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93. (WO2013093637) PLANT TREATMENT METHODS AND MEANS THEREFOR
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Title: PLANT TREATMENT METHODS AND MEANS THEREFOR

This application claims the benefit of priority to U.S. provisional application 61/577279, filed on December 19, 201 1 , which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The invention relates to the field of plant and agricultural technology. More specifically, the present invention relates to a method for regulation (increasing) of tree growth, development, biomass production, and stress tolerance and means therefor. In particular, the invention relates to a method for altering the biomass production, in optimal conditions and under water stress by genetically engineering plants to specific expression of PAD4, EDS1, LSD1 genes, alone or in combinations.

BACKGROUND

A potential solution to the growing problems of inadequate food supplies and the limited availability of renewable energy sources could be an increase in biomass production, particularly for plants that are used for food and/or fuel production. There is an increasing interest in ethanol and oils produced from plants as alternatives to fossil fuels. The interest is focused not only on the finite nature of fossil fuel reserves, but also on problems created by emission that result from their use. Since the 19th century, atmospheric concentration of CO2 has increased by more than 25%, primarily from the combustion of fossil fuels. Excess carbon dioxide in the atmosphere absorbs infrared energy and prevents it from leaving the atmosphere, and thus is often referred to as a "greenhouse" gas. Increasing levels of greenhouse gases in the atmosphere may contribute to an increase in average global temperatures, resulting in adverse climate changes known as global warming. From the wide range of options currently considered as long-term energy supply, the renewable tree biomass source offers a very good alternative to fossil fuels. Nowadays, biomass is the fourth largest energy source in the world after coal, petroleum and natural gas, accounting for 14% of the world's primary energy consumption.

SUMMARY

The object of the present invention is to obtain transgenic trees and crop plants with improved water use efficiency and biomass production in stress and semi-stress field (and laboratory) conditions. Increase in stress tolerance opens opportunities for successful cultivation of crop and other plant species under conditions that were not previously possible, e.g. in areas with poor irrigation. By "increased tolerance toward water stress" it is meant that under water deficiency conditions, the plant exhibits the ability to grow in a more successful manner than the control plant that is not genetically engineered as described herein. In other words, the genetically engineered plant is able to develop better traits with respect to growth rate, biomass, color, maturation, time of flowering, fruit production, etc. under stress conditions.

According to an aspect of the invention, an isolated nucleic acid molecule including a nucleotide sequence is provided. The nucleotide sequence includes at least two amino acid sequences chosen from among amino acid sequences having at least 60% identity with SEQ ID NO. 4, at least 60% identity with SEQ ID NO. 5, and at least 60% identity with SEQ ID NO. 6; or chosen from among amino acid sequences having at least 60% identity with SEQ ID NO. 7, at least 60% identity with SEQ ID NO. 8, and at least 60% identity with SEQ ID NO. 9; wherein SEQ ID NO. 4 and SEQ ID NO. 7 encode phytoalexin deficient 4 (PAD4) protein in a plant; SEQ ID NO. 5 and SEQ ID NO. 8 encode lesion simulating disease 1 (LSD1 ) protein in a plant; and SEQ ID NO. 6 and SEQ ID NO. 9 encode enhanced disease susceptibility 1 (EDS1 ) protein in a plant.

An allelic variant or a homolog of the nucleotide sequence or a DNA fragment of the nucleotide sequence is also provided, wherein one or more functional characteristics of the protein are retained.

A vector construct including the isolated nucleic acid molecule is provided wherein the nucleotide sequence is operably linked to an expression control sequence. A host cell including the isolated nucleic acid molecule is provided, wherein the nucleotide sequence is flanked by exogenous sequence. A host cell Including the vector construct or a progeny of the cell is provided, wherein the cell is operably linked to an expression control sequence and expresses the polypeptide.

According to another aspect of the invention, a plant, plant material, plant cell, or a seed of a plant is provided, which includes the isolated nucleic acid molecule, wherein the nucleotide sequence is exogenous or heterologous to the plant or the plant cell. A plant, plant material, plant cell, or a seed of a plant, further includes the host cell. A plant regenerated from a plant cell or seed that includes the isolated nucleic acid molecule is also provided.

According to yet another aspect of the invention, a method of regulating growth and increasing biomass production in a plant is provided. The method further includes over expressing at least two of phytoalexin deficient 4 (PAD4) gene, lesion simulating disease 1 (LSD1 ) gene, and enhanced disease susceptibility 1 (EDS1 ) gene in the plant, wherein the overexpression step further includes contacting the genome of the plant with an expression cassette comprising an isolated nucleic acid molecule comprising the nucleotide sequence encoding at least two of PAD4, LSD1 , or EDS1 proteins, wherein the nucleotide sequence is operably linked to an expression control sequence that is functional in plants under conditions that permit integration of the expression cassette into the genome of the plant; integrating the expression cassette into the genome of the plant; and overexpressing the nucleotide sequence in the plant.

According to another aspect of the invention, a method of regulating growth and increasing biomass production in a plant including attenuating the expression of at least two of phytoalexin deficient 4 (PAD4) gene, lesion simulating disease 1 (LSD1 ) gene, and enhanced disease susceptibility 1 (EDS1 ) gene in the plant according to claim 1 , wherein the attenuating step further includes contacting the genome of the plant with an expression cassette comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding at least two of PAD4, LSD1 , or EDS1 proteins, wherein the nucleotide sequence is operably linked to an expression control sequence that is functional in plants under conditions that permit integration of the expression cassette into the genome of the plant; integrating the expression cassette into the genome of the plant; and attenuating the expression of the nucleotide sequence in the plant.

In a preferred embodiment, the method further comprises an amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO. 10 that encodes PAD4 protein; an amino acid sequence set forth in SEQ ID NO. 2 or SEQ ID NO. 1 1 that encodes LSD1 protein; and an amino acid sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 12 that encodes EDS1 protein. The method may further include a nucleotide

sequence with at least 60% identity to the nucleotide sequence encoding at least two of PAD4, LSD1 , or EDS1 .

According to an aspect of the invention, a transgenic plant having a gene construct including a nucleotide sequence encoding at least two of PAD4, LSD1 , or EDS1 proteins is provided. The nucleotide sequence is operably linked to a promoter such that the nucleotide sequence is overexpressed or attenuated, thereby causing the transgenic plant to exhibit increased biomass, increased stress tolerance, efficient growth development, changes in tissue density, modified cell division to programmed cell death ratio, greater fruit or seed yield, changes in chemical composition, physical, mechanical, and technological properties, , efficient use of water or early appearance of fruit or seed

than a non-transgenic plant that does not overexpress or attenuate at least two of PAD4 LSD1 , or EDS1 genes, when the transgenic plant and the non-transgenic plant are cultivated under identical growth conditions and identical stress conditions.

