3000 leads to complex transcriptome changes , Mol. Plant-Microbe Interact. 21, 244-259; Fabro et al. 2008: Genome-wide expression pro fi ling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation, PlantPhysiol. 146,1421-1439), which makes it easier to make the pathogen available of simple sugars at the sites of infection. These branched homopolysaccharides are synthesized by starch / glycogen synthase using ADPglucose (ADPG) as a sugar donor molecule.

Starch and glycogen are the main storage carbohydrates in plants and bacteria, respectively, their metabolism being closely connected with that of amino acids by still poorly understood mechanisms. In Escherichia coli, amino acid deprivation triggers the response to strict conditions, a pleiotropic physiological change that causes the cell to pass in a growth-related way to a maintenance / survival / biosynthesis mode. Under conditions of limited supply of nutrients (amino acids), the cell division is separated, and there is a decrease in the demand for ATP-dependent proteins and the nuclei acid degradation and synthesis. The ATP process is then diverted from the nucleic acid / protein metabolism to glycogen biosynthesis if excess excess is present in the medium. carbon sources (Eydallin et al., 2007b: Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12, FEBS Lett 581, 2947-2953; Montero et al. 2009: Escherichia coli glycogen metabolism is controlled by the PhoP -PhoQ regulatory system at submillimolar environmentalMg2 + concentrations, andis highly interconnected with a wide variety of cellular processes, Biochem. J. 424, 129-141). The characteristic sign of this pleiotropic physiological response is the accumulation of the alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a nucleotide that binds to the bacterial RNA polymerase to potentiate the expression of genes (including those involved in the glycogen metabolism) expressed at the beginning of the stationary phase. are controlled by RaEL (a ppGpp synthase) and SPT (a bifunctional enzyme that shows activities of ppGpp synthase hydrolase) (Potrykusand Cashel 2008: (p) ppGpp: still magical ?, Annu. Rev. Microbiol. 62, 35-51). E. coli mutants that have damage to the relA function, and overexpressing spoT cells show a glycogen deficient phenotype (Montero et al. 2009: Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg2 + concentrations , and is highly interconnected with a wide variety of cellular processes, Biochem.

J. 424, 129-141). In contrast, E. coli mutants that have damaged amino acid synthesis such as cysteine show an excess glycogen phenotype as a result of strict response (Eydallin et al., 2007b: Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K -12, FEBS Lett 581, 29472953). These mutants show a normal glycogen phenotype when grown in medium supplemented with cysteine, which points to the existence of close connections between sulfur, nitrogen and carbon metabolisms.

Recent studies have shown that plants have a regulatory system mediated by ppGpp similar to that found in bacteria, which has been shown to play a crucial role in aspects such as plant fertility. The ppGpp accumulates in the chloroplast of stressed leaves through the regulation of the expression of RelA / SpoT (RSH) counterparts (Takahashi et al. 2004, Identi fi cation of the bacterial alarmone guanosine 5'diphosphate 3'-diphosphate (ppGpp) in plants, Proc. Natl. Acad. Sci. USA 101, 4320-4324).

Contrary to the fact that bacteria, where the degradation of the glycogen has a second phosphorolytic pathway, the degradation of starch in plants is mainly hydrolytic, playing important roles in the degradation of the endosperm and cereal grains of α-amylases and β-amylases, respectively (Scheidig et al. starch-excess phenotypeinleaves, PlantJ.30, 581-591; Fulton et al. 2008: β-amylase4, a noncatalytic protein required for starch breakdown, acts usptream of three active βamylases in Arabidopsis chloroplasts, Plant Cell 20, 1040-1058). Since the initial demonstration that ADPG serves as a precursor molecule for the biosynthesis of both bacterial glycogen and plant starch, the consideration that ADPG pyrophosphorylase (AGP) is the only enzyme that catalyzes the production of ADPG Genetic evidence that bacterial glycogen biosynthesis occurs only by the path of AGP (GlgC) has been obtained with glgC mutants. However, recent studies have shown that these mutants accumulate substantial amounts of glycogen and a normal ADPG content. In addition, evidence has been provided that demonstrates the existence of various important sources, other than GlgC, of ADPG linked to glycogen biosynthesis in different bacterial species.

Generally, the biosynthesis of leaf starch has been considered to take place exclusively in the chloroplast, and is segregated from the sucrose biosynthetic process that takes place in the cytosol (Fig. 1A). In accordance with this classic view, starch is considered the end product of a unidirectional route in which AGP exclusively catalyzes the synthesis of ADPG, and functions as the main regulatory stage of the biosynthetic process of starch (Neuhaus et al. 2005: No need to shift the paradigm on the metabolic pathway to transitory starch in leaves, Trends Plant Sci. 10, 154-156; Streb et al. 2009: The debate on the pathway of starch synthesis: a closer look at low-starch mutants lacking plastidial phosphoglucomutase supports the chloroplast-localized pathway, Plant Physiol. 151, 17691772). However, recent evidence has indicated the existence of an additional route in which de novo is produced in the cytosol, by means of SuSy, ADPG linked to the biosynthesis of starch. SuSolytic enzyme SuSy is the main determinant of the sump force that intensively controls the channeling of the incoming sucrose into starch and polysaccharides of the cell wall (Amor et al. 1995: Amembrane-associated formof sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proc. Natl. Acad. Sci. USA 92, 9353-9357). It catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding glycosayfructose nucleoside diphosphate. Although UDP is the preferred nucleoside diphosphate substrate for SuSy to produce UDPG, ADP also acts as an effective acceptor molecule to produce ADPG.

According to this alternative vision, both the sucrose and starch biosynthetic pathways are closely interconnected through SuSy's ADPG-producing activity (Muñoz et al., 2006: New enzymes, new pathways and an alternative view on starch biosynthesis in both photosynthetic and heterotrophic tissues of plants , Plant Cell Physiol. 46, 1366-1376; Baroja-Fernández et al., 2009: Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield, PlantCellPhysiol. 50,1651-1662), and through the action of an ADP translocator still identifies the membranes in the envelope of the chloroplasts. The "alternative" view of the starch biosynthesis in the leaves illustrated in Fig. 1B also assumes that both laAGP and plastidial phosphoglucomutase play an important role in the removal of glucose units derived from starch degradation.

Most of the studies on plant-microorganism interactions have taken place under conditions of physical contact between the host plant and the organism. However, little is known about how microbial volatile emissions can affect plant physiology in the absence of physical contact.It is known, however, that microorganisms such as Pseudomonas spp., Streptomyces spp., Botrytis cinerea and various truffles produce ethylene (Splivallo et al. 2007b : Truf fl evolatiles inhibitgrowthand inducing oxidativeburstin Arabidopsis thaliana, NewPhytologist175,417-424), a plant-based hormone that plays important roles in multiple aspects of plant growth and development, including seed germination, elongation of the hypocotyl, initiation of the seeds, the accumulation of the seeds, the accumulation of the seeds, the accumulation of the seeds of the plant, the accumulation of the various layers Only recently Splivallo et al. (Splivallo et al. 2009: Truf fl es regulate plant root morphogenesis via theproductionofauxinandethylene, PlantPhysiol.150,2018-2029) provide evidence that the ethylene produced by truffles induces alterations in the development of Arabidopsis plants, which are presumably accompanied by significant changes in metabolism.

As far as bacteria are concerned, the few works that describe the effect of microbial volatiles on plant growth revolve around a limited number of specialized strains of plant growth promoting rhizobacteria (PGPR: plant growth promoting rhizobacteria). Certain symbiont bacteria that exist in the soil and colonize the roots of the plants are termed rhizobacteria. Most of the strains whose cultivation gives rise to a positive effect on the growth of the plants grown in their presence, without the need for physical contact, belong to the genus Bacillus or a genus closely related to it. , Paenibacillus, to which belong bacteria that in the past were classified as belonging to the genus Bacillus. Thus, for example, it has been shown that volatiles emitted by rhizobacteria from strains belonging to the species Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus cepacia promote the growth of Arabidopsis plants, facilitating the intake of nutrients, photosynthesis and defense response, and decreasing the sensitized acid levels of glucosaylos abscisic (Ryu et al. 2003: Bacterial volatiles promote growth in Arabidopsis, Proc. Natl. Acad. Sci. USA 100, 4927-4932; Ryu et al. 2004: Bacterial volatiles induces systemic resistance in Arabidopsis, Plant Phylio 134, 1017- 1026; Vespermann et al. 2007: Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana, Appl. Environ. Microbiol. 73, 5639-5641, Xie et al. 2009: Sustained growth promotion in Arabidopsis with long-term exposure to the bene fi cial soil bacterium Bacillus subtilis (GB03), Plant Signal. Behav. 10, 948-953). Specifically, Ryu et al. (Ryu et al. 2003: Bacterial volatiles promote growth in Arabidopsis, Proc. Natl. Acad. Sci. USA 100, 4927-4932) describe an increase in the growth of Arabidopsis thaliana seedlings triggered by organic volatiles released by specific PGPR strains , specifically Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, commenting also that their data demonstrate that the release of organic volatile compounds is not the common mechanism of growth stimulation of all rhizobacteria. When grown in the medium rich in amino acids agar with soy tripticase, both bacteria release 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol, compounds not emitted by the other PGPRs tested whose volatiles did not affect the growth of Arabidopsis , but which are also released by other bacterial strains for which the ability to increase germination and growth of plants such as Brassica oleracea has been detected without physical contact between plant and bacteria, such as the Bacillus subtilis WG6 strain. 14 object of US patent application 2008/0152684 A1. However, there are many bacteria that release these substances (some belonging to the genus Bacillus) that do not promote plant growth. In addition to the aforementioned strains of the genus Bacillus GB03 and IN937a, Ryu et al. They only mention that the effect of increased growth by the release of volatiles will be detected for another of the bacteria tested, Enterobacter cloacae JM22, although no data is shown to support this last result nor is there any mention of the volatile profile emitted by This last bacterium. In addition, a subsequent article by the same research group (Ryu et al. 2004: Bacterial volatiles induces systemic resistance in Arabidopsis, Plant Phylio 134, 1017-1026) shows marked differences between the high capacity of volatiles emitted by the two Bacillus strains for Protect Arabidopisis thaliana plants from the effect of the Erwinia carotovora pathogen and the poor protective effect of volatile emitted by Enterobacter cloacae JM22.

Other strains of the genera Bacillus or Paenibacillus have also been described as emitting volatiles capable of promoting the growth of different plants but, in these cases, the effect seems to be mainly linked to the ability to control the growth of pathogens that are affecting the plant. Such is the case, for example, of the bacillus KyuW63 described in the Japanese patent. JP10033064, whose volatiles are capable of controlling pathopoiesis due to the presence of fungi of the Cercospora genus in cucumber leaves, thereby facilitating plant growth. The description suggests that the effect could be similar using other fibrous bacteria, provided that the culture is produced in a medium rich in sugars such as PDA agar, which is not defined in more detail; neither is it proven that they demonstrate the influence of the suggested medium or the applicability of the method for any lacteal bacteria.

Also the method for increasing the growth of plants, based on compositions comprising a volatile metabolite produced by a bacterium, which is claimed in the application of the Korean patent KR20090066412, refers in combination to the induction of protection against diseases and the attack of insects and to the promotion of the growth of different plants, monocotyledonous and dichotyled. Examples of possible useful metabolites are 3-acetyl-1-propanol, 3-methyl-1-butanol, indole, isobutyl acetate acetate butyl. The summary mentions that the possible microorganisms that have a volatile metabolite with the desired effect comprise bacteria belonging to the Bacillus or Paenibacillus genera, being a strain of the species Paenibacillus polymyxa the preferred microorganism.

As previously mentioned, it has also been detected that volatile compounds emitted by some bacteria have other effects on plants, in addition to activating the defense system and promoting growth. Thus, Zhang et al. (Zhang et al. 2008: Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisicacidlevelsin plant, ThePlantJournal56,264-273), describe how the exposure of Arabidopsis thaliana plants to the volatiles emitted by Bacillus subtilis GB03, cultivated again in the cultivation medium with agar they repress the sensitization of the plant glucose, simultaneously causing a slight increase in the accumulation of sugar and an increase in photosynthesis, the latter process that is normally inhibited when the levels of soluble sugars accumulated in the plants are increased. Plants that come into contact with the volatile emitted by B. subtilis GB03 show increases of 50-62% of the content of soluble sugars over the control plants, which, approximately, accumulate 2 micromoles of hexanes by fresh gram weight (cf. Fig. 2, Zhang et al. 2008: Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisic acid levels in plant, The Plant Journal 56, 264-273). The increase in soluble sugar content is generally associated with a decrease in intracellular starch levels (Caspar et al. (1985): Alterations in growth, photosynthesis and respiration in a starchless mutant of Arabidopsis thaliana (L) de fi cient in chloroplast phosphoglucomutase, Plant Physiol. 79: 11-17; Jones et al. (1986): Reduced enzyme activity and starch level in an induced mutant of chloroplast phophoglucose isomerase, Plant Physiol. 81: 367-371; Lin et al. (1988): A starch de fi cient mutant of Arabidopsis thaliana with low ADPglucose phosphorylase activity lack one of the subunits of the enzyme, Plant Physiol. 86: 1131-1135; Neuhaus and Stitt (1990): Controlanalysis of phosphosynthatepartioning. in Arabidopsis thaliana, Plant 182: 445-454; Szydlowski et al. (2009): Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthase, Plant Cell 21, 2443-2457). Therefore, it is expected that, under the conditions employed by Zhang et al., Plants that come into contact with the volatiles emitted by B. subtilis GB03 accumulate little starch. The method used by the group of Zhang et al. It only allows the content of glucose, fructose, fructose-6-phosphate and glucose-6-phosphate, although not accumulated starch, although the absence of variations in the levels of expression of genes involved in starch metabolism such as starch synthesized starch or enzymes of the starch developed by the plant shows that the non-volatile elevation of plants is indicated by the elevated elevation of the chloroplastic plant in the plant. This reserve polysaccharide when the plant is subjected to the effects of volatiles emitted by B. subtilis GB03. This interpretation is supported by the fact that trials referring to the inhibition of hypocotyl length and germination of seeds indicate that volatile B. subtilis GB03 do not cause a metabolic response to treatment, since they do not seem to affect the metabolism of sugars, but rather the sensitivity to said compounds .

