SE2230450A1 - Method of Obtaining Transgenic Plants with Modified Enzymes for Saccharification of Ligninocellulose, and Method for Production of Modified Enzymes - Google Patents

Abstract

The present invention relates to transgenic plants with changed physicochemical properties of wood, lower content of lignin, and cellulose with higher polymerization degree, in relation to non-transformed trees; more particularly to transgenic plants that are characterized by introducing a modified EDSl, LSDl, and PAD4 genes, in combination, which are responsible for production of enzymes leading to separation of cellulose fiber mass from lignin, and cellulose saccharification to soluble sugars. The invention also includes the method for production of modified enzymes from plant lines with suppressed LSD1 , PAD4 and EDS1 genes in combination.

Description

DESCRIPTION Plant lignocellulosic biomass is the most abundant and available raw material on the Earth. Cellulose is contained in nearly every natural, free-growing plant, tree, and bush, in meadows, forests, and fields all over the world without agricultural effort or cost needed to make it grow. Cellulose is used to produce pulp, paper, and biofuels. Among them, biofuels production from lignocellulosic biomass is relatively low due to limited knowledge on natural cell wall loosening and cellulolytic processes in plants. An industrial separation of cellulose fiber mass from lignin, its saccharification, and alcoholic ferrnentation is still cost ineffective. However, one of the benefits of cellulosic ethanol is it reduces greenhouse gas emissions (GHG) by 85% over reforrnulated gasoline. By contrast, starch ethanol (e.g., from com), which most frequently uses natural gas to provide energy for the process, may not reduce GHG emissions at all depending on how the starch-based feedstock is produced. According to the National Academy of Sciences as of 2011, there is no commercially feasible bio-refinery in existence capable of cost-effectively converting cellulosic biomass to fuel.

One of the most important challenges of the 2151 century is to provide reliable, renewable, and environmentally friendly sources of the energy (Ibrahimet al. Sci Rep 11, 9754, 2021). Geopolitical situation forces even more urgent an altemative for the traditional energy sources. Improving the production of renewable biofuels is one of the solutions. Nowadays, bioethanol is mostly produced as a first-generation biofuel from com and wheat grains, potato, sugar cane, sugar beets, sugarcane, soybean, grapes, and sunflowers. It reduces food sources and feed production, resource/land allocation is competitive with food production, and thus they usage is questionable. To circumvent a conflict between demand for biofuel and food sources, cellulose-containing food waste and wood chips have been used as substrates for the second- generation biofuels. Second-generation biofuels, which are derived from biomass, have attracted worldwide attentions as a renewable energy source, however, only ~3% of bioethanol is produced from this source as a second-generation bioethanol (Zoghlami and Paës, Frontiers in Chemistry 7, 2019; Hoengenaert et al. Science Advances 8, eabo5738, 2022). Breakdown of cellulose to sugar molecules is a required step before the ferrnentation process for ethanol production. However, a drawback in the development of this technology is that industrial-scale hydrolysis of cellulose is an expensive process due in large part to the cost of cellulases, the enzymes that degrade cellulose.

The main limitation of efficient, industrial production of second-generation bioethanol is a complex composition and structure of plant cell wall. Mostly composed of cellulose, cell wall is highly heterogeneous as it also contains hemicelluloses, pectin, and lignin. The outcome structure is deterrnined by the function of secondary cell walls (Turumtay, Bioenerg. Res. 8, 1574, 2015; Khan and Akhtar, Nat Prec 1-1, 2011; Alonso et al. Science Advances 3, e1603301, 2017). The chemical composition of wood depends on the type and variety of the tree. Typically, its solid part consists of about 45% cellulose, 25% hemicelluloses, 25% lignin and 5% other organic and inorganic substances. Cellulose, the most common naturally occurring polymer, is made up of numerous anhydroglucose groups linked by glycosidic bonds. Cellulose in wood reaches a degree of polymerization of 9,000-10,000, and even up to 15,000. Cellulose hydrolysis is a difficult process because cellulose fibers are stabilized by hydrogen bonds and surrounded by hemiecellulose polysaccharides (mannans and xylans) joined by covalent and hydrogen bonds. Hemicelluloses are polysaccharides with a lower degree of polymerization, consisting of hexoses (D-galactose, L-galactose, D-mannose, L-fructose) and pentoses (L-rhamnose, arabinose, xylose) and uronic acids (D-glucuronic acid). Lignin are phenolic macromolecules, which are the condensation product of three monomeric alcohols: trans-p-coumaryl, trans-p-coniferyl and trans-p-sinapyl. Lignin hydrolysis is a complicated process due to numerous ether and C-C carbon bonds. Lignin, as a polymer that ensures the structural integrity of the plant cell wall, is mechanically strong and resistant to chemical decomposition, as it deterrnines mechanical durability and resistance to pathogens. At the same time, the same characteristics are problematic when plants are used as raw material for some industrial applications.

During technological processes, the lignocellulosic biomass requires treatment separating its components, and break down complex polysaccharides into ferrnentable sugars (Sidana and Yadav, J. Cleaner Prod 335, 130286, 2022; Banu et al. Front. Energy Res. 9, 2021). In particular, lignin presents a great challenge because it affects the activity of cellulases and phenolic compounds are inhibitors of alcohol ferrnentation in Saccharomyces cerevísíae yeast (Lu et al. Biotech. Biofuels 9, 118, 2016; Fletcher and Baetz, Front. Bioeng. Biotech. 8, 2020). The pretreatments increase the cost of production and ne gatively impact environment with toxic wastes (Li et al. Chem. Sus. Chem. 10, 3445, 2017). However, pre-treatment of biomass is an important step in preparation for biomass processing in the process of enzymatic hydrolysis. In each case, the purpose of the pre-treatrnent is to increase access for the action of the enzymes. The main cost incurred for the initial processing and preparation of the (lignin) cellulose pulp is related to the mechanical fragmentation of the material and the final efficiency of technological processing. The cost deterrnines the economics of obtaining starting materials, e. g., for the production of paper and bioethanol from lignocellulosic pulp. All factors accelerating the process and reducing energy costs during biomass preparation reduce total costs. Because of the potentially high importance of this process, cellulose-degrading enzymes have received a great deal of attention (Beltrane et al., Bioresource Technol. 39, 165, 1992). To date, the conversion of plant cellulose into ferrnentable sugars is accomplished with commercial enzymes produced by large-scale ferrnentation. T ríchoderma reesei has been a target strain for the production of cellulase, and Bacíllus and Aspergíllus microorganisms have been a target for the production of xylanase. But, the concentration and activity of such enzyme is not enough to be used in industry. Similarly, the cost of producing these enzymes remains a significant barrier to the widespread utilization of this process (Cowan, Tibtech. 14, 177, 1996; Ho et al., Adv. Biochem. Eng. Biotechnol. 65, 163, 1999). Thus, various approaches have been taken to produce cellulose-degrading enzymes on a large scale and at low cost (Goddijin and Pen, Trends biotechnol. 13, 3790, 1995; Gidding et al., Nature Biotech. 18, 1151, 2000).