In a preferred embodiment, the transgenic plant is an annual plant or a perennial plant, wherein the annual plant is selected from a group consisting of Arabidopsis, lettuce, tobacco, soybeans, potato, tomato, canola, rice, corn, and wheat and wherein the perennial plant is a woody plant. The woody plant is a hardwood plant selected from a group consisting of acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple, and sycamore, wherein the hardwood plant is a plant of the Populus or Salicaceae groups.

DESCRIPTION

The use of biomass as energy source is of a great interest due to the following benefits: (i) biomass is a renewable, potentially sustainable and relatively environmentally benign source of energy; (ii) a huge array of diverse materials, frequently chemically defined are available from the biomass, giving the user many new structural features to exploit; (iii) increased use of biomass would extend the lifetime of diminishing crude oil resources; (iv) biomass fuels have negligible sulfur content and therefore do not contribute to sulfur dioxide emissions that cause acid rain; (v) the combustion of biomass produces less ash than coal combustion and the ash produced can be used as a soil fertilizer; (vi) the combustion of agricultural and forestry residues, and municipal solid wastes (MSW) for energy production is an

effective use of waste products that reduces the significant problem of waste disposal, particularly in municipal areas; (vii) the use of biomass could be a way to reduce carbon dioxide emission to the atmosphere (Cheila G., Mothe C, lara C. de Miranda. (2009) Therm Anal Calorim 97: 661-665). Biomass production in short-rotation woody crops plantations currently averages 8 Mg/ha per year of dry weight in Europe (e.g. Sweden) and 10 to 22 Mg/ha per year in the United States (Ragauskas et al. 2006. Science. Vol. 1 13, 484-489). The main factor limiting growth rate is low water availability which lowers from year to year, as an effect of climate changes. According to the United Nations, 30% of the world's population is going to face severe water shortage by the year 2025. Over 90% of global fresh water usage is for agriculture and irrigation purposes. The need of crop irrigation will increase since the global warming causes higher evaporation and changes in rainfall patterns. Therefore, the reduction in water supply for agriculture is one of the major challenges for both developed and developing countries in the 21 st century. Taking into consideration also the higher demands for water for non-agricultural purposes, any improvement in the effectiveness of managing water resources will be highly beneficial to society as a whole. The major goal is to reduce the amount of water used by crops without any loss of yield quantity and quality. The most promising way to avoid the decrease in growth rate under water limitation conditions is to provide safely and effectively transformed plants with improved water use efficiency.

Biomass production

The higher growth rate means that the transgenic plant increases its fresh and/or dry biomass at a rate that is more rapid than that of a control plant that has not been genetically engineered in the manner described herein. In other words, the plant with accelerated growth rate needs less time than the control plant to accumulate a particular amount of biomass. The yield (fruit, seeds) production may also be accelerated and/or appear sooner. Perennial plants are plants that have a life cycle which takes longer than 2 years and involves a long juvenile period with vegetative stage only. In contrast, annual plants such as Arabidopsis thaliana have life cycle which is completed within one year. Biomass accumulation in perennial woody plant species begin with cell divisions in the primary and secondary meristems. The primary apical meristems provide cells for root and shoot tip growth. A circumferential secondary meristem, called the vascular cambium (VC), is the

source of both the secondary xylem (inwards, towards the pith) and the secondary phloem (outwards), and is located between these tissues in the stem. Wood is produced by cells proliferation in the VC. Cells can divide in 2 ways: (i) periclinal division - cells divide along the longitudinal axis of the stem (responsible for diameter growth of cambium), (ii) anticlinal division - cells divide in radial direction (responsible for circumference growth of tree).

Although there has been a long tradition of anatomical and physiological analysis of the VC, still little is known of the molecular basis of its establishment. Even the cellular components that comprise the VC stem cell niche have remained unidentified. The increase in biomass of the above-ground part of a plant (e.g. stem) requires higher primary (height) and secondary (girth) growth. Growth may be improved by an overall increase of cell number of e.g. pith and cortex. However, there are also mechanisms that did not require cell division. For instance, many plants have avoided shading from the neighbouring plants by increasing height through intensified elongation of internodal cells. It was shown in both Poplar and Arabidopsis that the effect of the partial suppression of PtSHRI and AtSHRI, respectively (encoding SHORT-ROOT polypeptide; SHR), leads to the acceleration of the growth rate of shoots and increased biomass production. It was suggested that higher growth rate is a result of increased mitotic division rate in the VC and the shoot apical meristem, rather than the cell elongation.

In poplar transgenic lines, generated in our laboratory, the internodes were shorter than in the wild type (WT) T89 line and there was no significant difference in the length of tracheal elements between the WT T89 and transgenic plants. It indicates that, similarly to the increase in girth, their accelerated height was the result of intensified cell division (Pub. No.: WO/2008/125983, International Application No.: PCT/IB2008/001482, Publication Date: 23.10.2008, Inventors: JONES, Brian; WANG, Jiehua; SANDBERG, Goran); METHODS OF INCREASING PLANT GROWTH). In woody plants, the wood density may also increase as an effect of cell division and decrease in cell differentiation.

Regulation mechanisms in plant

In the natural environment, plants need to coordinate metabolism and growth between various cells, tissues and organs. Because of their sessile nature, plants are equipped in different defense mechanisms against both biotic and abiotic

stresses. In the course of evolution, plants have developed discrete, dynamic and emerging physiological responses that are able to process simultaneously various environmental stimuli and thereby optimize Darwinian fitness under short- and long-term changes in the natural environment (S. Karpinski et al., (1999) Science 284, 654-657; D. Peak, J. D. West, S. M. Messinger, K.A. Mott, (2004) Proc. Natl. Acad. Sci. USA 101 , 918-22; M. Szechynska-Hebda, J. Kruk, M. Gorecka, B. Karpinska, S. Karpinski, (2010) Plant Cell 22, 2200-2218; S. Karpinski, M. Szechynska-Hebda, (2010) Plant Signal. Behav. 1 1 , 1391 -1394). For instance, our results (Experiment 1 ) indicate that Arabidopsis seed yield efficiency can be genetically predetermined for optimal or suboptimal conditions. Our studies indicate that Arabidopsis plants in vegetative stage perform sort of molecular computations that aim at optimizing physiological processes to reach the best possible seed yield in permanently fluctuating environmental conditions. Therefore, the integration of a variety of stress signals with metabolic processes and the prioritization of responses according to the prevailing conditions, are dependent on genetic and physiological plant potential to balance the constructive/destructive processes within cell. Our research identified some elements of the biological hardware (predefined intra- and inter-cellular interactions) in plants that regulate systemic acquired acclimation and resistance (SAAR) to abiotic and biotic stress. This biological hardware includes quantum-redox sensing and modifications in photosystem II, e.g. changes in trans-thylakoid pH, in non-photochemical quenching (NPQ) (Karpinski et al., 1999; Mullineaux and Karpinski 2002; Szechynska-Hebda et al., 2010), redox status of the glutathione and plastoquinone pools, photoelectrophysiological signalling, reactive oxygen species (ROS), hormonal circuits (SA, JA, IAA) and the cellular light stress memory (Karpinski et al. 2003, Slesak et al. 2007, Karpinski and Szechynska-Hebda 2010). Such responses influence the balance between cell division and cell death, and therefore control plant growth, including secondary (girth) growth of woody plants. Our invention demonstrates that acclimatory and basal defense strategies are orchestrated via a genetic system that regulates programmed cell death and acts as a hub that perceives signals from specific stress factors and adjust the ROS/hormonal homeostasis. This basic genetic framework for regulation of plant responses, including cell death, to abiotic and biotic challenges has been established mainly from studies on Arabidopsis.