According to what was known so far, all these effects on plants are not common to the volatile emitted by any bacteria. Thus, for example, as discussed above, the tests carried out by Ryu et al. (Ryu et al. 2003: Bacterial volatiles promote growth in Arabidopsis, Proc. Natl. Acad. Sci. USA 100, 4927-4932) show that several strains of Bacillus species, such as Bacillus pumilus T4 or Bacillus pasteurii C-9, as well as bacteria belonging to other genera such as Pseudomonas fl uorescens 89B-61 or Serratia marcescens 90-166, were not able to increase the growth of Arabidopsis thaliana plants subjected to the effect of the volatile emitted by said bacteria, despite having been also grown in the same medium Decultive, rich in sugars and amino acids: elagarcon tripticasadesoja.Otradelasbacterias includingse the same test, Escherichia coli DH5α, was used in the same test as a control, because it is recognized as a strain that does not increase the growth of plants subjected to the action of volatiles emitted by it.

In addition, it has also been seen that volatile bacteria such as Pseudomonas spp., Serratia spp. And Stenotrophomonas spp., And some fungal species exert inhibitory effects on the growth of Arabidopsis plants (Splivallo et al. 2007b: Truf fl e volatile inhibit growth and induce and oxidative burst in Arabidopsis thaliana, .NewPhytologist 175, 417-424, Tarkka and Piechulla, 2007: Aromatic weapons: truf fl es attack plantsbythe productionofvolatiles, NewPhytologist 175, 381-383).

Due to the lack of knowledge about microbial volatile how they can affect the reprogramming of cellular metabolism, in particular the primary metabolism of carbohydrates, it is currently not possible to act on the metabolism of plants for the promotion of microbial volatile growths, since the mechanisms involved in promoting or inhibiting the growth previously activated by microorganisms are not clear. activate one or the other or the possible differences between microorganisms that give rise to one or the other effect. However, it would be interesting to know these mechanisms to be able to design a procedure to activate the growth and / or fl ow of plants, and increase their growth, biological and mechanical resistance through the use of microbial volatiles and, preferably, to increase the synthesis of starch in plants, as this is currently a product of great interest in Some industries The present invention provides a solution to this problem.

Description of the invention

The invention is based on the surprising discovery that, when plants grow in the presence of any type of microorganism (Gram-positive or Gram-negative bacteria, fungal yeasts), without any contact between the plant and the microorganism, the volatile emitted by the microorganism will occur that there is an alteration in the pattern of development and an increase in growth, fertility, dry weight and starch accumulation of plants. In addition, exposure to such volatiles induces the accumulation of a starch with structural characteristics different from those of starch accumulated by non-volatile plants, both in terms of the structure of the starch molecule itself and the size of the starches. accumulation granules. These effects are observed with both monocotyledonous and dicotyledonous plants (Arabidopsis, corn, barley, tobacco, potatoes ...), and are independent of the microorganisms being pathogenic for the plant and belonging to a species that does not live with the plant in natural conditions. These effects are observed both if the plants are grown in vitro and on land, as long as the plant is grown in the presence of a microorganism that emits volatile microorganisms have presence of the microbial volatile emitted by the microorganism.These seem to be responsible for the observed effects.Therefore, although there is no physical contact between the plant and plant microorganism, the latter must be sufficiently close to the plant, so that the volatile compounds emitted by the microorganisms come into contact with the plant and can exert their effect on it.

The alteration of the development pattern of the plant is manifested by increasing the number of leaves, the number of branches, the number of flowers and seed induced by fl owing.

This effect has been observed with all types of microorganisms and, particularly in the case of leaf studies, seems to be the consequence of a transition from source to sink status. Thus, the results presented in the present application show that the volatile emissions of all the microbial species analyzed promoted an increase in biomass in the plants and led to the accumulation of a high starch content, compared to the control plants grown in the same conditions, except for the absence of the microorganism culture. The observed effect is independent of the presence of sucrose in the culture medium and is strongly repressed by cysteine supplementation. The effect occurs in both monocotyledonous and dicotyledonous plants. These are only para-volatile emitted by rhizobacteria that promote growth of plants such as certain isolates of Bacillus subtilis, but also, surprisingly, for the volatile emitted for different fungal and pathogenic plant pathogens of species such as E. coli or Pseudomonas. found with these species some species of fungi, in which the opposite effect had been observed, giving rise to volatile growth inhibition of plants grown in their presence). In this way, the present invention demonstrates that the ability to emit volatiles that positively influence the biomass of plants in general and that their growth in particular is restricted to rhizobacteria, provided that the microorganism is grown in the appropriate medium.

The positive effect on the growth and accumulation of starch is observed especially when the microorganism is grown in minimal medium (understanding as such a medium that lacks amino acids but that contains several salts, which can vary according to the species of microorganism and growth conditions, which are those that provide essential elements such as magnesium, nitrogen , phosphorus and sulfur for the microorganism can synthesize proteins and nucleic acids) supplemented with a source of organic carbon (usually, a sugar, such as glucose or sucrose). It seems that the use of this type of medium avoids that microorganisms generate volatile ammonium from amino acids or other sources of organic nitrogen present in amino acid-rich media such as those used in assays those that other authors had observed inhibition of growth by microbial volatiles (such as the Middle Boborn medium), and this difference seems to be the cause that gives place where the cultivation of plants in the presence of microorganisms with which they do not have contact results in an increase in growth, fl owing, branching, fertility, robustness, biomass in general and the accumulation of starch in particular.

This is the first time that microbial volatiles are reported to be capable of inducing both growth and fl owing, branching and starch accumulation, as well as the structural alteration of this polymer in plants that grow under its effect. Thus, the authors of the invention seem to have found the culture conditions that allow any type of microorganism (pathogenic or not for the plant) to release a volatile mixture capable of exerting a positive effect on both growth and flowering, ramification and accumulation of starch.

The results obtained by the authors of the invention also contradict some previous ideas regarding the culture medium to be used for a bacterium to produce suitable volatiles to promote the growth of cultivated plants in the presence of said volatiles, such as the idea that seems It is suggested in Japanese patent JP10033064 that the culture in a medium rich in sugar could be sufficient for some bacteria to emit a volatile mixture capable of inducing plant growth and protecting them against pathogens. Thus, the tests described below in the Examples of the present application show volatile emitted by bacteria and other microorganisms, grown in LB with 50 mM glucose exerts a negative effect not only on growth, but also on the accumulation of starch in plants that come into contact with such volatiles.

As for the increase in the amount of accumulated starch, it is observed in different organs of the plant: leaves (not only when they are attached to the whole plant, but also in detached leaves of the plant, placed in the presence of volatiles emitted by microorganisms of different species); roots; stems; tubers (tighten the amount of starch accumulated, for example in potato plants, is greater than that accumulated in control plants ...).

The discovery that microbial volatiles induce starch over-accumulation in leaves and other plant organs constitutes a mechanism that had not been previously reported, which establishes an additional function for volatiles as signaling molecules that mediate plant-microorganism interactions and that helps in the elucidation of the process of induction of carbohydrate metabolism in plants by microorganisms. The increase in the amount of accumulated starch seems to be accompanied, in addition, by structural changes in the starch, both as regards the structure of the biopolymer, as well as that of the granules. Thus, it is observed, on the one hand, that the starch granules are larger than the control plants grown in the absence of volatiles. This is a fairly important characteristic, since the size of the starch granule is of great importance on an industrial level, because it is an important determinant of the physical and chemical properties of the starch granule suspensions, so that the size and shape of the starch granules of species such as potato, wheat, corn, etc. ., is what, to a large extent, determines that these starches have different industrial applications. In addition, it is observed that the starch accumulated by plants that grow in atmospheres that contain microbial volatiles have a significant reduction in the relative amylose content, so that the amylose / amylopectin ratio is lower than that of the control plants. This modification in the structure of the starch molecule is accompanied by changes in the degree of polymerization of the amylopectin chains, which is lower in plants treated with microbial volatiles. Thus, the cultivation of plants in volatile-containing atmospheres emitted by microorganisms makes it possible to obtain plants that not only have superior starch production but also give rise to a starch whose characteristics allow different industrial applications to those of starch synthesized by plants. grown in the absence of volatiles.

Transcriptome analyzes of, among others, leaves of potato plants exposed to volatiles of fungal origin (specifically, produced by fungi of the Alternaria genus) have revealed that changes in starch metabolism are accompanied by changes in multiple biological processes and in activated expression of different enzymes, such how:

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upward regulation of: sucrose synthase, invertase inhibitors, class IV starch synthase, starch rami fi cating enzyme, proteins involving endocytes and tetraphyllicles, stromaal glucose-6 phosphate transporter, enzymes involved in glycolytic, respiratory and fermentation pathways;

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Low-level regulation: invertase acid, plastidial thioredoxins, starch degradation enzymes, proteins involved in the conversion of plastidial triosaphosphate into cytosolic glucose-6-phosphate, proteins involved in the internal provision of amino acids such as nitrite reductase, glyceraldehyde-3-phosphate plastidial dehydrogenase, cysteine synthase, glucose-6-phosphate plastidial dehydrogenase, etc ...

Fig. 21 illustrates a suggested metabolic model for the process triggered by the microbial volatiles, deduced from the studies carried out in potato leaflets that are described later in Examples of the present application, which includes variation studies both enzymatic activities and the transcriptome, as well as analysis by means of RT-related transducts of the metabolites.

As discussed, Examples that appear later in the present memory, the regulation of sucrose synthase (SuSy) seem to be one of the determinants of starch accumulation in plants subjected to the effect of volatile microbials. But the effects observed on other enzymes, particularly the reduction of cysteine synthase, the reduction of nitrite reductase, the reduction of plastidial glyceraldehyde-3-phosphate dehydrogenase, the reduction of plastidial glucose-6-phosphate dehydrogenase, the over- Expression of the glucose-6-phosphate translocator and the overexpression of the protease inhibitor, alone combined in between, appear to be able to give place to the increase in starch content, without the need for an increase in sucrose synthase activity.

All these findings open the door to consider procedures to increase the growth of plants and / or their production of starch through their cultivation in the presence of microorganisms that produce volatile, without contact between them, or through the cultivation of plants in the presence of the volatile mixture produced by the microorganism previously cultivated in a different space from the plant's growth. Therefore, it leaves open the possibility of increasing the productivity of cultivated plants, for example, in greenhouses, co-cultivating with them microorganisms emitting volatile compounds; alternatively, the plants could come into contact with the volatiles because they were applied directly to the greenhouses, after the microorganisms were grown in large fermenters, in the appropriate media (in general, minimum media such as M9, MOPS, Murashige & Skoog (MS ) etc.). The fact that the leaves accumulate more starch in the presence of volatile microorganisms, even when these leaves are separated from the plant, allows the design of an alternative mechanism for obtaining starch in which leaves detached from plants are used, which can be waste products of the processing of them. The elucidation of the enzymes in whose activity / expression changes occur that promote the accumulation of starch allow to achieve the increase in starch production by alternative methods, based on the same inventive principle, in which changes in certain enzymes occur in the plant by the use of transgenic plants that overexpress the gene or genes of interest in which an inhibitor of the same whose activity is expressed by the presence of microbial volatiles is expressed.

In addition, the fact that the leaves separated from the whole plant are also capable of producing starch, when they maintain the presence of cultures of microorganisms that produce volatile, is very important from the point of industrial view. In a matter of 2-3 days, the leaves are capable of producing huge amounts of starch with such only 4 ingredients, which could be considered "cheap": a little water, natural CO2, natural light, volatile microbial. It can be considered that the leaf would act as a starch-producing biofactor powered by sunlight. In addition to the interest that the starch produced could have for the starch industry, the advantage of using separate leaves from the whole plants must be taken into account, as the remains of pruning that are normally destroyed (for example, potato leaves) , could be destined to the production of a type of starch of industrial interest.

Therefore, the invention refers to a method for increasing the size of a plant, its development pattern (including characteristics related to its fertility, the biomass in general and the starch in particular growing the same in the presence of volatile emitted by a microorganism , a microorganism that can be grown in the same space as the plant, so that the plant comes into contact with them because the microorganism releases these volatiles into the atmosphere in which the plant is growing, or that may have been previously collected and added artificially to the plant's growth atmosphere.

Thus, one object of the present invention is a method to increase the growth of a plant and / or alter its development pattern, characterized in that the plant is grown in the presence of a microorganism that produces volatile compounds, without any contact between the plant and the microorganism, or in the presence of volatile emitted by the microorganism, in that the microorganism is different from the isolated Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937. The increase in plant growth can be manifested in an increase in size (in length) of the plant and increase in the size of the leaves. As for the alteration of the development pattern, it can manifest itself as an increase in the number of leaves, increase in the number of branches, or in effects more closely related to fertility, such as the increase in the number of flowers and seeds of angiosperm plants, and / or the induction of flowering, or in combinations of the above effects.

The microorganism can be a bacterium, a yeast or a multicellular microscopic neuron. When the microorganisms are chosen among bacteria, a different genus can be chosen from Bacillus or Paenibacillus, which are the genus that belong to the specific strains of rhizobacteria in which a positive effect of their volatile emissions on the growth of the plant had previously been detected by growing in very rich media.