Plants are capable of efficient saccharification in vívo during induction of certain types of a Programed Cell Death (PCD) e. g., during lysigenous aerenchyma formation in response to roots hypoxia (Escamez and Tuominen, Curr. Opinion Plant Biol. 35, 124, 2017; Mühlenbocket al. Plant Cell 19, 3819, 2007; Mustroph, Agronomy 8, 160, 2018). According to our knowledge, plant molecular regulators and enzymes involved in natural saccharification of the cell wall have not been explored in the context of PCD. Similarly, the role of genes encoding molecular PCD regulators have not been studied during lignin related processes. However, native plant cellulases and cell wall loosening (CWL) enzymes could potentially be as efficient and specific as currently used microbial enzymes in industrial biomass processing (Kao et al. Biotech. Biofuels 12, 258, 2019), while PCD regulators involved in lignocellulosic cell wall modification.

Genetically engineered transgenic plants are one of the most economical systems for large scale production of recombinant proteins for industrial and medical uses, because a large quantity of enzymes can be produced with minimal input (Jensen et al. Proc. Natl. Acad. Sci. 93, 3487- 3491, 1996; Kawazu et al., J. Ferrnent. Bioeng. 82, 205, 1996; Nuutila et al., Plant Mol. Biol. 41, 777, 1999; Ziegelhoffer et al., Mol. Breed. 5, 309, 1999; Dai et al., Transgenic Res. 9, 43, 2000), the cellulase gene has been expressed in transgenic plants, but the expression levels obtained have been low. The purification of recombinant cellulases expressed in plants is also an expensive step, which contributes greatly to the cost of production of ferrnentable sugars from cellulose. Therefore, clearly there is a critical need in the art for more efficient methods for producing cellulases for the cost-effective mass production of second-generation biofuels. Other methods of improving tree varieties for their use in the cellulose, wood and energy industries are known. For example, from the description of the European patent EP 1261726, a transgenic modification of trees belonging to the Populus family is known, which show increased biomass production and greater fiber length compared to the wild variety of such a tree. The DNA sequence of a gene encoding a polypeptide having GA20-oxidase activity was introduced into the genome of the transgenic tree. In tum, from the description of the intemational patent application No. W02015060773, there is known a method of producing a transgenic tree with a reduced xylan content in relation to the wild tree, consisting in reducing the expression of one or more genes from the family of glycosyltransferases (GT43). The method of said application comprises introducing a vector into plant cells, said vector comprising at least one or more GT43 family genes in a down-regulating orientation of one or more GT43 family genes. Transgenic trees are characterized by improved growth parameters and better mechanical properties. Another example is the solution known from the description of the patent application WO2010062240, in which genes affecting the level of lignin in the cell wall were indicated. In 1997, the Gene Discovery Platform was also established, focused on identifying poplar genes that control wood formation and tree flowering. The effect of their work was the publication of a bioinforrnatics database (the Populus EST database) on the poplar. Therefore, there is great innovation potential for the targeted improvement of poplar and the practical application of lines with controlled genes involved in biomass growth and regulation of wood properties at the tissue and cellular level.

Usually, transgenic plants with modified wood composition suffered growth penalties, especially in the field. Downregulation of cinnamate 4-hydroxylase (enzyme taking part in an early stage of phenylpropanoid synthesis pathway) by RNAi resulted in ~30% less lignin content in hybrid aspen, but it affected tree growth and wood mechanical properties (Bjurhager et al. Biomacromolecules 11, 2359, 2010). Another widely used target for genetic manipulation of lignin content is 4-coumarate:coenzyme A ligase (4CL). Suppression of this enzyme leads up to 40-45% reduction in lignin content in aspen (Hu et al. Nat Biotechnol 17, 808, 1999; Jia et al. Chin. Sci. Bull. 49, 905, 2004; Li et al. Proc. Natl. Acad. Sci. USA 100, 4939, 2003), however phenotype traits were confirmed only in greenhouse conditions, and any information was provided about growth in field trial". In experiments carried out in the field, the decrease in lignin content was accompanied by stunted growth (Stout et al. Biomass and Bioenergy 68, 228, 2014; Leple et al. The Plant Cell 19, 3669, 2007; Van Acker et al. Proc. Natl. Acad. Sci. USA 111, 845, 2014). Although, Tian et. al. (Plant Physiol Biochemi 65, 111, 2013) achieved a 30% reduction in lignin content and transgenic plants were higher than wild-type plants, it is not clear if transgenic lines were planted in pots or in field soil. One of the most promising field experiments carried out on poplar (Populus tomentosa) with RNAi of 4CL and CCoAOMT resulted in 28% less lignin content and unchanged growth parameters, however the experiment was performed for l-year-old plants (one vegetation season). Summarizing, the reduction of lignin is considered often as defense trait against biotic stress, and is not correlated with the plant growth; or reduced-lignin phenotype of transgenic plants acceptable in the greenhouse, is strongly dismissed in fluctuating field conditions.

Thus, the present inventors produced transgenic plants With a biotechnological potential of the conditional PCD regulators i.e., LESION SIMULATING DISEASE 1 (LSD1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFFICIENT 4 (PAD4) (Wituszynska et al. Plant Physiol. 161, 1795, 2013; Czamocka et al. Plant Cell Environ. 40, 2644, 2017).

PAD4 Mutational screens in Arabic/Opsis identified several plant defense signaling genes that are components of plant PCD. For example, PHYTOALEXIN DEFICIENT 4 (PAD4) encodes a protein that operate upstream of pathogen-induced SA accumulation and therefore pad4 (phytoalexin def1cient 4) mutant demonstrates blocked biosynthesis of salicylic acid triggered by infection With avirulent pathogens (Feys et al. EMBO J. 20, 540, 2001; Jirage et al. Proc. Natl. Acad. Sci. USA 96, 13583, 1999). Moreover, PAD4 expression can be enhanced by exogenous applications of SA, suggesting that it is regulated by SA-dependent positive feedback. Although, PAD4 Was mainly characterized to be involved in defense response to bacteria, insects and other pathogens, its implication in many other processes e. g., regulation of hydrogen peroxide concentration, leaf senescence, aerenchyma formation in response to hypoxia Was also reported. PAD4 acts also as a positive regulator of cell death in response to abiotic stresses such as high light or Water stress. HoWever, there have been no publications that Would postulate to use pad4 mutation in other purpose than the enhancement of plant disease resistance to pathogens (e.g. Patent application number: US20100223690, Publication date: 09/02/2010, Inventors: Bachettira W. Poovaiah (Pullman, WA, US), Liqun Du (Pullman, WA, US), title: COMPOSITIONS AND METHODS FOR MODULATING PLANT DISEASE RESISTANCE AND IMMUNITY, Patent application number: US201001 15658, Publication date: 05/06/2010, Inventors: Mireille Maria Augusta Van Damme (Amsterdam, NL), Augustinus Franciscus Johannes Maria Van Den Ackerveken (Houten, NL), title: DISEASE RESISTANT PLANTS, Patent application number: US20090048312, Publication date: 02/19/2009, Inventors: Jean T. Greenberg (Chicago, IL, US), Ho Won Jung (Chicago, IL, US), Timothy Tschaplinski (Oak Ridge, TN, US), title: PLANT PATHOGEN RESISTANCE, Patent application number: US20090138981 , Publication date: 05/28/2009, Inventors: Peter P. Repetti (Emeryville, CA, US), T. Lynne Reuber (San Mateo, CA, US), Oliver Ratcliffe (Oakland, CA, US), Karen S. Century (Albany, CA, US), Karen S. Century (Albany, CA, US), Katherine Krolikowski (Richmond, CA, US), Robert A. Creelman (Castro Valley, CA, US), Frederick D. Hempel (Albany, CA, US), Roderick W. Kumimoto (Norman, OK, US), Luc J. Adam (HayWard, CA, US), Neal I. Gutterson (Oakland, CA, US), Roger Canales (Redwood City, CA, US), Emily L. Queen (San Leandro, CA, US), Jennifer M. Costa (Union City, CA, US), title: BIOTIC AND ABIOTIC STRESS TOLERANCE IN PLANTS).