PAD4

Mutational screens in Arabidopsis 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 (Glazebrook et al., 1997; Zhou et al., 1998) and therefore pad4 (phytoalexin deficient 4) mutant demonstrates blocked biosynthesis of salicylic acid triggered by infection with avirulent pathogens (Feys, B. J., Moisan, L. J., Newman, M. A., and Parker, J. E. (2001 ). EMBO J. 20, 5400-541 1 ; Jirage, D., Tootle, T. L, Reuber, T. L, Frost, L. N., Feys, B. J., Parker, J. E., Ausubel, F. M., and Glazebrook, J. (1999). Proc. Natl. Acad. Sci. USA 96, 13583-13588). Moreover, PAD4 expression can be enhanced by exogenous applications of SA, suggesting that it is regulated by SA-dependent positive feedback (Falk et al., 1999; Jirage et al., 1999; Feys et al., 2001 ). 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 SUSCEPTIBILITY1 (EDS1 ) blocks SA-mediated signaling and enhances disease susceptibility (Rusterucci et al. 2001 ). 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, J., Rogers, E. E., and Ausubel, F. M. (1997). Annu. Rev. Genet. 31 , 547-569; Jirage et al., 1999; Feys et al., 2001 ; Nawrath, C, Heck, S., Parinthawong, N., and Metraux, J-P. (2002)). Recent data provides evidence that EDS1 regulates chloroplast-derived O2 ~ during the photo-oxidative stress signaling which indicates a link between EDS1 and ROS (Mateo et al., 2004; Muhlenbock et al., 2008, (Straus MR, Rietz S, Ver Loren van Themaat E, Bartsch M, Parker JE (2010). Plant J. 62(4):628-40). EDS1 is suggested to have a 'master' role in the coordination of SA and ROS accumulation, and in regulation of individual cells death in response to abiotic and biotic stimuli.

LSD1

Knock-out mutant of LESION SIMULATING DISEASE1 gene (Isd1) belongs to one of the best characterized mutants deregulated in terms of programmed cell death (PCD) in Arabidopsis thaliana (Dietrich et al., 1994; Jabs et al., 1996, Hunt et al. 1997, Rusterucci et al. 2001 , Epple et al. 2003, Mateo et al., 2004; Torres et al. 2005; Muhlenbock et al., 2007; 2008). The Isd1 mutant was initially characterized for its ROS- and SA-dependent uncontrolled spread of PCD that develops under long (>16 h) or continuous photoperiods, supply of superoxide ion (O2 ~) or after infection with avirulent pathogen. We propose that the role of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked (Mateo A, Muhlenbock P, Rusterucci C, Chi-Chen Ch, Miszalski Z, Karpinska B, Parker JE, Mullineaux PM, Karpinski S (2004). Plant Phys. 136: 2818-2830). The Isd1 phenotype is indicative for failure to stop both the initiation and propagation of PCD, therefore it was named runaway cell death (red). LSD1 function was proposed as a negative regulator of PCD, acting as a ROS rheostat (Dietrich et al., 1994; Jabs et al., 1996) and preventing the pro-death pathway below certain ROS level (Dietrich et al., 1997; Kliebenstein et al., 1999). However, our results (Mateo et al., 2004; Muhlenbock et al., 2007; 2008) reported that LSD1 is predominantly required for acclimation to conditions that promote excess excitation energy (EEE) such as photooxidative, photorespiratory and root hypoxia stress conditions. We linked runaway PCD in Isd1 mutant to the amount of light energy absorbed in excess by PSII light harvesting complex, to the redox changes in PSII proximity, to stomatal conductance and ultimately to photorespiratory burst of H2O2 and ethylene (Mateo et al., 2004, Muhlenbock et al., 2007; 2008). Furthermore, non-photorespiratory conditions retard propagation of lesions in Isd1. All these results suggest that LSD1 can influence the effectiveness of EEE dissipation and consequently is a key determinant of acclimatory processes.

PAD4, LSDL EDS1 hub

We showed that EEE in Isd1 mutant causes overproduction of ethylene precursor (ACC) and ROS followed by runaway cell death. Using photosystem I (PSI)- and photosystem II (PSII)— specific light wavelengths together with photosynthetic electron transport inhibitors (Mateo et al., 2004), we found that accumulation of ROS and ACC in Isd1 mutant in response to stress as well as programmed cell death propagation require EDS1 and PAD4 activity. Moreover, activation of the SA signaling pathway in response to EEE was observed after an initial induction of ethylene/ROS signaling (Mateo et al., 2004). These results showed that LSD1 , PAD4, and EDS1 belong to the same signaling pathway that controls ROS and ethylene levels (Muhlenbock et al., 2007) and concomitant responses in Arabidopsis.

Our recent data demonstrate that the formation of lysigenous aerenchyma depends on LSD1, EDS1 and PAD4 that operate upstream of ethylene and ROS production. Moreover, null recessive mutations in EDS1 and PAD4 revert Isd1-conditioned runaway PCD. The obtained results indicate that programmed cell death during lysigenous aerenchyma formation in hypocotyls occurs in a similar, but

independent manner from the foliar programmed cell death. Thus, the aerenchyma formation is subjected to a genetic- and tissue-specific program.

All these results indicate that LSD1 , EDS1 and PAD4 constitute a molecular hub, which coordinates the signaling of programmed cell death, light acclimation, and holistic defense responses that are initiated, at least in part, by redox changes of the plastoquinone pool (Muhlenbock P, Szechynska-Hebda M, Plaszczyca M, Baudo M, Mateo A, Mullineaux PM, Parker JE, Karpinska B, Karpinski S., 2008, Plant Cell. 20(9): 2339-56). The LSD1/EDS1/PAD4 hub regulates many aspects of plant defense and acclimation by affecting SA, jasmonate, ethylene, and other yet undefined signal intermediates. All of these features point to an intrinsic activity of LSD1 , EDS1 , and PAD4 in the processing and transduction of redox-derived signals from different subcellular compartments and different plant organs and tissues during a variety of environmental stresses.