Due to the observed effect, specifically, on the increase of starch accumulation, another object of the invention is a method for increasing the production of starch in a plant, characterized in that the plant is grown in the presence of a microorganism that produces volatile compounds, without any contact between the plant and microorganism, or in the presence of volatiles emitted by the microorganism. It is especially preferred that, in addition, the starch produced has modifications with respect to the normal structure of the plant, which may refer both to the increase in the size of the starch granules, and to the structure of the starch molecule itself, specifically, in a reduction in the amylose / amylopectin ratio (which, as explained above, decreases with respect to that found in the control plants, because there is a significant reduction in the relative amylose content), and in the decrease in the polymerization of amylopectin chains, which is less than the plants treated with volatile microbials with respect to that observed in the control plants, such as shown below in the Examples referring to the structural analysis of the starch obtained by growing the plants in the presence of microbial volatiles.

Since the increase in starch, as demonstrated in the Examples of the present application, has been observed in different organs of the plant (leaves, stems, roots and tubers), are possible embodiments of the method of increasing the accumulation of starch, preferably with modified structure, those in which the increase in starch production occurs at least in one organ of the plant, preferably selected from leaf, stem, root, seed, in plants that present it, the tuber. As happens when looking for an increase in growth and / or alteration of the development pattern, when the specific objective is the accumulation of starch, the plant can be an angiosperm, monocotyledonous or dicotyledonous. Potato or corn plants are particularly preferred. Among the microorganisms, a possible option is the fungi of the genera Alternaria or Penicillium, whose utility is demonstrated later in the Examples of the present application.

Taking advantage of the knowledge acquired by the authors about the modifications in the metabolism of plants through which microbial volatiles cause an increase in starch accumulation, an alternative to the method of increasing starch accumulation is to cause accumulation directly through the use of transgenic plants, based on the same inventive principle, in The transgenerated transgene transgene (s) results in the overexpression of some of the enzymes regulated upwards by exposure to volatiles or consists of an inhibitor of activity or expression (by interfering RNAs, for example) of some of the enzymes regulated on the low. Thus, an alternative aspect of the invention is a method to increase the production of starch in a plant, characterized in that the plant is a transgenic plant in which at least one transgene is present whose expression gives rise to a product selected from the group of: one in plant protease hybridizer (such as, for example, the one that presents the accession number in GenBank DQ16832), the rami fi cant enzyme of the starch, an inhibitor of the acid invertase (such as, for example, the one that presents the access number in GenBank FN691928), a targeted RNA against cysteine synthase (which can be deduced, for example, from the sequence corresponding to the cysteine synthase of the potato plant, with accession number in GenBank AB029512), an antisense RNA directed against glyceraldehyde-3-phosphate dehydrogenase plastidial (such as the one with the accession number in GenBank FN691929), an antisense RNA directed against glucose-6-phosphate dehydrogenase plastidial (which can be deduced, for example, from the sequence corresponding to glucose-6 phosphate dehydrogenase of potato, with access number in GenBank X83923) or an antisense RNA directed against nitrite reductase (such as, for example, the one with no. access to GenBank access FN691930). Preferred embodiments are those in which the plant expresses at least one transgene whose expression results in a product selected from the group of: plant protease inhibitor, an antisense RNA directed against cysteine synthase or an antisense RNA directed against nitrite reductase. In the latter case, the coding sequences of the transgene can be derived, for example, from the corresponding potato genome, that is, the transgene can be one that expresses the protease inhibitor whose coding sequence is represented by SEQIDNO: 67oenelqueelRNA antisense expressed by the transgene whose cysteine synthase is directed against whose cysteine synthase coding sequence is represented by SEQIDNO: 69o against nitrite reductase whose coding sequence is represented by SEQIDNO:

71. It is especially preferred that the plant expresses more than one transgene of any of the aforementioned and / or those embodiments in which the plant, in addition to one or more transgenes of one of the above groups, also has at least one transgene that it gives rise to the ectopic expression of the sucrose synthase enzyme (SuSy) (such as a transgene that expresses the potato enzyme, whose mRNA has the accession number in GenBank AJ537575) or the expression of the glucose-6-phosphate transporter (such as a transgene express) Potato transporter, whose engine coding sequence has the access number in GenBankAY163867).

Finally, the fact that the increase in accumulated starch is also observed in leaves that are not part of whole plants, but separated from them, when the leaf is maintained next to a culture of a microorganism, allows us to contemplate one more aspect of the invention. It would be a method for obtaining starch from leaves of plants separated from them, comprising a stage in which the leaves are maintained in the presence (but without physical contact) of a culture of a microorganism or volatiles issued by this. Again, the microorganisms are pre-erected by a bacterium, a microscopic yeast that contacts the plant under natural cultivation conditions. A possible preference is the fungi of the Alternaria or Penicillium genera, especially when the leaves are potato leaves.

As it has been mentioned, the method of the invention, in any of its aspects (except that which refers to the use of transgenic plants) requires that the cultivation of the plant or the placement of the detached leaves on which it is desired to induce the Starch accumulation is done so that the volatiles emitted by the microorganism are present in the atmosphere in which the plant is grown. If this condition is fulfilled, the process of the invention can be carried out in different ways.A possibility that the plant and microorganisms simultaneously grow in a same container, or that leaves are introduced into a container in which the culture of microorganisms is produced; in this case, to favor that the plant comes into contact with the volatile ones, it is preferred that the container be closed, the container is preferred, specific vessel, such as a Petri dish, in which the microorganism is grown, preferably in a solid medium. The common culture vessel of the plant and the microorganism can be a greenhouse in which, preferably, the conditions of humidity, temperature and even air circulation speed are arti fi cially controlled.

Since the process of the invention does not require contact between the microorganism and the plant, but rather they are the volatile emitted by the microorganism, it is not necessary that the microorganism and the plant be cultivated in proximity, but that the plant can only be cultured in the presence of the volatile emitted by the microorganism without the need for it to be located near the plant. Thus, it is possible to cultivate the microorganisms beforehand, in which culture conditions are chosen, picking up the volatile emitted to, subsequently, make the plant grow in the presence of such volatile, making them present in the atmosphere in which the plant is grown.

As used in the present application, the term "microorganism" includes bacteria, yeast, algae and protozoa, all of them generally unicellular, as well as multicellular microscopic fungi such as molds, which can be propagated and manipulated in a laboratory.

The microorganism used can belong to a pathogenic or non-pathogenic species for the plant, which coexists

or not with the plant in natural conditions. Said microorganism can be a bacterium, a yeast or a microscopic fungus. Among them, there is particular preference for fungi belonging to the genus Penicillium (for example Penicilliumchartesii, Penicillium aurantiogriseium) or Alternaria (for example Alternaria alternata), for yeasts of the species Saccharomyces cerevisiae and for bacteria belonging to the genera Bacillus (especially Bacillus subtilis and, for example, Bacillus subtilis 168), Salmonella (for example, Salmonella enterica LT2), Escherichia (especially, Escherichia coli and, very particularly, Escherichia coli BW25113), Agrobacterium (especially, Agrobacterium tumefaciens and, particularly, Agrobacterium tumefaciens EHA105óGV2260) or Pseudomonas (especially, Pseudomonas syringae and, very particularly, Pseudomonas syringae 1448A9, 49a / 90 or PK2).

As previously mentioned, when the microorganism is chosen among bacteria, it can be chosen from a genus other than Bacillus or Paenibacillus, which are the genera to which the specific strains of rhizobacteria belong in which a positive effect of its volatile emissions on plant growth by growing in very rich media: both genera can be excluded from the group of eligible bacteria. In any case, it has been observed that the volatile emitted by bacteria belonging to these genera, from strains other than Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937, are capable of producing the growth effect of plants when bacteria are grown in a medium that lacks organic compounds that have amino groups, particularly amino acids and / or proteins, such as minimum media supplemented with an organic carbon source, also producing an increase in starch accumulation: none of these effects were expected from previous results obtained with rhizobacterial volatiles, which seemed to indicate that the effect on growth was exclusive of volatiles emitted by certain strains. The induction of starch accumulation, moreover, had not previously been described for volatile mixtures emitted by microorganisms and, as demonstrated below in the Examples of the invention, is observed for all microorganisms with which experiments were performed: thus, as previously mentioned, considers that any bacterium, microscopic fungus or yeast can be chosen to carry out the aspect of the method of the invention specifically referred to starch accumulation, either in whole growing plants or in detached leaves.

In any of the aspects of the method of the invention that do not refer to the specific use of transgenic plants, it is particularly preferred that the growth of the microorganism occurs in a medium that lacks organic compounds that include nitrogen in its formula or, at least , lacking organic compounds that present amino groups, such as amino acids and / or proteins. Very special preference is given to the culture in minimal medium containing an organic compound as a carbon source, which may be, for example, sucrose or glucose or other organic compounds such as succinate.

Among the plants, angiosperms (both monocotyledonous and dicotyledonous) are preferred, in which there has been an increase in the number of branches and the number of flowers with respect to the control plants. This effect also allows one more aspect of the invention to be a method for inducing fl owing and for increasing the number of branches and / or fl owers produced by a plant, in which the objective is achieved by cultivating the plant in the presence of a microorganism that produces volatile compounds. , without any contact between the plant and the microorganism. When the effect sought is the increase in the number of flowers, the plant will logically have a plant capable of producing them: an angiosperm, both monocotyledonous and dicotyledonous.

The invention will now be explained in more detail using the Examples and Figures below.

Brief description of the fi gures

Fig. 1: Suggested routes of starch synthesis in leaves of origin.

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Panel (A) illustrates the "classical model," according to which the starch biosynthesis process occurs exclusively in the chloroplast, separated from the sucrose biosynthesis process that takes place in the cytosol.

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Elpanel (B) illustrates the "alternative model" in which both the biosynthetic route of sucrose and that of starch are interconnected by means of SuSy ADPG-producing activity (sucrose synthase).

Compounds involved: FBP: fructose-1,6-bisphosphate; F6P: fructose-6-bisphosphate; G6P: glucose-6-phosphate; G1P: glucose-1-phosphate; ADPG; ADP-glucose; UDPG: UDP-glucose. Enzymatic activities: 1, 1 ’: fructose-1,6-bisphosphate aldolase; 2, 2 ’: fructose-1,6-bisphosphatase; 3: PPi: fructose-6-phosphate phosphotransferase; 4, 4 ’: phosphoglucose isomerase; 5, 5 ′: phosphoglucomutase; 6: UDPG pyrophosphorylase; 7: sucrose phosphate synthase; 8: sucrose phosphate phosphatase; 9: AGP; 10: SS (starch synthase); 11: starch phosphorylase; 12, SuSy (sucrose synthase).

Fig. 2: Cultivation conditions of Arabido plants and the effect of microbial volatiles on them.

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Panels (A), (B), (C) and (D): Photographs illustrating the growing conditions of Arabidopsis plants in the absence (AyC) and presence (ByD) of microbial returnable. For the volatile effect, Petrique plates contained plants completely developed were introduced in plastic boxes that previously included cultures of E. coli BW25113 grown in solid medium M9 supplemented with 50mM glucose (panel B) and Alternaria alternata grown in solid medium MS supplemented with 90 mM sucrose (panel D). The boxes were sealed and, at the indicated incubation times, the plants were collected for analysis.

Fig. 3: Effect of volatile products produced by Alternatira alternata on fresh weight (panel A), dry weight (panel B), number of flowers (panel C), number of pods (panel D), length of the shoot (panel E) and number of branches (panel F) of Arabidopsis. The photographs of the panels of G and Hilustranel the positive effect of the volatile (FVs) in the number of flowers, sheaths, bud length and number of branches on the plant exposed during the day to the PV. A. alternata was grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 4: Conditions of cultivation of tobacco plants and the effect of microbial volatiles on the development and growth of plants:

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PanelA: Photographs that illustrate the conditions of cultivation of tobacco plants in the absence (-FV) or in the presence (+ FV) of fungal volatiles (FV) emitted by a culture of Alternaria alternata. The plants were grown for 6 days, and their comparison was carried out.

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PanelsByC: Comparison of tobacco plants grown under the same conditions, except for absence (-FV)

opresence (+ FV) of the culture of Alternaria alternata. It is observed that plants grown in the presence of fungal volatiles emitted by A. alternata are larger, a fact that can be seen particularly in the leaves and even have a greater number of leaves (panel C). In addition, it is observed that plants treated with VF flourish earlier.

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Panel D: Photographs showing whole tobacco plants, including roots, grown in the absence (-FV) or presence (+ FV) of the Alternaria alternata crop. It can be seen that the size of the root is larger in the case of plants grown under + PV conditions. A. alternata was grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 5. Effect of volatiles produced by Salmonella enterica LT2 (panel A), the strains of Agrobacterium tumefaciens indicated next to the photographs (EHA105 or GV2260) (panel B), or the strains of Pseudomonas syringae 49a / 90yPK2 (panelC) on the growth of Arabidopisis, according to the bacterial culture medium: minimum medium (M9) or LB supplemented with 50 mM glucose.

It is observed that Arabidopsis plants grown in the presence of bacteria grown on LB medium have yellow areas (lighter spots on the leaves) and smaller size and / or appearance of being sick.

Fig. 6: Effect of volatile microbial species on starch accumulation in Arabidopsis leaves.

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Panel A: starch content in leaves of Arabidopsis plants grown 2 days in solid MS medium supplemented with 90 mM sucrose, in the presence or absence of: Alternaria alternata, Penicillium charlessi, Penicillium aurantiogriseum, Pseudomonas syringae PK2, Pseudomonas syringae 49a / 90, Pseudomonas syringae 1448A9, A. tumefaciens GV2260, A. tumefaciens EHA105, E. coli (BW25113), Salmonella enterica (LT2), B. subtilis 168, Saccharomyces cerevisiae NA33. All microorganisms, except S. cerivisiae, Penicillium aurantiogriseum, and Penicillium charlessi were grown in M9 solid medium supplemented with 50 mM glucose. P. aurantiogriseum, P.charlessi and A. alternatase grew in solid MS supplemented with 90 mM sucrose.