EDS1 Similarly to pad4 mutation, the disfunction of ENHANCED DISEASE SUSCEPTIBILITY] (EDS1) blocks SA-mediated signaling and enhances disease susceptibility. EDS1 is the important regulator of innate immunity. EDS1 together With and PAD4 are required for signal transfer of R genes- mediated resistance. Both proteins share homology to triglyceride lipases and both act upstream of SID2 and EDS5 in the regulation of SA accumulation (Glazebrook et al. Annu. Rev. Genet. 31, 547, 1997). Recent data provides evidence that EDS1 regulates chloroplast-ROS during the photo-oxidative stress signaling Which indicates a link between EDS1 and ROS (Straus et al. Plant J. 62, 628, 2010). EDS1 is suggested to have a 'master' role in the coordination of SA and ROS accun1ulation, and in regulation of individual cells death in response to abiotic and biotic stiniuli.

LSD1 Knock-out n1utant of LESION SIM ULA T ING DISEASEI gene (Isdl) belongs to one of the best characterized n1utants deregulated in terrns of progran1n1ed cell death (PCD) in Arabídopsís thalíana. The Isdl n1utant was initially characterized for its ROS- and SA-dependent uncontrolled spread of PCD that develops under long (>l6 h) or continuous photoperiods, supply of superoxide ion or after infection with avirulent pathogen. We propose that the role of LSD1 in light acclin1ation and in restricting pathogen-induced cell death are functionally linked (Mateo et al. Plant Phys. 136, 2818, 2004). The Isdl phenotype is indicative for failure to stop both the initiation and propagation of PCD, therefore it was nan1ed runaway cell death. LSD1 function was proposed as a negative regulator of PCD, acting as a ROS rheostat and preventing the pro-death pathway below certain ROS level. However, our results reported that LSD1 is predoniinantly required for acclin1ation to conditions that promote excess excitation energy (EEE) such as photooxidative, photorespiratory and root hypoxia stress conditions. We linked runaway PCD in Isdl n1utant to the an1ount of light energy absorbed in excess by PSII light harvesting complex, to the redox changes in PSII proxiniity, to ston1atal conductance and ultin1ately to photorespiratory burst of H20; and ethylene. Furtherrnore, non-photorespiratory conditions retard propagation of lesions in Isdl. All these results suggest that LSD1 can influence the effectiveness of EEE dissipation and consequently is a key deterrninant of accliniatory processes.

The DS1, PAD3, LSD1 regulators were earlier recognized, as involved in lysigenous aerenchynia forrnation in response to root hypoxia stress in Arabídopsís thalíana. Conditional PCD regulation by LSD1, EDS1, and PAD4 was proved by diversification of runway PCD during growth of Isdl n1utant in nonperrnissive laboratory conditions (PCD induction) and in the field (PCD inhibition) (Wituszynska et al. Plant Physiol. 161, 1795, 2013; Czarnocka et al. Plant Cell Environ. 40, 2644, 2017; Mühlenbock et al. Plant Cell 19, 3819, 2007; Schuetz et al. J. Exp. Bot. 64, 1 1, 2013). In the laboratory, the Isdl plant displayed deregulation in 2100 genes, whereas only 102 genes in the field (Wituszynska et al. Plant Physiol. 161, 1795, 2013). Seed yield was 4 tin1es lower in Isdl than wild type in laboratory conditions but sin1ilar in both genotypes in the field conditions. The present invention relates to the role of LSD 1 , EDS1, and PAD4 in cell wall niodification. Based on the results, stable transgenic aspen lines with deregulated PtEDSI , PILSDI, PtPAD4, and lower lignin content in the wood were generated. Higher efficiency of industrial bioethanol production fron1 PtEDSI, PtLSDI, PtPAD4 wood was found.

The aini of the present intention was to develop a technology in the field of genetic engineering enabling: i' *ÅS \" . E" Enzymatic system of transgenic trees was more efficient as substrate for bioethanol production than wild type enzymatic system. Presented invention can trigger development of novel biotechnologies in the biofuel industry.

Increasing cellulose while decreasing lignin content in the wood was the goal of present invention. Wood formation requires the occurrence of PCD (Schuetz et al. J. Exp. Bot. 64, ll, 2013). PCD regulators LSD1, EDSl, and PAD4 are conditional regulators of PCD and lysigenous aerenchyma formation in Arabídopsís thalíana. Earlier, Arabídopsís lsdl mutant, with a strong reduction of LSD] expression, was shown to display runaway cell death phenotype in ambient laboratory conditions, but PCD was not developed in the field conditions. Presented invention show their biotechnological potential for agriculture, forestry, and bioethanol industry. The present inventors proved important similarities between Arabídopsís proteomic and transgenic aspen transcriptomic results in the cambium and differentiating xylem tissue. The deregulation of PCD impacted lignin content in the range of 10 - 22%. Lower relative lignin content did not affect growth of the transgenic trees in comparison to the wild- type plant during four growing seasons in the field. Therefore, modif1cation of the conditional PCD regulators in combination rather than the lignin biosynthesis pathway might appear much more promising for bioethanol production. Lowering the lignin level up to 20% allowed for maintaining the growth of transgenic lines at the wild type level, but also was accompanied by a higher cellulose polymerization level. The higher degree of polymerization of cellulose facilitates the formation of hydrogen bonds among neighboring cellulose fiber and thus plays a pivotal role in increasing the mechanical performance of wood, allowing easier removal of lignin and hemicellulose from wood and simultaneously suppressing the decomposition of cellulose during lignin removal. These features for trees growing in the field for four subsequent seasons (optimal period for harvesting of fast-growing aspen plantation) are desirable for the production of bioethanol. Indeed, it was confirmed during experiments in a semi-industrial scale. Considering the above, the deregulation of well-known conditional PCD regulators (LSDI, EDSI and PAD4) and a well-known process (PCD) in a completely new context give unexpected effects. Importantly, the function of these proteins is similar in many plant species (Guan et al. BMC Genomics 17, 142, 2016; Guo et al. Plant Physiol Biochem 71, 164, 2013; Gao et al. Planta 231, 1037, 2010).