Laboratory versus field experiments

The gene function study based on the mutant phenotype is conventionally performed under highly controlled laboratory conditions. These conditions do not represent the multivariable signals that plants perceive in their natural environment. The phenotype of particular mutant may differ depending on condition. Thus, the study of specific gene function should be studied under multiple environments. So far only a few studies focused on the role of plant genes in natural, field conditions (Kulheim et al. 2002, Ganeteg et al. 2004, and Athanasiou et al. 2010).

Our work indicates that the function of LSD1 is condition-dependent. The results point to a role of LSD1 in coordinated and integrated regulation of abiotic and biotic stress responses, regulation of plant growth and development, and seed yield. These data emphasize the importance of gene functions examination under multiple environmental stresses that are typical for natural field conditions.

Examples:

Production of transgenic plants with improved biomass production and stress tolerance compared to non-transgenic plants can change breeding ability and survival of transgenics in field conditions. Functional analysis of genes LSD1, EDS1 and PAD4 and their role in physiological processes such as biomass production, resistance to abiotic stresses, cell wall composition and water use efficiency were analyzed using Arabidopsis mutants and poplar transgenic lines. Plant morphology, growth rate, stomatal number per leaf area, leaf transpiration for Arabidopsis and poplar as well as fiber quality for all transgenic lines were also analyzed.

Method:

Growth conditions of Arabidopsis mutant and wild type plants

Arabidopsis thaliana wild type (Ws-0) and five different mutants of the same accession: Isd1, edsl, pad4, eds1/lsd1 and pad4/lsd1 were grown in standard laboratory conditions (9 or 16 h photoperiod, PPFD: 100 ± 25 μηηοΙ m"V, 50% relative air humidity and temperature day/night: 22/18 °C). Arabidopsis thaliana Ws-0 and mutant plants were also grown in the field at two different locations (Cracow, 50°03'41 "N 19°56'18"E and Warsaw, 52°09'38"N 21 °02'52"E) in four independent experiments during several seasons (June-September, 2006-2010). The smallest experimental field unit was approximately 100 cm2 where all the six representative genotypes were grown together. For measurements of physiological and morphological parameters, 3- to 4-week-old plants were used, whereas for determination of seed yield, 8-week-old plants were harvested

Lines generation and growth conditions of transgenic and wild type poplar plants

Poplar (Populus tremula x tremuloides) transgenic lines were generated based on the stable plant transformation using Agrobacterium tumefaciens strains containing proper binary plasmids - pCAMBIA or pH7GWIWG2(l). This method is widely used in woody plant species transformation as discussed in Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al . (1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 16, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al. (1990) Bio/Technology 8, 736-740; Christou, et al. (1991 ) Bio/Technology 9, 957-962; Peng, et al. (1991 ) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant CeH Rep. 1 1 , 585-591 ; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al. (1993) Plant Molecular Biology 21 , 871 -884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2, 603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology 1 1 , 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio/Technology 10 , 1589-1594; WO92/14828; Nilsson, O. et al (1992) Transgenic Research 1 , 209- 220). pCAMBIA vectors were obtained from CAMBIA Institute, Australia. pH7GWIWG2(\) vector based on Gateway technology was a kind gift of the Department of Plant System Biology, Gent University, Belgium (M., Inze, D., Depicker, A. Trends Plant Sci. 2002 May;7(5): 193-195).

There are many plant transformation techniques well-known to workers in the art, and new techniques are continually becoming known. Any technique suitable for the target plant can be employed with this invention. For example, sequences may be introduced in a variety of forms such as a strand DNA, in a plasmid, to name a few. Those skilled in the art can refer to the literature for details and select suitable techniques without undue experimentation. Similarly, the method used for regenerating transformed cells can be obtained from literature and any suitable technique can be selected without undue experimentation.

To obtain gene silencing of poplar orthologues of AtLSDI, AtEDSI and

AtPAD4, T-DNA containing the antisense sequences of poplar LSD1, EDS1 or PAD4 was inserted into binary vectors pCAMBIA and pH7GWIWG2(l). During Agrobacterium-mediated tissue infection, T-DNA regions from vectors were incorporated into plant cell genome together with selective markers conferring resistance to antibiotics (kanamycin and hygromycin). Following transformation, plants were regenerated from leaf discs via callus formation (Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989). Transgenic lines of P. tremula x tremuloides with significantly diminished expression of poplar orthologues of AtLSDI, AtEDSI and AtPAD4 were selected (3-4 independent lines for each transgen).

Many plant transformation techniques and techniques for silencing genes in a plant are well-known to workers in the art, and new techniques are continually becoming known. Any technique suitable for the target plant can be employed with this invention. Those skilled in the art can refer to the literature for details and select suitable techniques without undue experimentation.

After initiation and establishment of rooting in tissue cultures, transgenic poplar lines were grown together with the wild-type control (T89) in a greenhouse

under a photoperiod of 18 h and a temperature of 22°C/15°C (day/night). The humidity was ca. 70%. The plants were grown for 4-9 months before harvest. During this time their positions within the glasshouse were altered every 2-3 days and their heights and diameters were measured as described in the results. The transgenic lines were also grown in natural field conditions near Warsaw.

Example IA

Vegetative growth and seed yield determination

3-week-old Arabidopsis plants were used for rosettes dry weight measurement. For determination of seed yield, 8-week-old plants were harvested.

Table 1. Regulation of vegetative growth by LSD1 , EDS1 and PAD4 in Arabidopsis thaliana.

Dry weight was determined for plants grown in controlled laboratory (lab) and natural variable field (field) conditions. Arabidopsis wild type plants (Ws-0) and five different mutants of the same accession: Isd1, edsl, pad4, eds1/lsd1 and pad4/lsd1 were tested. Mean values (±SE or SD) are average of at least 13 plants for each experiment per genotype (n = 50 - 89). Two independent field experiments were performed during two independent 2008-2010 summer seasons, tested in two different geographical locations (Warsaw and Krakow).

wt pad 4 Isd1 edsl pad4/lsd1 eds1/lsd1 lab

dry mass (mg) AVR 37.68 42.2 33.22 37.03 38.98 33.31

SD 9.84 1 1 .04 1 1 .22 6.99 5.91 7.77 field

dry mass (mg) AVR 134.60 150.5 34.53 134 139.1 124.94

SD 18.93 16.26 19.41 23.42 13.27 35.23

Table 2. Regulation of siliques number and seeds yield by LSD1 , EDS1 and PAD4 in Arabidopsis thaiiana.