Panel B: quantification of starch content in leaves of Arabidopsis plants, grown in the presence of different bacteria and fungi as indicated under the corresponding bars, bars that are grouped according to the microorganism culture medium: minimum medium (M9) solid supplemented with 90 mM glucose (unfilled bars) or solid LB supplemented with 90 mM glucose (dark filled bars). It is observed that the positive effect on the increase in starch only takes place when microorganisms grow in minimal medium.

Panel C: starch content in leaves of Arabidopsis plants grown under the same growing conditions as in panel A, in the presence of coal, a crop of A. alternata. A. alternata in the presence of coal, or an initial culture of Alternaria removed during the following 3 days, as indicated under the bars. The decrease in the inductive effect is observed in the presence of coal and the disappearance of the effect after 3 days outside of contact with fungal volatiles. A. alternata was grown in solid MS medium supplemented with 90 mM sucrose.

Panel D: quantification of starch content in stems of Arabidopsis grown in the absence (-FV) or in the presence (+ FV) of fungal volatiles produced by A. alternata. A very strong increase in starch is observed in the stalks under + PV conditions. A. alternata was grown in solid MS medium supplemented with 90 mM sucrose.

PanelE: starch content in roots of Arabidopsis grown in the absence (∅) or in the presence (+ FV) fungal devoltants produced by A. alternata. There is also a strong increase in starch in the roots in conditions + FV. A. alternata was grown in solid MS medium supplemented with 90 mM sucrose.

FyG Panels: comparison oflabelomasa observed in leaves (first single barred, stitched fill) and in roots (second bar of each pair, continuous darker filling) of Arabidopsis, in the absence (-FV) or in the presence (+ FV) fungal return produced by A. alternata. The effect is observed both if the fresh weight (panel F) is determined as the dry weight (panel G). A. alternata was grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 7: Effect of fungal volatiles on corn plants and Arabidopsis grown on land.

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PanelA: Photographs of spider plants grown on land, in the absence (-FV) or presence for 6 days (+ FV) of fungal volatiles produced by fungi Alternaria alternata grown in solid MS medium supplemented with 90 mM sucrose. It is observed that plants grown in the presence of volatiles are more robust. As demonstrated in Panel B, they accumulate more starch.

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Panel B: Starch content, expressed in glucose micromoles per gram of wet weight (FW), of the leaves of corn plants in panel A. Control: plants grown in the absence of fungal volatiles; + Fungus: plants grown in the presence for 6 days of a crop of A. alternata that emits fungal volatiles.

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PanelC: Photographs of Arabidopsis plants grown on land, in the absence (-FV) or presence (+ FV) fungal return produced by fungi Alternaria alternata grown in solid MS medium supplemented with 90 mM sucrose. As demonstrated in PanelPanel, they accumulate more starch.

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Panel D: Starch content, expressed in glucose micromoles per gram of wet weight (FW), of the leaves of Arabidopsis plants of panel C. -VF: plants grown in the absence of fungal volatiles; + FV: plants grown for 6 days in the presence of a crop of A. alternata that emits fungal volatiles.

Fig. 8: Visual and microscopic examination of tissues of plants grown in the absence or presence of volatiles emitted by Alternaria alternata grown in solid MS medium supplemented with 90 mM sucrose:

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Panels A and B: iodine staining of complete Arabidopsis plants grown in the absence or in the presence of PV, respectively.

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PanelsCyD: analysis of iodine stains by optical microscopy of cross sections of leaves of plants grown in the absence or in the presence of PV, respectively. Inserts: Where staining intensity and distribution of positive material for iodine in individual mesophilic cells.

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Panels E, F, G: confocal laser scanning microscopy of plant leaves expressing GBSS-GFP, grown in the absence (E) or presence (FyG) of PVs.

Bar = 5µm in E, F, G; Bar = 100 µm in CyD.

Fig. 9: Ubiquity of the effect of microbial volatiles between plants. Graphs depicting the amount of starch (expressed as micromoles of fresh glucogram, weight gram of the corresponding leaf extract) detected in Arabidopsis (A), potato (B), corn (C), barley (D) and (E) tobacco grown for 3 days in medium Solid MS, with or without 90 mM sucrose, as indicated below the bars, in the presence (bars with the legend "+ FV") or in the absence (bars with the legend "-FV") of volatiles emitted by a crop of A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 10: Graphs depicting the amount of starch (expressed as micromoles of glucose per fresh gram of the corresponding plant extract) accumulated in tubers (panel A) and tails (panel B) of potato plants grown in solid MS medium without sucrose in the presence or absence of VF emitted by A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 11: Accumulation of starch in leaves detached from the plant promoted by fungal volatiles emitted by a culture of A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

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Panels A, B, C, D: Graphs depicting the amount of starch (expressed as micromoles of fresh gram-positive glucose of the corresponding foliar extract) detected in Arabidopsis leaflets (A), potato (B), corn (C), barley (D) maintained for 2 days in solid MS medium (90mM sucrose conosin, as indicated below the bars), in the presence (bars with the legend “+ FV”) or in the absence (bars with the legend “-FV”) of volatiles emitted by a culture of A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

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Panel E: Photographs illustrating the conditions under which the leaves of potato plants were maintained in the absence (column labeled "-FV") or in the presence (column labeled "+ FV") of microbial volatiles.

Fig. 12: Accumulation of starch in detached leaves of the plant grown on wet paper surface in liquid MS or in water promoted by fungal volatiles emitted by a culture of A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

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Panel A: Graph depicting the amount of starch (expressed as micromoles of glucose per gram of fresh weight of the corresponding leaf extract) detected in tobacco leaves held for 2 days in paper in water or in liquid MS (90 mM sucrose, as indicated under bars) , in the presence (bars with the legend "+ FV") or in the absence (bars with the legend "-FV") of volatiles emitted by a culture of A. alternata.

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PanelB: Photographs that illustrate the conditions in which the leaves of cultivated tobacco plants were maintained on their wetted surface in liquid or in water in the absence (column with the “-VF” label) or presence (column with the “+ FV” label) of microbial volatiles emitted by A. alternata.

Fig. 13: Relative abundance, expressed as a factor of variation, of the levels of transcripts of the indicated genes in abscissa, measured by quantitative real time (RT) -PCR, in leaves of potato plants grown in the presence of fungal volatiles emitted by A. alternata grown in MS medium solid supplemented with 90 mM sucrose. Variation factors represented are relative to the control leaves of cultivated plants in the absence of PVs. The plants were grown for 3 days in the presence of solid FVsen medium medium supplemented with 90 mM sucrose, and collected at the end of the light period. The levels of their Sysy (sucrose synthase) and glucose-6-P translocator were measured both in the presence (+ sac) and in the absence (-sac) of sucrose.

Fig. 14: Functional categorization of differentially expressed transcripts in potato leaves grown in supplemented MS (panel A) or not (panel B) with 90 mM sucrose in the presence of fungal volatiles emitted by A. alternata grown in solid MS medium supplemented with 90 mM sucrose. The transcripts were identified using the 60-meter oligo array POCI 44K (http://pgrc.ipk-gatersleben.de/poci). Regulated transcripts significantly alzay and alabaja (difference of 2.5 times in plants grown with sucrose and difference of 1.9 times in plants grown without sucrose) compared to controls, were classified according to their theoretical functional category according to MapMan software. This category is indicated under the abscissa axis. The number of genes deregulated in each categorical group is indicated in ordinates. The upregulated genes appear on bars with lighter fill and the downward regulated genes appear on bars with darker fill. The genes of the category "no effect found" were not included in the graph.

Fig. 15: Analysis of AGP in Western blot transfer of potato plant leaves, under non-reducing conditions (without 10 mM dithiothreitol) and reducing (with 10 mM dithiothreitol: + DTT). Whole plants were grown for 3 days in solid MS supplemented with sucrose90mM in the presence ("+ FV") or in the absence ("- FV") of volatiles emitted by A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 16: Graphs showing that changes in AGP activity play a minor role in the accumulation of volatile induced starch in potato blades: (A) AGP activity, (B) starch content, and (C) PGD content in wild-type plant leaves ( WT) and AGP62 (antisense plants of the small subunit of the ADPG pyrophosphorylase) grown in the presence ("+ FV") and in the absence ("-FV") returned by A. alternata grown in solid MS medium supplemented with 90 mM sucrose. The whole plants were grown for 3 days in solid MS medium supplemented with 90 mM sucrose.

Fig. 17: Graphs depicting, in potato leaves grown in the presence (+ FV) or in the absence (-FV) of fungal volatiles emitted by Alternaria alternata grown in solid MS medium supplemented with 90 mM sucrose: the activity of Its producer of ADPG (panel A), and the intracellular contents of starch (panel B), ADPG (panel C) andUDPG (panel D), all expressed with reference to grams of fat. There is a correlation between the activity of SuSy (sucrose synthase) and the contents of the other compounds.

Fig. 18: Analysis of SuSy in Western blots on leaves of potato plants grown in the presence

(A) and absence (B) of sucrose treated and not treated with VF emitted by A. alternata grown in solid medium supplemented with 90 mM sucrose.

Fig. 19: Graph showing the starch content (expressed as µmoles of glucose per gram of fresh weight) measured in potato plants grown for 2 days in MS supplemented with sucrose90mM and the indicated concentrations of cysteine, glycine, serine and methionine in the absence (-FV) or in presence (+ FV) returned by A. alternata grown in solid MS medium supplemented with 90 mM sucrose.

Fig. 20: Fungal volatiles promote both the reduction of amylose content and changes in the composition of amylopectin.

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Panel (A): Percentage of amylose with respect to amylopectin in leaves of potato plants grown in the presence (+ FV) and in the absence (-FV) fungal return emitted by A. alternata grown in solid medium supplemented with 90 mM sucrose.

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Panel (B): Chain length distribution profiles (degree of polymerization: GP) in branched amylopectin purported to distribute cultivated potato plant leaves in the presence and absence of PVs (black and white bars, respectively).

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Panel (C): Difference between distributions of purified branched amylopectin chain lengths in the presence and absence of fungal volatile emitted by A. alternata grown in solid MS medium supplemented with 90 mM sucrose, calculated as the difference between the profile in the presence of less FVs the profile in the absence of PV.

Fig. 21: Schematic representation of the main carbohydrate metabolism pathways that occur during MIVOISAP according to the alternative vision of starch biosynthesis.The panel represents the situations that are likely to occur when plants are grown in heterotrophic conditions, while the panel It represents situations that are likely to occur when plants are grown under autotrophic conditions.The changes in the expression of genes that encode major enzymes of carbohydrate metabolism indicate by variations in the gray scale and in the continuity of the lines (dashed gray lines, increase; continuous gray lines, decrease; black lines, no differences signi fi cant).

Examples

Examples of the present application include tests performed with the following methodological materials and techniques:

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Plants, microbial cultures, growth conditions and sample collection

This work was carried out using Arabidopsis thaliana plants (cv. Columbia), potato (Solanum tuberosum

L. cv Desirée), tobacco (Nicotiana tabacum), maize (Zea mays, cv.HiII), barley (Hordeum vulgare cv. Golden promise) grown in PlatesPetrique contained sucrose medium with sucrose-free 90mMyla supplementation with amino acids indicated. The plants were grown in growth chambers with a photoperiod of 16h of light (300 µmol photons s − 1m − 2) and at a constant temperature of 24 ° C.

E. coli BW25113, A. tumefaciens EHA105 and GV2260, Salmonella enterica LT2, Bacillus subtilis 168 (Bacillus Genetic Stock Center, Ohio State University, Columbus) and Pseudomonas syringae 1448A9, 49a / 90yPK2 were grown in Petri dishes containing medium M9 solid (95 m solid) Na2HPO4 / 44 mM KH2PO4 / 17 mM NaCl / 37 mM NH4Cl / 0.1 mM CaCl2 / 2 mM MgSO4, 1.5% bacteriological agar) supplemented with 50 mM glucose. S. cerevisiae NA33 were grown on plates containing LB solid medium (1% tryptone, 1% NaCl, 0.5% yeast extract and 1.5% bacteriological agar) supplemented with 50 mM glucose. The colonies of Penicillium chartesii and Penicillium aurantiogriseiumo of Alternaria alternata were grown in Petri dishes containing solid MS medium supplemented with sucrose90mM.

Microbial cultures were placed in sterile plastic boxes and sealed. After two days, the Petri dishes containing fully developed plants were placed in boxes containing microbial cultures, as illustrated in Fig. 2.Lascajassesellaron and lassholes were collected after incubation times indicated for biochemical and transcriptome analyzes. Unless stated otherwise, the leaves were collected at the end of the light period. As a negative control, Petri dishes containing fully developed plants were grown in sealed plastic boxes along with Petri dishes that possessed sterile medium for the cultivation of microorganisms.

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Western Type Transfer Analysis

To produce polyclonal antisera against Sus4, a full-length Sus4 coding cDNA was cloned into the expression vector pET-28b (+) (Novagen) to create pET-SuSy. BL21 (DE3) cells transformed with pET-SuSy were grown in 100ml of liquid medium to an absorbance at 600nm of 0.5 and then added isopropyl-β-D-thiogalactopyranoside 1m. After 5h, the cells were centrifuged 6,000xg for 10 min. ), treated with ultrasound and centrifuged at 10,000xg for 10 min.The supernatant thus obtained sesometióa chromatography in His-bind (Novagen). Sus4 labeled His eluted was rapidly desalted by ultracentrifugation in a YM-10 Centricon (Aicon, Bedford, MA). The purified protein was electrophoretically separated by preparative PAGE with 12% SDS and stained with Coomassie Blue. A band of proteins of approximately. 90 kDa was eluted and used to produce polyclonal antisera by immunizing rabbits.