Importantly, molecular interaction between LSDl, EDS 1 , and PAD4 molecules deterrnines the outcome of lignin biosynthesis or cell wall saccharif1cation in response to hypoxia. Both processes are regulated in the presence of functional LSDl-EDSl-PAD4 trimer, but in both species i.e., Arabídopsís and aspen, reduction of EDS] expression led to the greatest changes. LSDl and PAD4 cannot interact directly, while EDSl binds to both LSDl and PAD4 (Feys et al. EMBO J 20, 5400, 2001; Czamocka et al. Plant Cell Environ. 40, 2644, 2017). Therefore, EDSl presence is necessary to control the proper functioning of protein trimer and thus effective lignin biosynthesis or cell wall saccharification in response to hypoxia. Important, it is enough to deregulate two genes in combination so that all there of these genes are silenced and the combination of two genes regulate the biochemical composition of wood and the enzymatic composition during hypoxia in the same way as three silenced genes. The deregulation of genes encoding conditional PCD regulators i.e., LSDl, EDSl, and PAD4, in combination of two or three genes, can be a breakthrough biotechnology for biofuel industries and can be applied directly to improve bioethanol production.

Examples Example 1. EDS1 and LSD1 are involved in lysigenous aerenchyma formation LSD1, EDS1, and PAD4 are involved in lysigenous aerenchyma formation (Fig. 1). Changes in content of reducing sugars were determine before and 7 days after root hypoxia stress to prove a cell wall lysis during the formation of lysigenous aerenchyma in hypocotyls. Elevated level of reducing sugars before hypoxia was measured in lsdl mutant when compared to wild- type plant (Fig. la). After stress, lsdl mutant exhibited elevated (compared to wild-type plant) but stable (as compared with lsdl plants before hypoxia) level of reducing sugars. edsl mutant exhibited the highest level of reducing sugars, whereas edsl lsdl mutant did not differ from Ws- 0 (Fig. lb). ggggg Fig. 1 is a set of graphs showing lysigenous aerenchyma formation in Arabidapsis thaliana roots and hypocotyls in response to hypoxia stress monitored by a reducing sugar content and in vitra activity of cellulolytic and xylanolytic enzymes. The reducing sugar content was measured before (a) and after (b) 7 days of hypoxia stress expressed as D-glucose content. Analysis of hydrolytic activity of protein extracts against carboxymethyl cellulose (c) and Xylan (d) Was measured in control conditions and after 7-day-hypoxia stress by a release of reducing sugars during 3-h incubation. Means values (iSD) were calculated from separate biological replicates (n = 3-6). Stars above the bars indicate statistically significant differences in comparison to the Ws-0 plants, according to Tukey HSD at levelp < 0.05 (*),p < 0.01 (**), andp < 0.00l (***). Model of experimental setup for lysigenous aerenchyma formation in response to hypoxia stress (e).

Example 2. In vitro cellulolytic and xylanolytic activities of edsl enzymes Two different substrates i.e., carboxymethyl cellulose (CMC) and beechwood xylan were added to proteins extracted from WS-0 and edsl hypocotyls. CMC is a substrate for ß-D-glucanase hydrolyzing ß-l-4 glycosidic linkages between glucopyranose units (in an amorphous cellulose), while xylan is a substrate for xylanase acting on ß-l-4 glycosidic linkages between xylopyranose units in xylan backbone. The Arabídopsís enzymes induced in roots and hypocotyls during hypoxia stress have a potential to break down both cellulose and xylan, however proteins extracted from edsl mutant exhibited higher activity than those extracted from WS-0 (Fig. lc, d). In Contrast, enzymatic extracts from lsdl and lsdledsl had lower activity against both CMC and xylan than edsl mutant and wild type plants (Fig. 2a-d). The high reducing sugar content after stress and stable enzymatic activity of extracts of edsl mutant indicated that cellulase and CWL proteins were intact and unaffected by endogenous proteolysis.

Activity against CMC ÅCÜVWY öâflífifif CMC before hypoxia stress b añfif "Yšlmflla 537955 D-Glucnse inn/X) ïêxnc-s fn) w _ Actívšw agalnst xylsn Activity against xylzm afieriwçloxia mess _ _ ~ before lwuoxia stress Tcmes iii) Tšxres (h) 'vVsC I :dl edsl lsdí/edsfi Fig. 2 is a set of graphs showing glucanase and xylanase activities in roots and hypocotyls measured after hypoxia stress. Protein extracts isolated before hypoxia stress exhibited lower activity against carboxymethyl cellulose (a) and xylan (c), compared to extracts isolated after hypoxia stress (b, d). Means values (iSD) are derived from separate biological replicates (n = 3-6). Stars above the bars indicate statistically significant differences in comparison to the WT plants, according to Tukey HSD at level p< 0.05 (*), p< 0.0l (**), and p < 0.00l (***).

Enzymes generating reducing ends were found in hypocotyls and roots undergoing lysigenous aerenchyma formation. In the protein fraction with and enzymatic activity that was isolated from zymographic activity assay many proteins in individual genotypes were identified during Maldi-TOF analysis. Some of them were found in two or more genotypes, but after removing duplicates, 563 proteins were found. Approximately 6.4% of all identified proteins were assigned/aligned to known or predicted proteins capable to generate reducing sugars (Fig. 2a, b). Some of them, like Beta-glucosidase 23 (BGL23), Were identified in more than one sample, other, like CELLULASE 2 (CEL2), Were found only in edsl mutant in Col-0 background. The identified enzymes Were also from different CAZY families, With known or predicted CWL activities (homogalacturonan, cellulose, xylan, mannan, callose, xyloglucan, and hydroxyproline-rich or arabinogalactan). Some of the enzymes are involved in generation of reducing ends from CMC or could contribute to the hydrolytic activity against xylan. The protein ontology analysis (Fig. 3c) using Panther (Pantherdb.org) softWare Was performed for edsl mutants in both ecotypes, due to exhibited saccharification potential. Four proteins Which abundance depend on EDS1 dysfunction (gene expression inhibition) Were found (Fig. 3d). These proteins represent cell Wall dismantling machinery and are interesting targets as plant enzymes for use in plant biotechnology. a of identified geoafating ends edsl (Vvs-O) 6180701 f 46311401 i 157806111 yxrseoezsoi ëïäeßsarsfz ooooooo - i T4G 342601 \ i-Pmsis as šßGALio exoT-Ai i 17 šPmEi-Pmszo íffiïfïšååêëâíï-š? ______________________________________________________ __ 115152101 BXL6 6135601 6267201 THEXZ 18 l39SO.l , msrgeszirr: Fig. 3 Identification of proteins isolated from roots and hypocotyls during lysogenic aerenchyma formation due to root hypoxia stress by Maldi-TOF analysis. Number of enzymes generating reducing ends (a) and their presence in particular genotypes (b). Protein classes analysis by the Panther shoWn as a pie chart presenting different protein classes taking part in root hypoxia stress response and their abundance among identified proteins in edsl (Col-O) and pie chart presenting division of categories metabolite interconversion enzyme (PC00262) into subcategories (c). EDS1 abundance dependent generating reducing ends enzymes (d) Data are based on six separate experiments (biological replicates) and tWo technical repetitions per sample. Results Were analyzed using Mascot softWare and trimmed so there Will be not more than 1% of false positives in each sample.