Siliques number and seeds yield was determined for 8-weeks-old plants grown in controlled laboratory (lab) and natural variable field (field) conditions. Arabidopsis wild type plants (Ws-0) and five different mutants of the same

accession: Isd1, edsl, pad4, eds1/lsd1 and pad4/lsd1 were tested. Mean values were calculated for two (siliques) and four (seeds) independent field experiments performed during two independent 2006-2010 summer seasons, tested in different geographical locations (Stockholm and Krakow) and two (siliques) and four (seeds) independent laboratory experiments (Krakow and Warsaw). At least 15 plants for each experiment per genotype were analyzed (n siliques = 40-145, n seeds = 50-89).

wt pad4 Isd1 edsl pad4/lsd1 eds1/lsd1 no. siliques, experiment l-lll

AVR 62.70 80.07 25.55 48.10 61.34 1 13.54

SD 20.56 39.82 26.33 26.17 6.19 35.27

AVR 128.00 107.63 1 14.60 1 12.75 165.77 192.74

SD 46.71 35.41 27.50 38.04 19.27 29.50 seeds yield (seeds number/plant), experiment l-IV

lab AVR 7615.8 7900.5 2028.4 8413.9 6586.3 7784.7

SE 618.2 609.6 275.6 729.5 581.0 678.3 field AVR 2517.2 2081 .7 2585.5 2560.6 2227.9 3016.3

SE 202.3 181.1 349.0 198.9 212.9 327.6

In laboratory conditions 3-week-old rosettes of Arabidopsis mutants: pad4 and pad4/lsd1 had higher dry weight than wild-type plants (Table 1 ). Isd1 mutant plants showed an unambiguous cell death phenotype that was associated with significant growth retardation.

In field conditions, most of tested lines exhibited higher dry mass production compared to the laboratory-grown counterparts. The highest dry mass was observed for pad4 mutant. Interestingly, field-grown Isd1 mutant did not demonstrate visible cell death phenotype, however dry mass was on similar level as observed for laboratory conditions.

The increase of siliques yield (Table 2) for pad4 and eds1/lsd1 and seed yield (Table 2) for pad4, edsl and eds1/lsd1 were observed in laboratory conditions. In field, higher generative growth (both siliques yield and seed yield) was observed for eds1/lsd1. Higher seed yield in field conditions were observed for Isd1 and edsl.

It can be concluded from these experiments, that vegetative growth is regulated by at least PAD4 solely (higher value of dry mass for pad4 mutant) or/and in cooperation with PAD4/LSD1 (higher value of growth parameters for pad4/lsd1), whereas generative growth in field conditions is coordinated by EDS1 (higher value of seed yield for edsl mutant) and can be aided by LSD1 (higher value of seed yield for eds1/lsd1). Moreover, these data reveal striking differences between the phenotypes of Arabidopsis defense and abiotic stress mutants challenged with single stresses in the laboratory and those same genetic backgrounds grown in nature. For example, runaway cell death phenotype of Isd1, observed in long photoperiod in highly controlled laboratory conditions did not appear in the natural long photoperiod conditions, and Isd1 mutant was actually growing similar or better in the field than corresponding wild type plant.

Example IB

Poplar growth measurements and morphology

Poplar plants exhibited an exponential growth pattern (plant height) up to an approximate height of 220-250 cm or a maximum of up to month 9 in the greenhouse. Height measurements were taken starting from apical part of plant (0.01 m) and subsequently on the levels corresponding to length measured at 0.1 m, 0.2m, 0.3m, 0.5m, 0.7m, 0.9m, 1 m, 1 .5m, 2.0m, 2.5m. Under the above defined growth conditions, stem width exhibited a comparatively linear increase over height. Morphology of main stems (length (cm), volume (cm3) and internodes (cm)), morphology of secondary stems (number and length (cm)) and leaves area (cm2) were determined.

Table 3. Regulation of tree biomass by the LSD1, EDS1 and PAD4 in poplar (P. tremula x tremuloides).

Morphology of leaves area (cm2), main stem volume (cm3) and internodes (cm) were determined for 4-9-month-old poplar wild type plants (t89) and three different transgenic lines: pad4, Isd1, edsl in l-lll greenhouse experiments from two different localizations (Stockholm and Warsaw). Values represent mean from different sublines (four for pad4, one for Isd1 and two for edsl) and replications (3-6 plant within sublines).

leaf area main stem volume internodes length exp.

(cm2) (cm3) (cm) t89 I 53.1 18.83 2.94

II 49.9

pad4 I 48.4 21 .68 2.82

II 48.2

Isd1 I 47.2 21 .62 2.83

II 44.5

edsl I 37.2 12.04 2.56

II 48.4

Table 4. Regulation of secondary wood accumulation by the LSD1 , EDS1 , and PAD4 in poplar (P. tremula x tremuloides).

Diameter of main stems (mm) was determined for 4 to 9-month-old poplar wild type plants (t89) and three different transgenic lines: pad4, Isd1, edsl in three (l-lll) greenhouse experiments. Diameter of main stems were measured starting from apical part of plant (0.01 m) and subsequently on the levels corresponding to length measured at 0.1 m, 0.2m, 0.3m, 0.5m, 0.7m, 0.9m, 1 m, 1 .5m, 2.0m and 2.5m. Values represent mean from different sublines (four for pad4, one for Isd1 and two for edsl) and replications (3-6 plant within sublines).

exp 0.01 m 0.1 m 0.2m 0.3m 0.5m 0.7m 0.9m 1 m 1.5m 2.0m 2.5m t89 I 1.27 1.53 2.40 2.87 3.73 5.00 5.00 - - - - II - 3.03 - 4.35 5.80 - - 7.55 9.38 10.41 13.35

III - 3.25 - 5.27 5.91 - - 8.34 9.54 9.84 12.74 pad4 I 1.40 2.00 2.50 3.20 4.45 5.13 6.20 - - - - II - 3.51 - 5.12 6.68 - - 8.40 10.04 10.75 13.29

III - 2.98 - 4.97 6.51 - - 8.03 8.87 10.41 12.08

Isd1 I 1.46 1.78 2.26 2.80 3.94 4.94 4.94 - - - - ii - 2.76 - 4.20 5.85 - - 7.78 9.78 1 1.08 14.61 edsl I 0.95 1.80 2.50 3.35 4.90 5.20 6.10 - - - - ii - 4.27 - 5.10 6.60 - - 8.57 9.38 10.43 14.32

Table 5. Regulation of stem cell division by the LSD1 , EDS1A and PAD4 in poplar (P. tremula x tremuloides).