For immunoblotting analyzes, the protein samples were separated on PAGE with 10% SDSal, nitrocellulose fi lters were transferred, and immunodecorated using antisera obtained against AGP of SuSy maize potato as primary antibody, and a IgG of goat anti-rabbit conjugated confosphatase as secondary antibody. In the case of Western-type AGP transfers, seextrajerony samples were separated on SDS-PAGE under reducing / non-reducing conditions essentially as described by Kolbe et al. (Kolbe et al., 2005: Trehalose 6-phosphateregulatesstarchsynthesisvia posttranslationalredoxactivationofADPglucosepyrophosphci.lst. 102: 11118-11123).

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Enzymatic assays

1 g of frozen leaf powder was resuspended at 4 ° C in 5 ml of 100 mM HEPES (pH 7.5) and 2 mM EDTA. When indicated, 5 mM DTT was added to the extraction buffer. The suspension was desalted and enzyme activities were tested therein. The activities test AGP, SuSy, acid invertase, total PPasaySS was performed as described by Baroja-Fernández et al. (Baroja Fernández et al., 2009: Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and totalyield, Plant Cell Physiol. 50: 1651-1662). The β-amylase assay was performed as described by Liu et al. (Liu et al., 2005: Toxicity of arsenate and arsenite on germination, seedling growth and amylolitic activity of wheat, Chemosphere 61: 293-301). SBE activity was measured as the decrease in absorbance of the amylose-iodine complex as described by Vos-Scheperkeuter et al. (Vos-Scheperkeuter et al., 1989: Immunological comparison of the Starch Branching Enzymes for potato tubers and Maize leaves, Plant Phylio. 90: 75-84). Nitrite reductase was measured following the method described by Rao et al. (Rao et al., 1981: Phytochrome regulation of nitrite reductase-a chloroplast enzyme-in etiolated maize leaves, Plant Cell Physiol, 22: 577-582). PG6PDH was measured according to Hauschild et al. (Hauschild et al., 2003: Differential regulation of glucose-6-phosphate dehydrogenase isozyme activities in potato, Plant Physiol. 133: 47-62). The cytosolic fructose-1,6-bisphosphatase assay was performed as described by Lee and Hahn (Lee and Hahn, 2003: Light-regulated differential expression of pea chloroplast and cytosolic fructose-1,6-bisphosphatase, Plant Cell Rep. 21: 611618). One unit (U) is defined as the amount of enzyme that catalyzes the production of 1 µmol of product per min.

-

Determination of ADPG, UDPG and 3-phosphoglycerate

An aliquot of 0.5g of the frozen powdered vegetable green tissue in liquid nitrogen was resuspended in 4ml of HClO41.0M, was set at 4 ° C for 2h and was centrifuged at 10,000g for 5min. The supernatant was neutralized with K2CO35M, 10,000g was centrifuged and subjected to a measurement of nucleotide measurement as it had been carried out by the nucleotide. (Muñoz et al., 2005: Sucrose synthase controls the intracellular levels of ADPglucose linked to transitory starch biosynthesis in source leaves, Plant Cell Physiol. 46: 1366-1376) by HPLC in a system obtained from P.E.Watersand Associates coupled to a columnPartisil -10-SAX. 3-fosfogliceratose measured as has been described by Muñoz et al. (Muñoz et al., 2005: Sucrose synthase controls the intracellular levels of ADPglucose linked to transitory starch biosynthesis in source leaves, Plant Cell Physiol. 46: 1366-1376).

-

Analytical procedures

Starch was measured using a test kit based on amyloglucosidase (BoehringerMannheim, Germany). The amylose content of starch was measured household and according to Hovenkamp-Hermelink et al. (Hovenkamp-Hermelink et al., 1988: Rapid estimation of the amylose / amylopectin ratio in small amounts of tuber and leaf tissue of the potato Potato Res. 31: 241-246). The side chain distribution analysis of isolated starch was carried out by HPAEC-PAD essentially as described by Abel et al. (Abel et al., 1996: Cloning and functional analysisofa cDNAencodinga novel 139 kDa starch synthase from potato (Solanum tuberosum L.), Plant

J. 10: 981-991) using a DX-500 (Dionex) system coupled to a CarboPacPA10 column.

-

Microarray

The total RNA was extracted from frozen potato leaves using the Trizol method following the manufacturer's procedure (Invitrogen), followed by purification with the RNeasy kit (Qiagen). The amplification, labeling and analysis of statistical data related to RNA were carried out basically as described by Adie et al. (Adie et al., 2007: ABAis an essential signal for plant resistance to pathogens affecting JAbiosynthesis and the activation of defenses in Arabidopsis, PlantCell19: 1665-1681) .For hybridization, Agilent POCI4 x44 (015425) slides were used containing 246,000 sequence tags corresponding to the sequence 46,345 unigenes (http://pgrc.ipk-gatersleben.de/poci) (Kloosterman et al. 2008: Genes driving potato tuber initiation and growth: identification based on transcriptomal changes using the POCI array, Funct Integr. Genomcs 8: 329340) . The conditions of labeling and hybridization were those described in "The manual two color microarray based gene expression Analysis" of AgilentTechnologies. For the leaflets treated with microorganisms and control plants, three independent biological replicas were hybridized. with respect to the differences in intensity and were captured with a GenePix 4000B scanner (Axon). The points were quantified using GenPix software (Axon) and normalized using the Light method. The means of the logarithms of the intensities ratios for the three replicas were calculated and their deviations are in the expression data analyzed statistically using the LIMMA software package (Smyth and Speed, 2003: Normalization of cDNAmicroarray data, Methods31: 265-273). Functional characterization of differentially expressed genes was done using the Mapman tool (http://gabi.rzpd.de/projects/MapMan/).

-

Quantitative PCR in Real Time

Total RNA was extracted from potato leaves as was done for microarray experiments. RNA was treated with RNAase-free DNAase (Takara). Reverse transcription of 1.5 µg of RNA was performed using depoliTyelkitExpandReverseTranscriptase (Roche) primers following the manufacturer's instructions.RT-PCR reaction was carried out using a 7900HT (Applied Biosystems) sequence detection system with the SYBR Green PCR MasterMix (Applied Biosystems) according to the main protocol. Each reaction was carried in triplicate with 0.4 µL of the first cDNA chain in a total volume of 20 µL. The specificity of the PCR ampli fi cation was checked with a dissociation heat curve (from 60 ° C to 95 ° C). The comparative threshold values were normalized with respect to an internal control of RNA18S and were compared to obtain relative levels of expression. The specificity of the RT-PCR products obtained was monitored in 1.8% agarose gels. The primers used for the RT-PCRs are listed below in Table 1:

TABLE 1

Real Time Quantitative PCR Primers

-

Iodine staining and microscopic location of starch granules

The leaves collected at the end of the light period were fixed by immersion in formaldehyde at 3.7% of the phosphate. Then the foliar pigments with 96% ethanol were removed. The rehydrated samples were stained with iodine solution (KI2% (w / v) I21% (w / v)) for 30min, quickly immersed in deionized water and photographed. The sheets for microscopic starch observation were mounted on microscope slides and seexaminated by confocal microscopy using excitation laser of Ar 488. Samples from which sections were to be obtained were immersed in OCT cryoprotectant medium (Tissue-Tec, USA) and frozen - 50 ° C. 10 µm thick cryosections were obtained on an AS620 cryotome (Shandon, England). After defrosting, the sections were stained with iodine solution for 2 min at room temperature, mounted on microscope slides and observed using an Olympus MVX10 stereo microscope (Japan). The photomicrographs were captured with a DP72 video camera (Olympus, Japan) and CellD software (Olympus, Japan).

-

Confocal microscopy

The GBSS-GFP subcellular location was performed using a D-EclipseC1 (NIKON, Japan) microscope equipped with a standard 488 excitation laser, a BA515 / 30 filter for green emission, a BA650LP filter for red emission and a transmitted light detector for bright field images.

Example 1

Volatiles emitted by different microbial species promote changes in plant development, growth growth and accumulation of starch in leaves of different plant species

1.1. Effect on growth

To check the possible effects that volatile chemical compounds released by microorganisms could have on plant metabolism, trials were first conducted with Arabidopsis plants grown in MS medium in the presence or absence of Escherichia coli BW25113, Agrobacterium tumefaciens EHA105 and GV2260, Saccharomyces cultures. cerevisiae NA33, Bacillus subtilis 168, Penicillium charlesiio Penicillium aurantiogriseum, Salmonella enterica LT2, Alternaria alternata, Pseudomonas syringae 1448A9, 49a / 90oPK2 and in the absence of physical contact between the plant and the culture medium. controls with plates with culture medium in which microorganisms had not been seeded analogously to that presented in panels A and Cdela Fig. 2.

Preliminary visual analyzes revealed that volatiles emitted by these microorganisms promote plant growth, as can be observed by comparing the first four panels of Fig. 2. This effect was confirmed by checking the fresh and dry weight of Arabidopsis plants grown in the absence or presence of volatile emitted by Alternaria alternata fungi, as well as other parameters related to the development of plants such as the number of fl owers, branches, or the height of the bud. As can be seen in Fig. 3, all These parameters were increased by the effect of volatiles, an effect that was also observed on the starch content.

This effect is not exclusive to Arabidopsis plants, but is observed in other species, such as in tobacco plants, as can be seen in Fig. 4, in which the plants subjected to the effect of volatiles emitted by fungi Alternaria alternata show larger size and greater number of leaves. Also the roots of the tobacco plants grew more in the presence of volatile Alternaria alternata.

In the specific case of some microorganisms, such as E. coli, that this bacterium promotes plant growth seems to contradict the observations made by Ryu et al. (Ryu et al. 2003: Bacterialvolatiles promote growth in Arabidopsis, Proc. Natl. Acad. Sci. USA 100, 4927-4932), who showed that E. coli DH5α does not promote the growth of Arabidopsis plants. In addition, these authors showed that volatiles emitted by Pseudomonas syringae have a negative effect on the growth and development of Arabidopsis. Therefore, it was thought that variations in growth conditions and test systems could justify the different results. Specifically, it was raised whether the results obtained in Arabidopsis by other authors could be due to the use of media containing yeast extracts, rich in amino acids, such as the BobKornberg media, while the growth of microorganisms in minimal medium could result in no volatile metabolite harmful to the plant.

To test this hypothesis, the test was repeated, using Salmonella enterica as a microorganism, culturing it or in minimal medium M9 or in LB medium. The results, shown in Fig 5A, show that the volatile produced by Salmonella enterica grown in LB causes the plants to turn yellow and yellow, while, when comparing the Arabidopsis plants grown in minimal medium M9 compared with the control plants maintained in identical conditions , but without having planted any microorganism in the corresponding Petri dish, it is observed that the plants grown in the presence of the largest microorganisms and present a large number of leaves.

Similar results were obtained with other plant pathogens, such as Agrobacterium tumefaciens, with either strain EHA105 or GV2260 (see Fig. 5B) or Pseudomonas syringae (see Fig. 5C): the positive effect on growth has always occurred and when the pathogen grows in a minimal medium, such Like MO9, which has its inorganic salts composition as sources of Na, K, Ca, Mg, P, Ny and Syque, except for the sugar used as a carbon source supplement (sucrose-glucose in the Examples of the present application) and the bacteriological agar itself, lacking other organic compounds, in particular compounds containing nitrogen-organic. Volatiles emitted by microorganisms grown in media such as LB, Kornberg or any other medium that contains a hydrolyzate of yeasts, proteins, or that are rich in amino acids, exert a negative effect (onotan positive as observed when minimum media are grown) on the growth and production of starch in the plant, so that plant plants are exposed such as only volatile, develop. All of this is probably due to the toxic effect of ammonium produced by the deamination of amino acids present in the environment.

1.2. Effect on starch accumulation

Next, the starch content of the leaves of the Arabidopsis plants, which had been grown in MS medium in the presence or absence of cultures of S. cerevisiae NA33, B, was measured. subtilis 168, Salmonella en-terica (LT2), E. coli (BW25113), A. tumefaciens EHA105, A. tumefaciens GV2260, Pseudomonas syringae 1448A9, Pseudomonas syringae 49a / 90, Pseudomonas syringae PK2, Penicillium aurantlternaria. microorganisms, except S. cerivisiae, were grown in M9 solid medium supplemented with glucose. All, in turn, grew in the absence of physical contact between the plant and the culture medium.

The results obtained show that all the microbial species tested emit volatile compounds that favorably affected starch accumulation, such as with quantitative measurement of starch using a test kit based on amyloglucosidase / hexokinase / glucose-6P dehydrogenase (Fig. 6A). This effect is due to volatile because (a) the lower inductive effect in the presence of activated carbon and (b) the effect disappears after 3 days out of contact with the volatile.

The difference in the accumulation of starch was also verified when the microorganisms Agrobacterium tumefaciens, Pseudomonas syringae, Penicillium charlesii, E. coli and Salmonella enterica were cultivated well in minimal medium M9, medium medium LB.As can be verified in Fig. 6B, the “positive” effect of microorganisms on the accumulation of solid starch it only takes place when they are grown in a minimum (M9, MOPS or even MS), while the effect is inferior or zero when the microorganisms grow in a medium rich with amino acids.

Additionally, it was found that starch accumulation also occurred in other organs of Arabidopsis plants, such as the stem or roots, when they were grown in the presence of fungal volatiles emitted by fungi Alternaria alternata. Samples of the accumulation in these tissues can be observed in the DyEdelaFig panels.6 The FyG panels show the increase in the accumulation of biomass produced by the fungal volatile in both leaves and roots.

The starch accumulation effect is not exclusive to Arabidopsis plants. In addition, the positive effect is confirmed if the plants are grown on land instead of in vitro culture plates: tests carried out with corn plants and Arabidopsis grown on land show an increase in size and vigor of the leaves when they grow in the presence of fungal volatiles emitted by Alternaria fungi alternata (Fig. 7A and 7C), a positive effect that is also reflected in an increase in starch accumulation (Fig. 7B and 7D).