Example 3. Enzymes generating reducing ends Were found in hypocotyls exposed to hypoxia In the protein fraction With and enzymatic activity that Was isolated from zymographic activity assay, 563 proteins Were identified during Maldi-TOF analysis. Some proteins Were assigned to knoWn or predicted proteins capable to generate reducing sugars (Table 1). Some of them, like Beta-glucosidase 23 (BGL23), Were identified in more than one sample, other, like CELLULASE 2 (CEL2), Were found only in edsl mutant in Col-0 background. Identified enzymes from different CAZY families, had (known or predicted) activities against homogalacturonan, cellulose, Xylan, mannan, callose, Xyloglucan, and hydroxyproline-rich or arabinogalactan. Some of the enzymes, e.g., CEL2 (GH9) and ß-glucosidase clade of GH3 are involved in generation of reducing ends from CMC. Moreover, ß-xylosidase clade of GH3 could contribute to the hydrolytic activity against Xylan. The enzymes involved in degradation of pectin such as a putative polygalacturonase (GH28), several pectin acetyl and methyl esterases (CE13 and CE8, respectively), and Xyloglucan degrading/modifying enzymes, including XTH4 and XTH7 (GHl6), Xyloglucan ß-galactosidase BGALl0 (GH35) or Xyloglucan ot-fucosylase AXY8 (GH95), Were identified.

Table 1. List of genes encoding cell Wall loosening proteins and enzymes generating reducing sugars identified in each ecotype (Col-O, Ws-0) and eds] mutants. Proteomic experiments in hypocotyls and roots samples collected 7 days after start of root hypoxia stress. Gene hierarchy in the table corresponding to the number of peptides identified for a given proteins in the Maldi-TOF analysis.

No ATG code Genotype and ecotype; Biological function; Protein C01-0 1 AT3G54420.1 Endochitinase EP3; Fungal defense; chitinase 2 AT4G15210.1 Beta-amylase 5; Starch degradation; ß-amylase 3 AT3G47000.1 Beta-glucosidase; XG modification; ß-glucosidase Ws-0 1 AT5G13980.1 Probable alpha-mannosidase; Protein modification 2 AT5G34940.2 Heparanase-like protein 3; AGP modification; glucuronidase 3 AT4G16260.l Probable glucan endo-1ß-beta-glucosidase; Callose degradation; ß-lß-glucanase 4 AT5G64570.1 Beta-D-Xylosidase 4; Xylan modification; ß-Xylosidase AT4G31140.1 Glucan endo-lß-beta-glucosidase; 5 Callose degradation; ß-lß-glucanase 6 AT3G09260.1 Beta-glucosidase 23; Deglycosylation; ß-glucosidase 7 AT5G083 70.1 Alpha-galactosidase 2; Mannan/raffiose degradation; oL-galactosidase 8 AT5G10560.1 Probable beta-D-Xylosidase 6; Xylan modification; oL-L-arabinofiiranosidase/ ß-1,4- xylosidase 9 AT3G12500.1 Basic endochitinase B; Fungal defense; chitinase AT5G25980.1 Myrosinase 2; Deglycosylation; myrosinase 11 AT1G61820.1 Beta-glucosidase 46; Deglycosulation; ß-glucosidase 12 AT3G263 80.1 Alpha-galactosidase; AGP degradation; ß-L-arabinopyranosidase 13 AT3G13560.1 Glucan endo-lß-beta-glucosidase 4; Callose degradation, ß-lß-glucanase 14 AT3G61490.1 Pectin lyase-like superfamily protein; Pectin degradation; polygalacturonas edsl (C01-0) 1 AT5G34940.2 Heparanase-like protein 3; AGP modification; ß-glucuronidase 2 AT5G07830.1 Heparanase-like protein 1; AGP modification; ß-glucuronidase 3 AT5G083 70.1 Alpha-galactosidase 2; Mannan/raffiose degradation; a-galactosidase 4 AT5G13 690. 1 Alpha-N- acetylglucosaminidase AT3G09260.1 Beta-glucosidase 23; Deglycosylation; ß-glucosidase 6 AT3G12500.1 Basic endochitinase B; Fungal defense; chitinase 7 AT4G31140.1 Glucan endo-lß-beta-glucosidase 5; Callose degradation; ß-lß-glucanase 8 AT3G263 80.1 Alpha-galactosidase; AGP degradation; ß-l-arabinopyranosidase 9 AT4G37800.1 Probable Xyloglucan endotransglucosylase/hydrolase protein 7; XG modification; XTH AT2G06850.1 Xyloglucan endotransglucosylase/hydrolase protein 4; XG modification; XTH 11 AT1G35580.1 Alkaline/neutral invertase ClNVl; Sucrose degradation; neutral/alkaline invertase 12 AT5G63810.1 Beta-galactosidase 10; XG modification; Xyloglucan ß-galactosidase 13 AT1G78060.1 Probable beta-D-Xylosidase 7; Xylan modification; ß-1,4-Xylosidase 14 AT3G18070.1 Beta-glucosidase 43; Deglycosylation of metabolites AT4G16260.l Probable glucan endo-1ß-beta-glucosidase, Callose degradation; ß-lß-glucanase 16 AT1G02800.1 Endoglucanase 1 Cellulase 2; Cellulose degradation; Endo-glucanase edsl (Ws-0) 1 AT5G13980.1 Probable alpha-mannosidase; Protein deglycosylation 2 AT3G26720.1 Alpha-mannosidase; Protein deglycosylation 3 AT3G09260.l Beta-glucosidase 23; Deglycosylation of metabolites; ß-glucosidase 4 AT5G64570.1 Beta-D-Xylosidase 4; Xylan modification; ß-1,4-Xylosidase 6 AT5G34940.2 Heparanase-like protein 3; AGP modification; ß-glucuronidase 8 AT3G12500.1 Basic endochitinase B; Fungal defense; chitinase 9 AT3Gl3560.l Glucan endo-1,3-beta-glucosidase 4; Callose degradation; ß-l,3-glucanase 10 AT4G3 l 140.1 Glucan endo-1,3-beta-glucosidase 5; Callose degradation; ß-l,3-glucanase 11 AT5G083 70.1 Alpha-galactosidase 2; Mannan/raffiose degradation; oL-galactosidase 12 AT5G07830.1 Heparanase-like protein 1; AGP modification; ß-glucuronidase 13 AT4G3 4260.1 Alpha-L-fiicosidase 2; XG modification; a-fucosidase ,_i 4 AT3G10740.1 Alpha-L-arabinofuranosidase 1; Pectin/Xylan/AGP modification; oL-L- arabinofuranosidase / ß-1,4-xylosidase AT3G55260.1 Beta-hexosaminidase 1 16 AT5G63 810.1 Beta- galactosidase 10; XG modification; Xyloglucan ß-galactosidase 17 AT1G52400.1 Beta-D-glucopyranosyl abscisate beta- glucosidase; Deglycosylation; ß-glucosidase 18 AT3 G263 80.1 Alpha- galactosidase; AGP degradation; ß-L-arabinopyranosidase Most importantly, some proteins were involved in polysaccharide catabolite processes and proteins involved in lignin precursor catabolic processes (Table 2).