Cells number, tracheids number and tracheids size in cross section of 8-month-old poplar stem of wild type plants (t89) and three different transgenic lines: pad4, Isd1, edsl were measured. Wood tissues were analyzed starting from apical part of plant (1 cm) and subsequently on the levels corresponding to length measured at 10 cm and 30 cm (stem length were tissue are effectively formed). Values represent means from replications (3-6 plant within sublines) and 3-5 cross sections per plant.

cells number tracheids number tracheids diameter (mm)

1 cm 10cm 30cm 1 cm 10cm 30cm 1 cm 10cm 30cm t89 25.25 20.00 18.67 4.67 5.00 3.67 0.018 0.023 0.020 pad4 28.58 29.50 22.75 4.67 7.00 4.00 0.016 0.014 0.019

Isd1 26.50 26.00 20.67 5.00 9.00 4.00 0.016 0.023 0.026 edsl 30.08 21 .25 17.25 5.67 6.00 3.00 0.016 0.020 0.030

Similarly to Arabidopsis laboratory and field experiments with rosette size determination, single genes LSD1, EDS1 and PAD4 did not influence green biomass accumulation in poplar (leaf area of trees correspond to rosette size of Arabidopsis). Moreover, like in Arabidopsis, PAD4 increases dry mass accumulation, which corresponds in trees to stem volume and wood accumulation. The higher volume did not resulted from lengthen of interrnodes (values for transgenic lines even lower then values for wild type plants, Table 3), and therefore must be an effect of faster (more effective) cell division, rather than cell differentiation and changes of cell volume.

Both pad4 and Isd1 transgenic lines exhibit higher secondary wood accumulation, when compared to wild type t89 (higher values of stem diameter, Table 4), and consequently, higher total volume of main stem (Table 3). Interestingly, pad4 plants more effectively increased stem diameter of younger tissue (higher diameter of apical part of stem, up to 0.90 m), whereas Isd1 had higher diameter of older part of stem (higher diameter of stem part from 1 up to 2.5 m). Microscopic analysis of stem cross section and measurements of cells number, tracheal elements number and tracheal elements size (Table 5) showed higher cell numbers for pad4 and Isd1 per volume unit of stem. Nevertheless, diameter increase of older part of Isd1 stem was results of higher cell number as well as higher tracheids number and size.

Due to tissue-(age)-dependent changes of stem diameter size in pad4 and Isd1 and cell division-dependent improvement of stem length in Isd1, we concluded that silencing of LSD1 gene caused the imbalance of both processes in main stem of Isd1: cell division and programmed cell death. Moreover, PAD4 gene cooperates with LSD1 in control of cell division/secondary growth of stem. Therefore, functional PAD4 and LSD1 genes seem to be required to control of trees size. Whereas, PAD4 regulate number of cells only, LSD1 influence balance of division/death of cells and control the formation of tracheids and other wood tissues. Consequently PAD4/LSD1 influence tissue properties e.g. wood accumulation, its physical and technological usefulness. This is a novel discovery with great implications for plant development, energy production and for sustainable agriculture.

Example IIA

Determination of water use and stomatal density

Water use was determined as water used during 4 weeks of plant growth in closed system. Plants were grown in 50 ml tubes filled with perlite and soil in 1 :1 proportion and 35 ml of water. Seeds were placed in a hole (0 ca. 1 .5 mm) made in the cap. After germination, the system was weighed. The water lost corresponds to water transpired by plants.

Numbers of stomata per mm2 of leaf area of each mutant and wild type were calculated for leaves area. Microscopic analysis was performed on the bottom layer of epidermal cells.

Water use efficiency experiment

5-week-old Arabidopsis thaliana plants: Ws-0 and mutant lines grown in 9 h photoperiod were transferred to high light (HL) conditions PPFD: 500 ± 50 pmol/nnV and the watering was stopped. Survival rate, defined as the percentage of plants that survived was measured after 1 1 and 15 days of water deficiency stress. Moreover, seed production, defined as number of seeds per plant was determined.

Table 6. Regulation of water use and stomatal density by the LSD1 , EDS1 and PAD4 in Arabidopsis thaliana.

Water use (ml of water used during 4 weeks) were determined for 4-week-old Arabidopsis thaliana wild type (Ws-0) and mutant plants grown in laboratory and field conditions. All data are means ± SD from at least 6 independent experiments. Stomata numbers per 1 mm2 of leaf area were calculated for Arabidopsis thaliana wild type (Ws-0) and five different mutants of the same accession: Isd1, edsl, pad4, eds1/lsd1 and pad4/lsd1. Results was obtained from field experiments (May-July 2005) performed near Krakow for 100 plants randomly planted for each genotype. Stomata were calculated under microscopy. Mean values were calculated for at least 30 different plants (n = 30).

wt pad4 Isd1 edsl pad4/lsd1 eds1/lsd1 used water (ml), experiment l-VI

lab AVR 22,31 23,54 18,31 21 ,29 22,18 19,77

SD 5,89 5,84 6,29 3,50 3,93 4,66 field AVR 32,14 32,17 13,53 33,39 33, 82 31 ,99

SD 4,21 5,48 5,47 4,07 1 ,81 4,13 stomata no per mm2 of leaf area

field AVR 249,5 148,6 180,5 255,8 244,2 215,5

SD 23,1 23,1 28,1 42,4 20,2 37,6 lab AVR 332 185 286 291 414 314

SD 30 65 152 59 41 34

Table 7. Regulation of survival rate and seeds production by LSD1 , EDS1 and PAD4 in Arabidopsis thaliana.

Survival rate and seeds production (seeds no./ plant) were determined in laboratory conditions for Arabidopsis thaliana wild type (Ws-0) and five different mutants of the same accession: Isd1, edsl, pad4, eds1/lsd1 and pad4/lsd1. Mean values were calculated for at least three to eight different plants from two independent experiments (n = 10-15).

pad4 Isd1 edsl pad4/lsd1 eds1/lsd1

11 days after water limitation and high light stress

Survival rate (%) AVR 100 93 100 93 93 79

AVR 5996 5002 5629 6488 5864 3783

Seed yield (Ys)

SE 897 729 708 809 616 625

15 days after water limitation and high light stress

Survival rate (%) AVR 0 0 45 26 0 0

AVR 0 0 1612 1288 0 0

Seed yield (Ys)

SD - - 762 697

The observation that the LSD1/EDS1/PAD4 regulate seed production in laboratory and field condition (Example I) tempted us to hypothesize that they can optimize their water use (transpiration rate) to obtain the predetermined seed yield. In the laboratory and field conditions water use was reduced in Isd1 and eds lsdl mutants. Lower water consumption was dependent on stomatal density per leave surface and further on plant capabilities to transpiration. The most visible differences in comparison to wt in stomatal number were observed for pad4, Isd1 and eds1/lsd1. Numbers of stomata guard cells per leaf area were reduced for these mutants. Such capacities determined the survival rate and seed yield in stress conditions (for Isd1 and edsl mutants). 15 days of water limitation and simultaneous high light stress was critical in case of all lines except Isd1 and edsl. Values of survival rate for these lines were at 45 and 26%, respectively.