To confirm that it was being measured was really starch, the authors of the invention characterized leaves that had been previously dyed with iodine solutions. In addition, a single analysis of confocal fluorescence fluorescence microscopy was carried out, expressing the granido starch synthesized starch (GBSS) of Arabidopsis fused with the green fluorescence protein (GFP) (Szydlowski et al., 2009: Starch granule initiation in Arabidopsis class III or the presence of either class III either of the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III or the presence of either class III required Plant Cell 21, 2443-2457) cultured in the presence and absence of PV (fungal volatile) emitted by Alternaria alternata. As shown in Fig. 8A and 8B, these analyzes revealed that the iodine staining of leaves of crop plants in the presence of PVs was much darker than the plants control. Microscopic analysis (Fig. 8C and 8D) of leaf sections showed that iodine staining was located within the chloroplasts of mesophilic cells. In addition, the laser scanning microscopy analysis of transgenic sheets expressing the GBSS-GFP starch granule marker showed that the starch granules were much higher when the plants were grown in the presence of PVs that were in the control conditions (Fig. 8E, 8Fy8G). These analyzes show that the increase in starch content is not due to the increase in the number of granules per plastide, but to the dramatic increase in size of the starch granules.

It was confirmed if the existence of the starch accumulation process induced by microbial volatiles (volatile microbial induced starch accumulation process: MIVOISAP) was extended in the plants by measuring the starch content in leaves of Arabidopsis plants, potato (Solanum tuberosum L.), maize (Zea mays), tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) grown in the presence or absence of FVs transmitted by Alternaria alternata. These experiments were carried out using only cultivated plants in the middle of solid MS with no supplementation with sucrose90mM. As shown in Fig. 9, these analyzes revealed that, regardless of the presence of sucrose in the culture medium, the starch content in the leaves of the five species was excessively higher when the plants were grown in the presence of PV with respect to PV. absence of PV. It is worth noting that the levels of starch in potato plants grown in the presence of VF (approximately 500-600 µmol glucose / g fresh weight) were comparable to those known for potato tubers (Baroja-Fernández et al., 2009 : Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield. Plant Cell Physiol. 50, 1651-1662). In addition, as shown in Fig. 10, the presence of PVs stimulates starch accumulation in tubers and potato potatoes. Thus, the global data demonstrated that MIVOISAP occurs ubiquitously between plants and in different organs.

Example 2

FVs promote the accumulation of starch in detached leaves

To investigate whether the volatile microbials that promote the accumulation of starch in the leaves are perceived in the leaves or in other parts of the plant, the starch content in leaves detached from potato, corn, Arabidopsis and barley tobacco grown in medium was measured Solid MS (with or without sucrose) in the presence or absence of VFs emitted by the fungal species Alternaria alternata.

Regardless of the presence of sucrose in the culture medium, the leaves treated with VF accumulated much higher levels of starch than the control plants (Fig. 11), thus showing the global data that the VFs that promote the accumulation of starch are perceived on the sheets. This effect was observed not only when the leaves were grown in solid MS medium, but also when kept on liquid MS medium or water (Fig. 12). It should be noted that the detached leaves of the five species analyzed accumulated more starch than the unidasa leaves in the whole plant, which indicates that (a) abiotic stress can promote, to some extent, the accumulation of starch in leaves and (b) exert a positive effect on MIVOISAP.

Example 3

Profile of transcriptomes of potato leaves grown in the presence of VF

To better understand the phenomenon of starch accumulation in leaves promoted by microbial volatiles, high-performance transcriptome analysis of potato plant leaves grown in MS medium (conosin sucrose) was carried out in the presence and absence of PVs emitted by Alternaria alternata using the array of oligos 60-mer POCI 44K (http://pgrc.ipk-gatersleben.de/poci) (Kloosterman et al. 2008: Genes driving potato tuber initiation and growth: identification based on transcriptomal changes using the POCI array, Funct. Integr. Genomcs8, 329-340).

When the plants were grown in MS supplemented with sucrose, 3019 genes were found to be deregulated in the presence of PVs (more than 2.5 times of difference with respect to the control; P value <0.05), 1203 of which were genes classified as “no assigned function”. Among this population, 1192 genes were regulated up to 1827 genes were down regulated.

When the plants were cultured in MS without sucrose, it was found that 2856 genes were deregulated in the presence of PVs, 1109 of which were genes classified as "no function assigned." Among this population, 1671 genes were regulated alzay and 1185 genes were reguladosala low. The PCR analyzes with quantitative time transcription, (RT) -PCR, of some of the identi fi ed genes (Fig. 13) validated the results of the array analysis.

To determine the biological processes affected by the microbial volatiles, an analysis of the genes was carried out using the MapMan tool (Thimm et al. 2004: MAPMAN: a user-driven toolto displaygenomics data sets onto diagrams of metabolic pathway and other biological processes , Plant J. 37, 914-939) (http://gabi.rzpd.de/ projects / MapMan /). As reflected in the graphs in Fig. 14, this study revealed that VFs promoted drastic changes in expression of genes involved in multiple processes such as carbohydrate metabolism, amino acids, sulfurylipids, cell redox status, development, cell wall biosynthesis, photosynthesis, secondary metabolism, translation and protein stability, traffic of vesicles, signaling, energy production and stress responses. Fig. 14 gives an overview of the metabolic processes involved in the changes, both in the presence (panel A), and in the absence (panel B) of sucrose.

It should be noted that there were no changes in the expression of numerous genes encoding proteins that are thought to be involved in the metabolism of sucrose starch such as β-glucan / water dikinase, plastidial adenylate kinase, plastidial hexokinase, plastidial phosphoglucose isomerase, plastid phosphoglucomutase , the small catalytic subunit of laAGP, ADPG pyrophosphatase, starch synthase (SS) classes I and II, sucrose transporters, UDPglucose (UDPG) pyrophosphorylase, cytosolic phosphoglucose isomerase, cytosolic phosphoglucomutase, sucrose phosphate synthase and alkaline invertase.

Example 4

Detailed analysis of functions linked to starch metabolism

The transcriptome analysis studies were complemented with variation analysis studies on the enzymatic activity of different enzymes, performed as previously described in the section “Enzymatic assays.” The enzymes studied and the results obtained (expressed in milliunities per grams of fresh weight) are summarized below in Table 2:

TABLE 2

Enzymatic activity of starch metabolism enzymes

These data were considered in combination with the data obtained from the transcriptome analysis, to study in detail some functions that were affected by the treatment with VF, which are directly or indirectly linked to the metabolism of starch. In the following sections the analysis performed for each of these functions is exposed.

4.1. Changes in AGP activity are not decisive in MIVOISAP in potato plants

It is widely assumed that AGP is the main limiting step in the biosynthesis of starch, and the only enzyme that catalyzes the production of ADPG linked to starch production (Neuhaus et al. 2005: No need to shift the paradigm the metabolic pathwayotransitorystarchinleaves, TrendsPlantSci.10,154-156; Streb et al. 2009: Thet et al. 2009: Thet debate on the pathway of starch synthesis: a closer look at low-starch mutants lacking plastidial phosphoglucomutase supportsthe chloroplast-localizedpathway, PlantPhysiol. 151, 1769-1772). In these leaves, heterotetrameric enzyme is activated allosterically by 3-phosphoglycerate and is inhibited by Pi. It comprises two types of subunits, homologous but distinct, the small one (APS) and the large one (APL), which is coded by three different genes (APL1, APL2 and APL3) (Crevillén et al. 2005: Differential pattern of expression and sugar regulation of Arabidopsis thaliana ADPglucosepyrophosphorylase -encoding genes, J. Biol. Chem. 280, 8143-8140).

Therefore, the possible influence of the GAP activity (ADP-glucose pyrophosphorylase) was studied, and changes in its two subunits, on the MIVOISAP. The studies were conducted on potato plants grown in the presence of Alternaria alternata crops in a manner similar to that described in previous Examples.

Transcriptome analyzes revealed that, of the two subunits of the AGP, the levels of minor subunit transcripts (APS) remained unchanged after treatment with VF. On the contrary, the expression of one of the genes that encodes the major subunit (APL1) was regulated upwards (14.98 times increase), while another of the genes also coded the larger subunit, APL3, was regulated under the low (decrease of 8.8 times) as It was also confirmed by quantitative RT-PCR analysis (Fig. 13).

As reflected in Table 2 above, the total activity of the AGP was slightly altered by the PV treatment (increase of 1.5 times and decrease of 1.5 times when the AG activity was measured in the presence or absence of 5 mM dithiothreitol (DTT), respectively). In addition, Western type transfer analyzes reveal no apparent difference in the amount of AG between the treated and untreated leaves with PVs (Fig. 15).

The treatment with PVsdio resulted in an increase of approximately 35% in the levels of intracellular 3-phosphoglycerate (494.0 ± 36.0 µmol / g FW in the absence of VF vs. 674.1 ± 127.5 µmol / g FW in the presence of VF), which It would indicate that the AG is slightly activated during the MIVOISAP.

Since AGP catalyzes the reversible conversion of ATP and glucose-1-P into ADP and G-pyrophosphate (PPi), it is considered that alkaline pyrophosphatase (PPase) plays a crucial role in starch biosynthesis, since it displaces the reaction of the AGP from equilibrium by rapid withdrawal of PCR, quantitative PCR-analysis. (Fig. 13) and transcriptome analyzes revealed that treatment with fungal volatiles resulted in down-regulation of the PPase (reduction of 3.72 times), which was accompanied by a reduction in PPase activity (see Table 2 ). It is thus conceivable that, with the PV treatment, PPi will accumulate in the chloroplast, thus preventing the production of ADPG mediated by AGP.

The synthesis of the starch is regulated by the post-translational redox activation of AGP by thioredoxins (Ballicora et al. 2000: Activationofthe potato tuberADP-glucosepyrophosphorylaseby thioredoxin, J. Biol. Chem. 275, 1315-1320), which is promoted by the trehalose formed in the Kololite-6-al-Kolosetum (6-Kol-et-Kol) 2005: Trehalose 6phosphateregulates starch synthesisvia posttranslational redox activationofADP-glucosepyrophosphorylase, Proc. Natl. Acad. Sci. USA 102, 11118-11123). It is worth noting that the quantitative RT-PCR analyzes (Fig. 13) and the transcriptome analyzes revealed that trehalose-6-phosphate synthase and plastidial thioredoxins were strongly regulated when the plants were cultivated in the presence of PVs (reduction of 7,57 and 8,31 for the thioredoxin myf, respectively, and reduction of 4.57 times of trehalose-6-phosphate synthase in the presence of sucrose; reduction of 3.71 and 2.74 times for thioredoxin myf, respectively, and reduction of 4.89 times of trehalose-6-phosphate synthase in the absence of sucrose) .To test whether this situation affects the redox status of the PGP, extracts from potato leaves controlled and treated with FVs are separated by means of non-reducing SDS-PAGE and subsequently subjected to Western blot analysis. It is important to note that when leaf extracts were separated into non-reducing gels with SDS, the PGP is present as a mixture of 50 k active monomers Inactive dimer of 1 00 kD formed by intermolecular bonds that involve cysteine bridges. These dimers can be reactivated in vitro by incubating extracts with DTT (Hendriks et al. 2003: ADPglucosepyrophosphorylaseisactivatedby posttranslational redox-modi fi cationin responsetolightandtosugars in leaves of Arabidopsis andotherplantspecies, PlantPhysiol.133, Trev. redox activationof ADP-glucosepyrophosphorylase, Proc. Natl. Acad. Sci. USA 102, 11118-11123). As shown in Fig. 15, potato leaves treated with PVs accumulate much larger amounts of inactive 100 kD dimers than control leaves under non-reducing conditions. These 100kD dimers were able to convert 50kD monomers when the extracts were obtained and separated under non-reducing conditions (including DTT) (Fig. 15), thus indicating the overall data that the AGG is mostly oxidized (inactive) in the VF treated sheets.

To further assess the relevance of the PGP in the MIVOISAP, the PGP activity, the starch content and the ADPG in potato plant leaves were measured AGP62 (which have the APS subunit inactivated by antisense elements (Müller-Röber et al. 1992: Inhibitionofthe ADPgluoringpyrophosphorestoral sugar-spider-sugar-spider-sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar-spider sugar and sugar-sugar-tubers) in fl uences tuber formation and expression of tuber storage protein genes, EMBO J. 11, 12291238)) cultured in the presence and absence of VF. As illustrated in Fig. 16A, the AGG activity on AGP62 sheets was 30% of that of the wild type leaves (WT). In contrast to WT sheets, AGP activity in AGP plants did not increase with PV treatment. It is worth noting that the treatment with PVs resulted in a drastic enhancement of starch accumulation (Fig. 16B), and an increase of approx. 70% in the content of ADPG (Fig. 16C) in sheets AGP62. The starch and ADP content in sheets AGP62 treated with PV were comparable to those observed in WT sheets treated with PV.

The global data would indicate, thus, that changes in AGP activity play a minor role, if any, in the MIVOISAP in potato leaves.

4.2. Fungal volatiles strongly regulate the SuSyyregulanala rise below the expression of acid invertase

As mentioned in the "Background of the invention" section, it has generally been considered that the biosynthesis of starch in leaves is exclusively in the chloroplast, and is segregated from the biosynthetic sucrose process that takes place in the cytosol, while recent evidence foresees the existence of (one) additional route (s) in the one that is de novo produced in the cytosol, by means of SuSy, ADPG linked to the biosynthesis of starch.