Table 2. Overrepresentation test of genes encoding proteins found in Maldi-TOF, Analysis Type: PANTHER Overrepresentation Test (Released 20221013). Annotation Version and Release Date: GO Ontology database DOI: 10.5281/Zenodo.6799722 Released 2022-07-01, Analyzed List: upload_1 (Arabídopsís thalíana). Reference List: Arabídopsís thalíana (all genes in database). Test Type: FISHER. Correction: FDR.

GO biological process; upload_1 (fold Enrichment); upload_1 (raw P-value); upload_1 (FDR) protein hexamerization (GO:0034214) 61.78 1.49E-03 l3.50E-02 cinnamic acid biosynthetic process (GO:0009800) 37.07 2.l2E-04 6.33E-03 cinnamic acid metabolic process (GO:0009803); 30.891 3.14E-04 8.84E-03 arabinan catabolic process (GO:0031222) 23.17 6.02E-04 1.58E-02 arabinan metabolic process (GO:0031221) 20.59 7.94E-04 2.05E-02 L-phenylalanine catabolic process (GO:0006559) 20.59 7.94E-04 2.04E-02 secondary alcohol metabolic process (GO: 1902652) 20.59 9.93E-05 3.27E-03 fructose 1,6-bisphosphate metabolic process (GO:003 03 88) 20.59 9.93E-05 3.25E-03 hexose biosynthetic process (GO:00l93 19) 16.63 7.4lE-07 3 .03E-05 Xylan catabolic process (GO:0045493) 15.44 2.5 1E-04 7.32E-03 hemicellulose catabolic process (GO:2000895) 15.44 2.5lE-04 7.29E-03 cell Wall polysaccharide catabolic process (GO:0044347) 14.54 3.07E-04 8.72E-03 Example 4. Cell walls of aspen with deregulated PCD growing in the field contain less lignin and it allow high fermentation efficiency.

Four transgenic lines with reduced expression of PtLSDI, PtEDSI, and PtPAD4 in different combinations were generated. All of three genes, i.e., PtLSDI, PtEDSI, and PtPAD4 were downregulated regardless of the double transgene combination (even if third gene was not modified in double transgenic line), because these genes are conditionally co-regulated, while LSDl, PAD4, and EDSl proteins interact With each (Fig. 4). Trees randomly grown during 4 years on experimental field had significantly lower lignin content in the wood (Fig. 4b). The reduction of lignin was in the range of 10% - 22% (Fig 4d). Cellulose content was lower; however, its polymerization degree was significantly higher (Fig. 4d).

Lignin is an inhibitor of alcoholic ferrnentation. Generated lines were suitable for bioethanol production. Higher glucose yield after enzymatic hydrolysis (Fig. 4e) and ethanol yield after ferrnentation (Fig. 4f) was obtained in comparison to the wild type plants. The high effectiveness of the alcohol ferrnentation process was confirrned, as almost whole glucose was converted to ethanol for wood (Fig. 4g). a b c C l z: C __ g .9 -.. 1.: r _ c.. Cl 51 . , 'v ß O, f; Du Q. w? .me 1un»21_finf= sim» 4 1- (9 i) K 131 å: (D -fi GD Aí1s-'ís-vr.c~r.t»x:c-r.

Glucoi-e ccmïc-sït igüimï Emanoi content lífdmfïš Glue-use come Fig. 4 is a set of graphs showing integrated deregulation of PtLSDl, PtEDSl, and PtPAD4 affected lignin content and other wood chemical properties. Transgenic aspen trees grew for four subsequent seasons in the field conditions. Relative expression level of PtLSDl, PtEDSI and PtPAD4 in leaves (a-c), relative lignin, cellulose, and holocellulose content and degree of cellulose polymerization (d), glucose content after saccharification of Wood (e), the amount of ethanol in the fermentation process (f), and the amount of un-fermented glucose in the pulp after fermentation (g). Mean values (iSD) are derived for 3 different biological replications and 3 technical replications for each transgenic line on graphs a-c (n = 9) and for three to nine different biological replications (n = 3-9) for data B, C, D and E. Stars above the bars indicate statistically significant differences in comparison to the WT plants, according to Tukey HSD at level p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Example 5. PtLSDl, PtEDSl, and PtPAD4 strongly influence cambium and xylem transcriptome Analyzing a transcriptome of cambium-developing xylem tissue 2648 deregulated genes were found, of which 2299 were known, and 342 were unknown. For all known genes, gene ontology analysis was performed considering their molecular function, or biological process. Many differentially expressed genes encoded putative enzymes. The overrepresentation of genes was assigned to regulation of disaccharide, sucrose, oligosaccharide, carbohydrate, polysaccharide processes. The overrepresentation of genes was also assigned to phenylpropanoid metabolic process, which is related to lignin biosynthesis was found (Table 3).

Table 3. Enrichment of biological process analysis of differentially expressed genes (DEGs) detected in cambium and differentiating xylem tissues isolated from 4-years-old transgenic aspen line 4 growing in the field condition using PNTHER.ORG software, Uniquely Mapped IDS: 42532 out of 42532 2296 out of 2296, displaying only results for FDR P < 0.05 Analysis Type: PANTHER Overrepresentation Test (Released 20220712), Ontology database DOI: l0.528l/Zenodo.6799722 Released 2022-07-01, Reference List: Populus tríchocarpa (all genes in database), Test Type: FISHER, Correction: FDR GO biological process complete; Populus tríchocarpa - REFLIST (42532); upload_1 (fold Enrichment); upload_1 (raW P-value); upload_1 (FDR) disaccharide biosynthetic process (GO:0046351) 43 4,31 3,14E-04 1,17E-02 sucrose metabolic process (GO:0005985) 52 3,92 3,25E-04 1,19E-02 disaccharide metabolic process (GO:0005984) 82 3,84 l,09E-05 6,16E-04 oligosaccharide biosynthetic process (GO:0009312) 56 3,31 1,86E-03 4,74E-02 oligosaccharide metabolic process (GO:0009311) 96 3,28 6,32E-05 2,95E-03 cellular carbohydrate biosynthetic process (GO:0034637) 219 2,45 3,45E-05 1,77E-03 cellular carbohydrate metabolic process (GO:0044262) 485 2,25 7,05E-08 5,99E-06 carbohydrate biosynthetic process (GO:0016051) 296 2,13 1,27E-04 5,64E-03 cellular polysaccharide metabolic process (GO:0044264) 274 2,03 5,94E-04 1,99E-02 polysaccharide metabolic process (GO:0005976) 460 1,81 3,3 8E-04 1,21E-02 carbohydrate metabolic process (GO:0005975) 1339 1,63 1,15E-06 8,03E-05 regulation of phenylpropanoid process (GO:2000762) 12 7,72 1,29E-03 3,87E-02 Some of the genes Were involved in lignin and cellulose Synthesis/metabolism, cell Wall biogenesis, cell Wall organization (Table 4).