Example MB

Determination of water use and stomatal density

Poplar leaves were cut from plants and immediately transferred into 1 ml Falcon tubes filled with water. Leaves were kept in greenhouse conditions (experiment I) or in laboratory conditions (experiments ll-IV; in 22 °C, ca. 60% humidity, light intensity - 100 μιτιοΙ m2 s"1). Tubes were weighted (or water volume changes were measured) at 0 and 24 hours of incubation. Leaves area was measured after experiment with ImageJ 3.0 software.

Stomatal density

Numbers of stomata per mm2 of leaf area of each transgenic line (Isd1, edsl, pad4 and wild type) were calculated for poplar (P. tremula x tremuloides) leaves. Microscopic analysis was performed on the bottom epidermis layer.

Table 8. Regulation of water use and stomatal density by the LSD1 , EDS1 and PAD4 in poplar (P. tremula x tremuloides).

Water use and stomata number was calculated for fully developed leaves of 8-month-old poplar stem of wild type plants (t89) and three different transgenic lines: pad4, Isd1, edsl were measured. Leaves were collected from part of plant about 1 m above ground. Values represent means from replications (3-6 plant within sublines) and 3-5 places o leaf surface.

Water use Water use Stomatal density

(ml/ H20/24h/cm2 of leaf) (ml/ H20/24h/cm2 of leaf) (no./mm2)

AVR SD AVR

t89 0,177 0.032 &7Λ

pad4 0,163 0.027 63.5

Isd1 0,164 0.028 81 .3

edsl 0, 146 0.066 71 .9

LSD1 poplar gene modifications resulted in the changes of water use similarly to Isd1 Arabidopsis plants. Moreover, transgenic line pad4 and edsl had also reduced water consumption. Although lower transpiration rate changes were observed in comparison to wild type for all transgenic lines, the relation between stomata number and water use was determined only in case of Isd1. Therefore, the internal control of stomata opening seems to be more important factor than total stomata number.

Example III

Regulation of wood properties by the LSD1 , EDS1 and PAD4 in poplar (P. tremula x tremuloides).

Wood formation

Percentage of wood tissue in cross section of 8-month-old poplar stem was determined via microscopic analysis. Cross sections were prepared by stem cutting at 0.1 m 0.2m, 0.5m and 1 m starting from apical part of the stem. Calculation of cross section area of wood and other tissue was preformed with ImageJ 3.0 software.

Water binding capacity of wood

Wood samples of poplar were cut from green stem in the form of 5 (radial) mm x 5 (tangential) mm χ 30 (longitudinal) mm pieces, samples were oven dried at 80 °C to constant weight and incubated and fully hydrated 48h in water. Radial, tangential and longitudinal dimensional changes were measured before and after water incubation and the data are presented as a volumetric swelling of samples. However, the volumetric swelling curves are mainly the effect of the tangential and radial swelling. Samples were also weighted and then dry and watered wood biomass (weight changes (%)) and wood density (density=mass/volume) were calculated. Water binding capacities were also determined by dilatometrical analysis. Dimensional changes under heating at 1 °C/min from 30 to 150 °C (shrinkage) were recorded and parameters characterized the dilatometric curves obtained for wood samples are presented. Data are presented as a ratio of sample length changes to initial sample length (dL/Lo/%) at 100 °C due to the stabilized values for such temperature.

Mechanical wood properties

The three points bending flexural test preformed with hydraulic power supply provides values for the characteristic of elasticity of wood: maximal force (N) required to wood rupture and maximal bending of wood. The maximum force capacity was 0.6 kN and the speed of loading was 1 mm/min in the flexural test. Wood sample were cut from air-dried stem in the form 5 (radial) mm χ 5 (tangential) mm x 30 (longitudinal) mm pieces.

Cell wall thickness

Investigation of cell wall structure was carried out with a scanning electron microscope JEOL JSM 840A at the 20 kV. Small sticks from the wood blocks were cut out (3 x 3 x 7) mm. The samples were air dried and attached to the stubs with double-sided adhesive tape. Then the samples were coated with gold by ion sputtering. Cell wall thickness (μιτι) was calculated with ImageJ 3.0 software.

Thermal analysis

Thermal analysis techniques include: simultaneous differential scanning calorimetry (DSC), thermogravimetry (TG) combined with quadruple mass spectrometry (QMS). It is a useful tool to obtain complete information on physical and chemical properties of plant cell wall.

Minimum of 20 mg of dry powdered wood tissues were introduced into sealed aluminum pans with lids and heated to 600 °C. Experiment was performed at Hel atmosphere with flow 80 ml/min. The samples were heated at 5 °C /min to 650 °C. Thermograms were recorded against an empty aluminum pan placed in the reference vessel. The final thermogram of the sample was obtained after the baseline was subtracted. Total ion current at temperature corresponded to maximum of sample weight changes was detected by quadruple mass spectrometer coupled with DTA/TG.

Table 9. Regulation of wood formation by the LSD1 , EDS1 and PAD4 in poplar (P. tremula x tremuloides).

Percentage of wood tissue in cross section of 8-month-old poplar stem of wild type plants (t89) and three different transgenic lines: pad4, Isd1, edsl was determined. Percentage of wood tissue was measured starting from tip of plant (0.1 m) and subsequently on the levels corresponding to length measured from tip: 0.2m, 0.5m and 1 m. Values represent means from replications (3-6 plant within sublines). Average data (AVR) represent mean calculated for sublines.

0.1 m 0.2m 0.5m 1 m t89(1) 20.2 37.1 40.2 59.3 t89(3) 21 .4 46.3 46.8 59.3 t89 AVR 20.2 37.1 40.2 59.3 pad 4(1) 28.2 42.9 55.4 72.2 pad4(9) 25.4 30.8 34.1 66.3 pad4(10) 25.5 44.4 54.6 64.7 pad4(11) 27.1 42.4 49.6 70.3 pad4 AVR 26.5 40.1 48.4 68.4

Table 10. Regulation of wood properties by the LSD1 , EDS1 and PAD4 in poplar (P. tremula x tremuloides).

Wood density (g/cm3), wood abilities to water binding (weight changes (%) and wood swelling (%) during tissue hydratation, dimensional changes under heating (wood shrinkage)) and mechanical properties of wood were determined for 8-month-old poplar wild type plants (t89) and three different transgenic lines: pad4, Isd1, eds1. Values represent means from different sublines (four for pad4, one for Isd1 and two for edsl) and replications (3-6 plant within sublines).

wood maximal dilatometrical maximal weight wood

density force (N) changes bending changes swelling

(g/cm3) (dL/Lo/%) (mm)

(%) (%)

air-dried hydrated

t89 0,421 297.27 27.78 35,79 -0.29 -0.74 5,69 pad4 0,441 304.44 21 .47 33,65 -0.22 -0.56 6,05

Isd1 0,420 300.34 22.86 44,05 - 6,09 edsl 0,437 296.97 37.99 32,41 - 7,88

Table 11. Regulation of cell wall material properties by the LSD1 , EDS1 and PAD4 in poplar (P. tremula x tremuloides).