In connection with these theories, it is worth noting that the transcriptome analyzes of leaves of plants grown in the presence or absence of PVs revealed that the treatment with PVSdio resulted in a drastic potentiation of the expression of Its4 (increase of 29.4 and 31.62 times when the plants were grown in the presence or absence of sucrose, respectively), a controlled accumulation of the plant. leaves as in potato tubers (Muñoz et al. 2005: Sucrose synthase controls the intracellular levels of ADPglucose linked to transitory starch biosynthesis in source leaves, Plant Cell Physiol. 46, 1366-1376; Baroja-Fernández et al. 2009: Enhancing sucrose synthase activityin transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield. Plant CellPhysiol. 50, 1651-1662). In fact, analyzes of the intracellular levels of starch and sugar-nucleotides in leaves of potato plants grown in the presence and absence of VF revealed a positive correlation between the activity patterns of SuSy and the starch content, UDPG and ADPG (Fig. 17). This drastic potentiation of the expression of Fys and by means of transfer through Fig. .18), quantitative RT-PCR (Fig. 13), and through measurement of enzymatic activity (10 times increase: see Table 2).

Acid invertase in a saccharolytic enzyme whose activity is regulated post-translationally by a protein inhibitor (Bracho and Whitaker, 1990: Puri fi cation and partial characterization of potato (Solanum tuberosum) invertase and its endogenous proteinaceous inhibitor, Plant Physiol. 92, 386-394 ) in the tubers. These two saccharolytic enzymes compete for the same supply of sucrose, acting as one of the main determinants of starch accumulation the balance between SuSy and acid invertase (Baroja-Fernández et al. 2009: Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield, Plant Cell Physiol. 50, 1651-1662). It is worth noting that the analysis of the RNA profile revealed that treatment with VFs resulted in a down-regulation of the expression of acid invertase (decrease of 2.61 and 2.24 times the presence and absence of sucrose, respectively), and a dramatic potentiation of the transcripts encoding the inhibitor of this saccharolytic enzyme (17.78 and 18.1 fold increase in the presence and absence of sucrose, respectively), which was further confirmed by quantitative RT-PCR (Fig. 13) and by enzymatic activity analysis (see Table 2).

Global data indicate that (a) SuSy and acid invertase-mediated saccharolytic pathways are coordinated in response to identical signals, and (b) the balance between these routes is a major determinant of starch accumulation in potato leaves exposed to microbial volatiles. Due to the clear correlation between SuSy activity patterns and the starch, ADPGyUDPG contents in leaves of plants treated and not treated with FVs (Fig. 17), the global data also indicated that the high levels of ADPG, UDPGyalmidon produced in leaves of Potatoes treated with VFs are ascribed, at least in part, to the enhancement of SuSy activity during MIVOISAP in potato.

4.3. Fungic volatiles regulate downward main routes of internal amino acid provision

As previously mentioned in the "Background of the invention" section, recent studies have shown that plants possess a regulatory system mediated by ppGpp similar to that found in bacteria, which has been shown to play a crucial role in aspects such as plant fertilization. The ppGpp accumulates in the chloroplast of stressed leaves through the regulation of the expression of RelA / SpoT (RSH) homologs.

It is worth noting that quantitative RT-PCR analyzes (Fig. 13) and transcriptome analyzes previously described in the Examples of the present application revealed that, regardless of the presence of sucrose, treatment with VFs resulted in downregulation. of RSH (decrease of 2,6 and 2,42 times in the presence and absence of sucrose, respectively).

It is possible for plants to develop similar responses to bacteria in bacteria that regulate glycogen synthesis as a consequence of the deprivation and / or provision of amino acids. Consistent with this presumption, quantitative RT-PCR analyzes (Fig. 13), and transcriptome analyzes revealed that treatment with VFs results in a drastic reduction of GAPDHypPGK expression (32.68 and 5.32 fold decrease, respectively). In addition, these analyzes revealed that treatment with VFs resulted in a marked downward regulation of G6P dehydrogenase (pG6PDH) plastidial (reduction of 6.17 times) (see also Fig. 13 and Table 2), an enzyme of the phosphate pentose oxidative pathway (OPPP) that is involved in the production of reducing power required for the biosynthesis of amino acids in heterotrophic organs in leaves during the night period.

In addition, treatment with PVdio resulted in a marked reduction in the expression of genes that encode a set of plastidial proteins involved in the assimilation of nitrogen such as those that encode the transporter denitrite, nitrite reductase, glutamate syntheses and the glutamate / malate translocator (decrease of 3.88, 9.85, 3.86 and 3.22 times, respectively) (see also Fig. 13 and Table 2).

The first step in the conversion of sulfate into sulfoamino acids cysteine and methionine is catalyzed by ATP sulfurylase. This plastidial enzyme catalyzes the reversible conversion of ATPysulfate into adenosine-5'-phosphosulfate and PPi, which is displaced from equilibrium by PPase by rapid withdrawal of PPi. As discussed above, treatment with VF results in regulation down from the PPase. Thus, it is conceivable that with the PV treatment PPi accumulates in the chloroplast, thus preventing the biosynthesis of sulfoamino acids by inhibiting the ATP sulfurylase. It is worth noting that the treatment with PVSda results in a low regulation of the serine acetyltransferase plastidial cysteine, cysteine synthase and gamma-synthase (reduction of 3.43, 2.85 and 2.53 times, respectively), all of them enzymes that are necessary for the synthesis of cysteine and methionine in the chloroplast.

Therefore, it was investigated whether defects in the provision of plastidial cysteine are directly implicated in MIVOISAP, measuring the starch content in detached potato leaves grown in the presence and absence of VF emitted by Alternaria alternata, and in the presence of different concentrations of cysteine. Most importantly, these analyzes revealed that, contrary to what happens with other amino acids, MIVOISAP was strongly repressed by exogenously added cysteine (Fig. 19). Other amino acids, on the other hand, did not inhibit MIVOISAP.

Thus, the global data strongly indicated that MIVOISAP is the consequence of a response triggered by the inadequate internal provision of cysteine. Since sulfoamino acids constitute the main metabolic input of reduced sulfur in cellular metabolism, the inventors considered the hypothesis that the high starch content of the leaves treated with VFs is the result, at least in part, of a triggered response. by deprivation of both nitrogen and sulfur.

Proteases play a leading role in the control of protein quality, being responsible for the degradation of damaged and damaging polypeptides as well as the recycling of amino acids for biosynthesis of de novo proteins. Proteolysis also provides the necessary amino acids to maintain cell homeostasis, being a process that involves a significant portion of the energy requirements for cell maintenance. Along these lines, it is worth noting that the transcript analyzes of the present invention revealed that treatment with VFs dramatically enhanced the expression of many protease inhibitors. Thus it is highly conceivable that

(a) the resulting proteolytic activity failure results in a defect in the internal supply of amino acids that triggers a response that leads to starch accumulation, and (b) the decrease in the demand for ATP for protein degradation results in the availability of an excess of energy for starch biosynthesis.

4.4. Regular fungal volatiles boost starch synthases of classes III and IV

Five different kinds of starch synthases (SS) are known in plants: the GBSS, which is responsible for the synthesis of amylose, and the SSSclassesI, II, III, and IV (SSI, SSII, SSIII, and SSIV, respectively) .Abel et al. (Abel et al., 1996: Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.), Plant J. 10, 981-991) have shown that the reduction of SSIII leads to starch synthesis with structural modifications in transgenic potato plants. On the other hand, Roldan et al. (Roldan et al. 2007: The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the controlof starch granule formation, Plant J. 49, 492-504) have shown that the elimination of SSIV determines that the chloroplasts accumulate only one large starch granule in Arabidopsis. In addition, using different combinations of SS mutations in a mutant fund for SSIV, Szydlowski et al (Szydlowski et al 2009: Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthase, Plant Cell 21, 2443 -2457) have recently shown that Arabidopsis double mutants that lack SSIII and SSIV functions show a starch-free phenotype.

Global data (a) indicated that both SSIII and SSIV play a key role in starch accumulation, although SSIV is mandatory to give rise to the regular number of starch granules found in wild-type plants, and (b) suggested that SSIV play a significant role in starch granule initiation process.

Consistent with the idea that SSIIIySSIV are major determinants of starch accumulation in potato leaves, transcriptome analyzes revealed that, regardless of the presence of sucrose in the culture medium, treatment with VFs resulted in a large increase in the expression of SSIV (increase of 7.00 and 4.68 times in the presence and absence of sucrose, respectively) and a moderate increase in the expression of SSIII (increase of 2.53 times in the presence of sucrose), results that they were confirmed by quantitative RT-PCR analysis (Fig. 13). No changes were observed in the levels of expression of SSI class I and

II. Enzymatic activity analysis revealed that treatment with FV was as a result of a 3-fold increase in Total S activity (see Table 2) .Global data indicates that the MIVOISAP can be assigned, at least in part, to the potentiation of the activity of SSIII and SIV.

4.5. Fungal volatiles promote both the reduction of amylose content and structural changes in amylopectin by increasing the balance between branching and branching activities of starch

The starch granule is composed of two structurally different homopolymers: amylose, which is essentially linear, and amylopectin, which is a moderately branched macromolecule. Potato leaf starch contains 10-15% amylose. While amylose is produced by GBSS, amylopectin is synthesized by the combined actions of soluble SS and starch branching enzyme (SBE), of which the latter catalyzes the formation of α-1,6 bonds in the molecule. starch. According to the "cut-out model" of starch granule formation, the biosynthesis of amylopectin is also the result of "cuts" by the de-branching enzymes (isoamylasasypululanases which hydrolyse α-1,6 bonds within the highly branched molecule of glucans that They are synthesized by the SSS soluble and the SBE.

In this line, it is worth noting that quantitative RT-PCR analyzes (Fig. 13) and transcriptome analyzes revealed that treatment with VFs resulted in a drastic increase in SBE expression (increase of 32.66 and 2.5 times in the presence of sucrose absence, respectively) and a moderate increase in expression. both pululanase and GBSS when the plants were grown under heterotrophic conditions (increase of 3.4 and 2.98 times, respectively). Consistently, the activity of the BSS in sheets treated with PV will be markedly higher than in untreated sheets (Table 2). It is worth noting that the changes in the expression of these genes were accompanied by a significant reduction in the relative amylose content (Fig. 20A).

To investigate the changes promoted by the Fs and the SS and S activities, as a result of structural changes in the amylopectin, enzymatic demystification was subjected to purified amylopectin blades treated and not treated with FVs, and the distribution of chain lengths was determined by high-density, high-density C-type A-metric chromatography. These analyzes revealed that treatment with FVs exerts an important effect on the structure of amylopectin, since the amylopectin of leaves treated with VFs contained more chains with a degree of polymerization (GP) less than 20 monomers, than amylopectin of untreated leaves (Fig. 20B, Fig. 20C). The global data thus indicated that the structural changes that occur in the starch molecules of plants treated with PVs can be ascribed, at least in part, to the potentiation of the SSS and SSE activities.

4.6. Fungal volatiles strongly regulate starch degradation enzymes

Using the antisense technique, Scheidig et al. (Scheidig et al., 2002: Downregulation of a chloroplasttargeted β-amylase leads to a starch-excess phenotype in leaves, Plant J. 30, 581-591) demonstrated that the chloroplast β-amylase BMY1 used as a target exerts a strong control over starch degradation in potato leaves. Consistently with this, the transcriptome analyzes described in this application revealed that the treatment with PVs resulted in a drastic regulation of the low CPT-BMY1 (decrease of 5.89 and 3.66 times in the presence of sucrose absence, respectively, which was further confirmed by quantitative RT-PCR (Fig. 13) and through enzymatic activity analysis Table 2).

The upper plants contain both cytosolic and plastidial starch phosphorylases. Contrary to what happens with plastidial β-amylases involved in starch degradation, the precise in vivo function of the plastidial isoform is not yet evident, although it has been generally accepted that it may be involved in starch degradation. Zeeman et al. (Zeeman et al. 2004: Plastidial α-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress, Plant Physiol. 135, 849858) argued that this enzyme is involved in abiotic stress tolerance in Arabidopsis, providing OPPP starch substrates for stress relief. It is worth noting that the RT-PCR analyzes (Fig. 13) and the transcriptomas analysis revealed that the treatment with PVs resulted in a marked regulation of the low expression of the phosphorylase of the plastid starch when the plants were grown under heterotrophic conditions (decrease of 5.14 times).

The global data thus indicated that MIVOISAP can be ascribed, at least in part, to the regulation of low plastid isoforms of β-amylasayla and phosphorylase delamidon.

4.7. The volatile fungalregular alzagenes involved in endocytosis and trafic code vesicles

Synthesized distributing phosphatidylinositol (PI) by means of IP-3-phosphate (PI3P) kinase (PI3K) and by means of IP-4 phosphate (PI4P) kinase (PI4K), PI3PyPI4P have been implicated in various physiological functions, including endocytosis, plasma membrane, endocytosis and endocytosis. a process involved in the internalization of molecules from the plasma membrane and the extracellular environment, and plasma membrane recycling. The increase in phosphoinositide metabolism results in an increase in the use of medium sugars (Im et al., 2007: Increasing plasma membrane phosphatidylinositol (4,5) biphosphatebiosynthesis increasesphosphoinositide metabolism in Nicotianatabacum, Plant Cell 19, 1603-1616), What is a strong indication that phosphoinositid-mediated signaling plays an important role in the potentiation of absorption, internalization and storage of extracellular sugars in vacuums. It is worth noting that recent studies have provided strong evidence that an important part of sucrose incorporated in heterotrophic cells is absorbed by means of the process of endocytosis mediated by PI3Ky / or PI4Kyel trafficking of vesicles prior to their conversion into starch (Baroja-Fernández et al. 2006: An Important Pool of Sucrose Linked to Starch Biosynthesis is taken up by endocytosis in Heterotrophic Cells, Plant Cell Physiol. 47, 447-456). Consistent with these observations, the RT-PCR analyzes (Fig. 13) and the transcript analysis revealed that the genes encoded by PI4K and PI3K are regulated by the treatment of PVs (increase of 3.55 and 3.12 times, respectively). PI is synthesized in the cytosol from G6P in a three-stage process, which involves inositol phosphate synthase, inositol monophosphate and PI synthase. It is worth noting that the RT-PCR analyzes (Fig. 13) and the transcriptome analyzes reveal that the genes that encode inositol phosphate syntheses and inositol monophosphates are regulated to ALP for treatment with PVs (increase of 3.78 and 4.62 times, respectively).