Table 4. The list of genes differentially regulated in line 4 cambium-differentiating Xylem tissues involved in cellulose and lignin biosynthesis, cell Wall construction and organization and other processes that may have contributed to better utility values.

Mapped IDs POPTR_010G1023 00v3 POPTR_014G0253 00v3 POPTR_009G142000v3 POPTR_009G141800v3 POPTR_00 8G05 7100v3 POPTR_019G125000v3 POPTR_002G023900v3 POPTR_009G03 1800v3 POPTR_014G164700v3 POPTR_003 G072700v3 POPTR_006G13 5100v3 POPTR_006G13 4600v3 POPTR_006G1 17200v3 POPTR_012G0145 00v3 POPTR_00 8G060100v3 POPTR_014G162900v3 POPTR_007G00 85 00v3 POPTR_010G2025 00v3 POPTR_012G109200v3 POPTR_001G240900v3 POPTR_005G117500v3 POPTR_015G013 700v3 POPTR_003 G072800v3 POPTR_006G071200v3 POPTR_013G125000v3 POPTR_005G218900v3 POPTR_009G1423 00v3 POPTR_002G0605 00v3 POPTR_014G15 7600v3 POPTR_009G0065 00v3 POPTR_002G2465 00v3 POPTR_005G22 8700v3 POPTR_0 1 9G0 693 00v3 POPTR_009G1 133 00v3 POPTR_009G063 3 00v3 Gene Xyloglucan endotransglucosylase/hydrolase WOX family protein;POPTR_014G0253 00 Chitin-binding type-1 domain-containing protein Acidic endochitinase WlN6.2C (Fragment) Expansin Xyloglucan endotransglucosylase/hydrolase Endoglucanase Expansin Histidine kinase Pectinesterase Pectinesterase Pectinesterase Protein kinase domain-containing protein Pectinesterase NTP_transferase domain-containing protein FASl domain-containing protein Xyloglucan endotransglucosylase/hydrolase Expansin Exostosin domain-containing protein Expansin Uncharacterized protein Pectinesterase Pectinesterase Xyloglucan endotransglucosylase/hydrolase Chitin-binding type-1 domain-containing protein Hexosyltransferase Chitin-binding type-1 domain-containing protein Xyloglucan endotransglucosylase/hydrolase Endoglucanase Exostosin domain-containing protein Hexosyltransferase Nn1rA domain-containing protein Endoglucanase Glycosyltransferase PKS_ER domain-containing protein POPTR_008G132700V3 POPTR_013 G152400V3 POPTR_006G199100V3 POPTR_010G160800V3 POPTR_005G195600V3 POPTR_007G099800V3 POPTR_005G10 8900V3 POPTR_002G0653 00V3 POPTR_015G003 5 00V3 POPTR_006G13 9100V3 POPTR_01 1 G0 623 00V3 POPTR_015G060000V3 POPTR_00 1 G03 3 800V3 POPTR_00 1 G0 89100V3 POPTR_005G0015 00V3 POPTR_010G004400V3 POPTR_005G03 8600V3 POPTR_007G091900V3 POPTR_005G0765 00V3 POPTR_005G109100V3 POPTR_002G120400V3 POPTR_010G063 000V3 POPTR_013 G019800V3 POPTR_003 G1425 00V3 POPTR_001G13 6200V3 POPTR_010G074700V3 POPTR_014G125100V3 POPTR_009G060800V3 Endoglucanase Xyloglucan endotransglucosylase/hydrolase PKS_ER domain-containing protein Uncharacterized protein Peroxidase; PRX36 Beta- galactosidase Peroxidase Peroxidase Peroxidase Beta- galactosidase Peroxidase COBRA-like protein Uncharacterized protein Fn3_like domain-containing protein Pectin acetylesterase Pectin acetylesterase Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein PMEI domain-containing protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Uncharacterized protein Cellulose synthase Moreover, compared genes encoding enzymes identified in Maldi-TOF experiment in Arabídopsís thalíana With their orthologs among genes deregulated in transgenic aspen; 19 similar genes (Table 5), including ALPHA-L-FUCOSIDASE 2 and BETA-D-XYLOSIDASE 7 Were found. These genes are targets for biotechnology in the second generation of biofuels.

Table 5. List of aspen differentially regulated genes in aspen and their corresponding orthologs found in Arabídopsís proteomic experiment using Maldi-TOF.

POPTR ID; Changes in expression in transgenic aspen in comparison to Wild type (Log2 fold change); Gene encoding enzymes found in MaldiTOF in Arabídopsís; Cell Wall loosening proteins and enzymes generating reducing sugars; Short description.

POPTR_001G299000V3 POPTR_004G162400V3 POPTR_013 G061800V3 homocysteine methyltransferase 2 i, iwfl POPTR_004G167600V3 POPTR_015G03 4700V3 POPTR_010G2195 00V3 POPTR_004G1913 00V3 POPTR_007G099800V3 POPTR_006G0 85400V3 POPTR_00 8G051600V3 transferring) POPTR_004G213400V3 POPTR_007G013 000V3 POPTR_018G03 6900V3 POPTR_014G0 863 00V3 = AT4G34260 * AT2G213 3 0 AT3G03780 AT4G3 0150 AT1G66200 AT1G07920 AT1G045 80 AT5G63 810 = AT4G26870 * AT5G65750 AT2G28000 AT2G18020 AT1G15250 -« i w AT3G61440 synthase C1, mitochondrial POPTR_004G13 8200V3 åš AT1G22530 Yes ><><><>< Yes ><><><>< Alpha-L-fiicosidase 2 Fructose-bisphosphate aldolase 1, chloroplastic 5-methyltetrahydropteroyltriglutamate-- Urb2/Npa2 family protein Glutamine synthetase cytosolic isozyme 1-2 Elongation factor 1-alpha 3 Aldehyde oxidase 4 Probable beta-D-xylosidase 7 Aspartate--tRNA ligase 1, cytoplasmic Oxoglutarate dehydrogenase (succinyl- Chaperonin 60 subunit alpha 1, chloroplastic 60S ribosomal protein L8-1 60S ribosomal protein L37-1 Bifunctional L-3-cyanoalanine synthase/cysteine Patellin-2 POPTR_007G108500v3 POPTR_008G1793 00v3 GAPC2, cytosolic POPTR_017G0 8 8600v3 POPTR_006G2 64600v3 AT1G77120 x Alcohol dehydrogenase class-P AT 1 G1 3 440 Yes Glyceraldehyde-3 -phosphate dehydrogenase AT4G12 8 80 x AtENODL19 AT2G25060 X Early nodulin-like protein l Example 6. Growth is unaffected, but photosynthesis is modified in transgenic aspen lines.

Slight(insignif1cant) differences in trees height, stem fresh Weight, stem diameter during annual growth Were measured. In contrast, C02 assimilation Was changed in transgenic lines, even if differences Were not found in chlorophyll content (Fig. 5).