The actin cytoskeleton of plants is a dynamic support structure that plays a crucial role in the movement of organelles, vesicle traffic, cytoplasmic currents, plant defenses against pathogens, etc. in response to internal and external signals. Evidence has recently been provided that the actin cytoskeleton is also involved in endocytic absorption and sucrose trafficking linked to starch biosynthesis in sycamore culture cells (Baroja-Fernández et al. 2006: An Important Pool of Sucrose Linked to Starch Biosynthesis is taken up by endocytosis in Heterotrophic Cells, Plant Cell Physiol. 47, 447-456). It is worth noting that SuSy, a key enzyme in the starch biosynthesis process (see above), has been shown to be associated with the actin cytoskeleton (Dunkan and Huber, 2007: Sucrose synthase oligomerization and F-actin association are regulated by sucrose concentration and phosphorylation, Plant Cell Physiol. 48, 1612-1623), which is further support for the view that the actin cytoskeleton determines to some extent the metabolism of starch. Actin depolymerizing factors are modulators of the dynamic organization of the actin cytoskeleton, which modulate the turnover rate of the filaments and the interconnection of cellular signals with the cytoskeleton-dependent processes. Consistent with the vision of endocytosis and / or tetraphyllofilms in the middle of the cytosphere that can play an important role in MIVOISAP, the analyzes of RT-PCR (Fig. 13) and transcriptome analyzes revealed that the expression of the depolymerizing factor of the actinaseveregulated at the increase of 3.2 times of the treatment.

The global data thus shows that, as schematically illustrated in Fig. 21, endocytic absorption of sucrose and traphylovesicles may be involved in MIVOISAP, especially when plants are grown in the presence of sucrose.

4.8. Fungal volatiles promote regulation of low photosynthetic genes when plants are grown under heterotrophic conditions.

One of the most striking alterations of the transcriptome of plants treated with VF cultured in the presence of sucrose involves the repression of genes that encode proteins that function in light reactions of photosynthesis. In addition, when plants are grown in the presence of sucrose, they are also severely repressed by treatment with PV genes that encode key enzymes of the Calvin cycle and photorespiration. These include pPGK, pGAPDH, triosa-P-isomerase, transketolase, pentose-P-epimerase, ribose-P-isomerase, fructose bisphosphate aldolase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, Rubisco, glycopy oxidase, catalase, serine hydroxymethyltransferase, and hydroxypyruvate reductase (reduction of 5.32, 32.68, 3.69, 3.65, 4.79, 6.43, 14.97, 17.99, 11.09, 46 , 62,9,24,4,01,6,6 and 7,79 times, respectively) (see also Fig. 13) .In addition, the PV treatment of plants grown with sucrose resulted in repression of the gene encoding the photochlophilic oxide reductase (reduction of 7.73), which is necessary for the biosynthesis of chlorophil. Under these conditions it is highly conceivable that, as schematically illustrated in Fig. 21, much of the starch accumulated by plants grown under heterotrophic conditions is produced from the metabolic degradation of sucrose taken from the culture medium (see below). ).

4.9. FVs strongly promote aerobic and anaerobic metabolism when plants are grown under heterotrophic conditions.

Simultaneously with the repression of genes that encode proteins that function in light reactions of photosynthesis (see above), treatment with VFs resulted in the regulation of lower ATP synthase plastidial when plants were grown under heterotrophic conditions (9.09 fold reduction). . Under these conditions, the FVs promoted the transcription of genes that encode glycolytic enzymes such as enolase, pyruvate kinase, phosphoenolpyruvate (PEP) carboxyquinine and PEP carboxylase (increase of 4.94.5,48.19.64 and 6.03 times, respectively). This effect was much less pronounced when the plants were grown under autotrophic conditions. Since PEP carboxyquines and PEP carboxylase are involved in the conversion of PEP into oxaloacetate, the global data indicates that PVs promote glycolysis and carbon flux towards the tricarboxylic acid (TCA) cyclode, as outlined in Fig. 21, especially when plants are grown under heterogeneous conditions. It is worth noting that the treatment with VFs resulted in an increase in the expression of the genes that encode enzymes of the TCA cycle such as succinate dehydrogenase and isocitrate dehydrogenase (increase of 2.8 and 2.57 times, respectively). Some genes involved in fermentation, including those that encode alcohol alcohol dehydrogenase (AD ), decarboxylase and an aldehyde dehydrogenase, were strongly regulated upwards by treatment with VF, both under heterotropic conditions (increase of 51.43 and 9.92 times for ADH and aldehyde dehydrogenase, respectively), and autotrophic conditions (increme 9.92 and 3.77 times for ADHypyruvate decarboxylase, respectively) (see also Table 2). The increase in activity of the ethane fermentation route from pyruvate allows re-oxidation of NADH produced in glycolysis, thus allowing the plant to generate ATP independently of oxidative phosphorylation. Therefore, the promotion by the VFs of the potentiation of the expression of genes that encode enzymes involved in glycolysis, the TCA cycle and fermentation is consistent with the notion that both aerobic and anaerobic metabolisms are upregulated during MIVOISAP to generate energy in production conditions of reduced photosynthetic ATP.

The upper floors have ATP / ADP transporters, both in heterotrophic plastids and in autotrophospar- cytosolic cytolic ATP by exchanging it with plastidial ADP. As heterotrophic plastidiums lack the machinery that produces ATP through photosynthesis, the ATP / ADP transporter is necessary to supply energy in such processes to the localized energy in such processes. production of amino acids, starch and fatty acids. In chloroplasts, ATP / ADP transporters have been shown to be important for nightly importation of ATP and to prevent damage caused by photooxidation (Reinhold et al. 2007: Limitation of nocturnal import of ATP into Arabidopsis chloroplasts leads to photooxidative damage. Plant J. 50, 293304). It is worth noting that the regulatory VF increases the expression of the plastidial transporter of ATP / ADP when the plants are cultivated under heterotrophic conditions (3.16 times increase), which is consistent with the notion that the extra-plastid ATP supports in large measure anabolic pathways that happen in the chloroplast plants of conditions treated with VF heterotrophic growth.

4.10. Fungal volatiles promote the exchange of glucose-6-phosphate and repress the exchange of triosaphosphate between chloroplast and cytosol

The main membrane protein of the chloroplast shell, the triosa-P / 3-phosphoglycerate / P (TPT) translocator, is essential for communication between chloroplastoy cytosol, since it exports the primary products of the Calvin cycle (i.e., triosa phosphates and 3-phosphoglycerate) outside the chloroplast in a strict exchange of units with respect to Pi. Potato plants with the TPT inhibited by antisense molecules accumulate in their leaves 2-3 times more starch and more 3-phosphoglycerate than the leaves of the type, and have reduced plant vigor. It is worth noting that the analyzes of RT-PCR (Fig. 13) and transcripts described that, regardless of the presence of sucrose in the culture medium, the treatment with PVs resulted in the reduction of the expression of the TPT (reduction of 3.17 and 2.25 times in the presence and in the absence of sucrose, respectively). 3-phosphoglycerate levels (see above), which can probably be attributed to the reduction of TTP-3-phosphoglycerate transport from chloroplast to cytosol.

Plastids of heterotrophic tissues depend on the provision of G6P through the G6P / Pi anti-carrier system. The imported G6P can be used for starch synthesis and acid grades. The G6P can also be used to activate OPPP which, as discussed above, is the main source of reductive power. nitrite reduction and for the biosynthesis of fatty acids and amino acids. It is worth noting that, although the expression of the G6P / Pi translocator is mainly restricted to heterotrophic tissues, treatment with PVs strongly enhanced the expression of the G6P / Pi translocator in leaves when the plants were grown in the presence and absence of sucrose, such as confirmed both by RT-PCR analysis (Fig. 13) and by transcriptome analysis (increase of 30.23 and 22.08 times when plants were grown in the presence and absence of sucrose, respectively).

The involvement of the G6P / Pi translocator in the importation of cytosolic G6P into chloroplast when plants were treated with questionable FVses, since, as we show above, the enzymes involved in G6P plastid metabolism are drastically regulated downwards. This and the reduction of the expression of the proteins involved in the synthesis of G6P in the cytosol from products of the Calvin cycle such as TPT (see above), cytosolic fructose-1,6-bisphosphatase (see Table 2 and Fig. 13) (reduction of 9,87 and 3,61 times in the presence of yen absence of sucrose, respectively), and fructose-6-phosphate2kinase / fructose-2,6-bisphosphatase (reduction of 3.39 and 2.2 times present in the absence of sucrose, respectively) (see also Fig. 13) suggest that, as outlined in Fig. 21, under conditions of VF treatment, the G6P / Pi transporter would play a major role in transporting G6P molecules from the chloroplast to the cytosol to later be channeled into the cycle of the TCAs and / or fermentative orifices, and / or converted into compounds such as sacrosy and PIquees that are necessary for endocytes and tetraphyllicles.

4.11 Influence of PVs in other interest areas

All the microbial species analyzed in this work emitted volatiles that promoted plant growth, which would indicate that the machinery involved in biosynthesis is regulated upwards during MIVOISAP. Consistent with this presumption, analysis of transcripts of potato plant leaves revealed that treatment with VFs resulted in the regulation of cellulose synthase and callose synthase (increase of 9.78 and 2.1 times, respectively).

MIVOISAP implies changes in the expression of a multitude of genes that encode fundamental enzymes in carbohydrate metabolism and energy production / consumption, suggesting that MIVOISAP is a highly coordinated and regulated process. It is worth noting that the transcript analyzes described in the present application revealed that treatment with VF strongly promoted the expression of SNF4 (6.64 fold increase) (see also Fig. 13), an activator of the protein kinase SnRK1, which It is a global regulator of carbon metabolism in plants. It is thus likely that (a) SNF4 exerts a positive effect on starch accumulation through the activation of SNRK1, and (b) SnRK1 plays a regulating role during the MIVOISAP.

Claims (24)

1. A procedure to increase the amount of starch accumulated in different organs of a plant, characterized in that the plant or the leaves detached from it are maintained in the presence of a culture of a microorganism that produces volatile compounds, without contact between the microorganism and the plants detached leaves , or in the presence of volatiles emitted by the microorganism. 2. Methods according to claim 1, in which additionally there is an increase in the size of the starch granules and a modified starch is obtained, in which the relative content of amyls and the degree of polymerization of the amylopectin chains are smaller than those of the starch produced by the control plants grown in the absence of microbial volatiles. 3. Methods according to claim 1 or 2, in which the microorganism is a bacterium, a yeast or a microscopic multicellularunhongo.

Four.

Method according to claim 3, wherein the microorganism is a yeast.

5.

Method according to claim 4, wherein the yeast belongs to the species Saccharomyces cerevisiae.

6.

Method according to any one of claims 1 to 5, in which the growth of the microorganism occurs in a medium lacking organic compounds having amino groups.

7.

Method according to claim 6, wherein the growth of the microorganism occurs in a medium that lacks amino acids and / or proteins.

8.

Method according to claim 6 or 7, wherein the growth of the microorganism occurs in a minimal medium supplemented with an organic carbon source.

9.

Method according to claim 8, wherein the microorganism is a bacterium belonging to a genus of the group Salmonella, Agrobacterium, Bacillus, Escherichia or Pseudomonas.

10.

Method according to claim 9, wherein the microorganism is selected from the group of: Bacillus. subtilis 168, Salmonella enterica (LT2), Escherichia. coli (BW25113), Agrobacterium tumefaciens EHA105, Agrobacterium tumefaciens GV2260, Pseudomonas syringae 1448A9, Pseudomonas syringae 49a / 90, Pseudomonas syringae PK2.

11. Methods according to claim 6, in which the microorganism is a fungal belonging to the genus Penicillium or Alternaria. 12. Method according to any one of the preceding claims, wherein the plant is an angiosperm, monocot or dicot.

13.

Method according to claim 12, wherein the plant is selected from the group of potato plants, corn plants, tobacco plants, barley plants or plants of the Arabidopsis thaliana species.

14.

Method according to claim 13, wherein the plant is a potato plant, a corn plant, a tobacco plant or a plant of the Arabidopsis thaliana species that is grown in the presence of a fungus belonging to the Alternaria or Penicillium genus that is allowed to grow in a minimum medium supplemented with a source of organic carbon, without physical contact between the plant and the microorganism.

fifteen.

Method according to claim 13, wherein medium is supplemented with sucrose.

16.

Methods according to claim 15, in which the plant is a potato plant or a seed plant.

17.

Methods according to any one of claims 1 to 16, in which they are organized in order to increase the amount of accumulated starch are leaves, roots, stems and / or tubers.

18.

Methods according to any one of claims 1 to 17, in which the whole plant is grown in the presence of a microorganism culture that produces volatile compounds, without any contact between the plant and the microorganism, or in the presence of the volatile emitted by the microorganism.

19. Method according to claim 18, wherein the plant is grown in vitro or on land. 20. Methods according to any one of claims 1 to 19, in which the plant is grown in an atmosphere in which volatile compounds emitted by a microorganism that has been cultivated in a different place are present at the plant's cultivation.

twenty-one.

Method according to any one of claims 1 to 17, wherein the organ in which the increase in the amount of starch is produced are separate leaves of the whole plant.

22

Method according to claim 21, wherein the microorganism is a fungus of the Alternaria genus

2. 3.

Methods according to claim 21 or 22, in which the leaves are potato plant leaves.

24.

Methods according to any of claims 21-23, in which the sheets are kept for less than 2 days in the presence of the microorganism culture, in contact with the volatiles emitted by the microorganism.