Mun mn: »weight (m) Fig. 5 is a set of graphs showing biometric parameters, C02 assimilation, and chlorophyll content in transgenic lines. The height of the main stem (a), Weight of the main stem (b), diameter of the main stem (c), average annual growth of the main stem (c), C02 assimilation (d), total chlorophyll content in leaves (f). Stars above the bars indicate statistically significant differences in comparison to the WT plants, according to Tukey HSD at level p<0.05 (*), p<0.0l (**), and p<0.00l (***). Mean values (iSD) for f1fe - eleven different biological replication for data on graph A and B (n = 5-l l), for three different biological replications on graph C (n=3), for four different biological replications on graph D (n = 4), for 30 different leaves per genotype on graph E (n = 30) and from nine different technical replication per genotype on graph E (n = 9).

Claims (10)

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence comprising at least two amino acid sequences chosen from among amino acid sequences having at least 60% identity With SEQ ID NO. 1 or SEQ ID NO. 2 (encode enhanced disease susceptibility 1 (EDS1) protein, at least 60% identity With SEQ ID NO. 3 or SEQ ID NO. 4 (encode phytoalexin deficient 4 (PAD4) protein, and at least 60% identity With SEQ ID NO. 5 or SEQ ID NO. 6 (encode lesion simulating disease l (LSDl) protein in a plant.

2. An allelic variant or a homolog of the nucleotide sequences of claim

3. A DNA fragment according to any one of claims l - 2, Wherein one or more functional Characteristics of the protein are retained.

4. A Vector construct comprising the isolated nucleic acid molecule according to any one of claims 1 - 3, operably linked to an expression control sequence.

5. A plant, plant material, plant cell, or a seed of a plant, Which comprises the isolated nucleic acid molecule according to any one of claims 1 - 3, Wherein the nucleotide sequence is exogenous or heterologous to the plant or the plant cell and is operably linked to an expression control sequence and expresses the polypeptide.

6. A plant regenerated from a plant cell or seed according to claim

7. A transgenic plant having a gene construct comprising a nucleotide sequence encoding at least two of PAD4, LSDl, and EDSl proteins, operably linked to a promoter such that the nucleotide sequence is attenuated, thereby causing the transgenic plant to exhibit one or more differentially regulated genes in comparison to Wild type plant, Wherein differentially regulated genes and their corresponding orthologs are those from the list: 1 AT5G34940.2 Heparanase-like protein 3; ß-glucuronidase 2 AT5G07830.1 Heparanase-like protein 1; ß-glucuronidase 3 AT5G083 70.1 Alpha-galactosidase 2; a-galactosidase 4 AT5G13 690. 1 Alpha-N- acetylglucosaminidase 5 AT3G09260.l Beta-glucosidase 23; ß-glucosidase 6 AT3G12500.l Basic endochitinase B; chitinase 7 AT4G31140.1 Glucan endo- 1 ß-beta-glucosidase 5; ß- 1 ß-glucanase

8.AT3 G263 80.1 Alpha-galactosidase; ß-l-arabinopyranosidase 9 AT4G37800.1 Probable Xyloglucan endotransglucosylase/hydrolase protein 7; XTH 10 AT2G06850.1 Xyloglucan endotransglucosylase/hydrolase protein 4; XTH 1 1 AT1G35580.1 Alkaline/neutral invertase CINVI; neutral/ alkaline invertase 12 AT5G63 810.1 Beta- galactosidase 10; Xyloglucan ß-galactosidase 13 AT1G78060.1 Probable beta-D-xylosidase 7; ß-IA-xylosidase 14 AT3G18070.1 Beta-glucosidase 43; Deglycosylation of metabolites 15 AT4G16260.1 Probable glucan endo-1ß-beta-glucosidase, ß- 1 ß-glucanase 16 AT1G02800.1 Endoglucanase 1 Cellulase 2; Endo-glucanase 17 AT5G13980. 1 Probable alpha-mannosidase; 18 AT3 G26720.1 Alpha-mannosidase; 19 AT5G64570.1 Beta-D-xylosidase 4; ß-IA-xylosidase 20 AT3G13560.1 Glucan endo-lß-beta-glucosidase 4; ß-lß-glucanase 21 AT4G34260.1 Alpha-L-filcosidase 2; a-fucosidase 22 AT3G10740.1 A1pha-L-arabinofuranosidase 1; a-L-arabinofuranosidase / ß-IA-xylosidase 23 AT3G55260.1 Beta-hexosaminidase24 AT1G52400.1 Beta-D-glucopyranosyl abscisate beta-glucosidase; ß-glucosidase 25 POPTR_001G299000v3 AT4G34260 Alpha-L-fucosidase26 POPTR_004G162400V3 AT2G213 3 0 Fructose-bisphosphate aldolase27 POPTR_0 1 3G061 800V3 AT3G03 7 80 5 -methyltetrahydropteroyltriglutamate- -homocysteine methyltransferase28 29 31 32 33 34 36 37 38 Cl 39 41 42POPTR_004G167600V3 POPTR_015G034700V3 POPTR_010G2195 00V3 POPTR_004G1913 00V3 POPTR_007G099 800V3 POPTR_006G085400V3 POPTR_00 8G051600V3 POPTR_004G213 400V3 POPTR_007G013 000V3 POPTR_018G03 6900V3 POPTR_014G0863 00VPOPTR_004G13 8200V3 POPTR_007G10 85 00V3 POPTR_00 8G 1 793 00V3 POPTR_017G08 8600V3 POPTR_006G264600VAT4G30150 AT1G66200 AT1G07920 AT1G04580 AT5G63 810 AT4G26870 AT5G65750 AT2G28000 AT2G18020 AT1G15250 AT3GAT1G22530 AT1G77120 AT1G13440 AT4G12880 AT2GUrb2/Npa2 family protein Glutamine synthetase cytosolic isozyme 1-2 Elongation factor 1-alphaAldehyde oxidaseProbable beta-D-xylosidaseAspartate--tRNA ligaseOxoglutarate dehydrogenase (succinyl-transferring) Chaperonin 60 subunit alpha0S ribosomal protein L8-60S ribosomal protein L3 7-Bifunctional L-3-cyanoalanine synthase/cysteine synthase Patellin-Alcohol dehydrogenase class-P Glyceraldehyde-3-phosphate dehydrogenase GAPC2 AtENODLlEarly nodulin-like protein8. The transgenic plant of claim 7, Wherein the transgenic plant is an annual plant or a Woody plant, Wherein the annual plant is selected from a group consisting of Arabídopsís, and Woody plant is selected from a group consisting of aspen plant.

9. A method of isolation of enzymes generating soluble sugars from a ligninocellulose material, Wherein enzymes are isolated from a plant comprising genetically modified: at least tWo of phytoalexin deficient 4 (PAD4) gene, lesion simulating disease 1 (LSDI) gene, and enhanced disease susceptibility 1 (EDSl) gene in the plant, according to claim 1-

10. A method of producing a cellulosic ethanol from reducing sugar, Wherein said method comprises enzymes isolated from a plant comprising genetically modified: at least tWo of phytoalexin deficient 4 (PAD4) gene, lesion simulating disease 1 (LSDI) gene, and enhanced disease susceptibility 1 (EDSl) gene in the plant, according to claim 1-8.