Microscopic analysis of cell wall thickness (μιτι) and DSC/TA QMS analysis of cell wall thermal properties were preformed for 8-month-old poplar wild type plants (t89) and three different transgenic lines: pad4, Isd1, edsl. Data of DSC/TA/QMS analysis are presented as: temperature of maximum peaks (°C) (indicating temperature of maximal thermal decomposition of individual cell wall components), mass loss (%) (mass loss during thermal decomposition), area under DSC curve (J/g) (area integration below peaks representing total energy from exothermal reaction) and total ion current determined for atomic mass 18, corresponded to H2O. DSC peaks correspond to individual cell wall components: I - hemicelulose, II -cellulose, III - lignin.

cell wall

thickness mass loss (%)

(μιπ)

peak I II III total

t89 0,81 1 -25,54 -34.1 1 -10.76 -70.41

pad4 1 ,020 -31 .46 -29,50 -1 1.68 -72.64

Isd1 0,861 -28.95 -30.37 -12.07 -71 ,39

edsl 0,694 -28.45 -29.53 -9.20 -67.18

ions current for m/z 18

area below DSC curve (J/g)

(x10"9 A)

peak I II l+ll III I II III

t89 1 15.2 57.48 172,68 234.1 2.96 8.97 0.77

pad4 139.9 27.6 167.5 544.6 0.68 1.77 0.18

Isd1 145.1 24.47 169.57 103.6 3.12 8.41 0.77

edsl 86.5 18.97 105,94 318.5 0.93 1.74 0.36

The density of poplar lines varied from 0.420 g/cm3 for Isd1 to 0.441 g/cm3 for pad4 (Table 10). The highest wood density of pad4 confirms the compact wood structure consisting higher number of small cells per volume unit. Presence of tracheids in Isd1 results in similar to wild type wood density. Higher tissues compaction determines structure stability e.g. lower volumetric swelling during wood tissue hydratation and higher parameters of dilatometrical test (wood shrinking). The small dimensional changes during the shrinking and swelling indicate that transgenic line pad4 was able to retain less "free" (non-chemically bounded water) water in wood tissue than wild-type t89. In general, shrinkage does not occur in wood until the moisture content drops below what is termed the "fiber saturation point" (fsp). Specifically, the fiber saturation point is defined as that moisture content at which all of the liquid water, essentially sap, has been removed from the cell cavities but the cell walls are still saturated with adsorbed water. Therefore small pad4 cells form compact wood, what prevent filling tissue with water. However this process occurred in more loose structure of tissue of wild type plant.

Chemical and physical properties lead to alteration in technological properties of wood e.g. density, cell compaction change, and moreover water status influence a plasticizing effects. Wood collected from pad4, Isd1 and edsl was less fragile in comparison to wild type plants, and it suggests different compaction and structure not only on cellular level but also different properties of cell wall material.

Due to differences in cell wall thickness among transgenic lines, the different accumulation of secondary cell wall components was expected. Therefore, for determination of the cell wall composition poplar lines have been examined using thermal analyses under non-isothermic conditions (Table 1 1 ). The analysis based on the evaluation of the thermal behavior of individual cell wall materials. At linear heating rates under a dynamic, inert gas atmosphere (Hel), the DSC signal clearly indicated three-step degradation between 30 °C and 600 °C. In this way it was shown that the peak corresponded to degradation of pectins was not presented on DSC curve. The hemicelluloses degraded first (peak I), then the celluloses (peak II) and finally lignins (peak III). Mass loss during heating indicates the higher degradation of hemicelluloses (lower thermal stability) and lower degradation of celluloses for all tested transgenic lines (the lowest degradation for celluloses was for pad4).

Hemicelluloses exhibit lower molecular weights than cellulose and decompose at temperatures of 200-260°C. They are a mixture of various polymerized monosaccharides such as glucose, mannose, galactose, xylose, arabinose, 4-O-methyl glucuronic acid and galacturonic acid residues. Different thermal behaviors (higher mass loss and higher energetic effects, indicate different hemiceluloses composition and the higher amount of easily depredated components for transgenic lines, when they are compared to wild type (with exception of edsl).

Cellulose degradation occurs at 240-350°C and the reaction is complete at 360°C. Cellulose forms long chains that are bonded to each other by a long network of hydrogen bonds. Groups of cellulose chains twist in space to make up ribbon like microfibril sheets, which are the basic construction units for a variety of complex fibers. These microfibrils form composite tubular structures that run along a longitudinal tree axis. The crystalline structure resists thermal decomposition better than hemicelluloses. Amorphous regions in cellulose exist that contain waters of hydration, and free water is present within the wood. This water, when rapidly heated, disrupts the structure by a steam explosion-like process prior to chemical dehydration of the cellulose molecules. Therefore lower degradation of celluloses (peak II - lower mass loss, lower energetic effects and lower ion current for gaseous H2O derived form bounds breaking) indicates their higher stability. Due to dense compaction of cell material, lower abilities to water binding observed for pad4 and Isd1 (Table 1 1 ) and according to results of DSC analysis we conclude that cell walls of such transgenic lines are build with celluloses forming compacted and crystalline structure of fibers.

Lignin is more difficult to dehydrate than cellulose or hemicelluloses and decomposes when heated at 300-600°C. The mass loss of lignins suggest their slightly higher level in pad4 and Isd2, however their decomposition was the most exothermic for pad4 (area below DSC curve). It results also in the highest total mass loss.

In one aspect of the invention there is provided a method of producing a perennial plant (woody plant) comprising the incorporation of a heterologous nucleic acid encoding a PAD4, LSD1 , EDS1 polypeptides alone or in combinations, into a perennial plant cell by means of transformation and; regenerating a perennial plant from one or more transformed cells. A woody plants produced by such a method may accumulate biomass more effectively than control plants. Preferably, the nucleic acid recombines with the cell genome nucleic acid such that it is stably incorporated therein.

In some preferred embodiments, a PAD4, LSD1 , EDS1 polypeptide may have the amino acid sequence of SEQ ID NO: 1 to 3 (respectively) or may be a fragment or variant of the SEQ ID NO: 1 to 3 sequences, which retains PAD4, LSD1 , EDS1 activity (respectively), including for example induction of biomass accumulation, and regulation of cell division and programmed cell death.

A PAD4, LSD1 , EDS1 polypeptides which are variants of SEQ ID NO: 1 to 3 (respectively) may comprise an amino acid sequences which shares greater than 60% sequences identity with the amino acid sequences of SEQ ID NO: 1 to 3 (respectively), preferably greater than 65%, greater than 70%, greater than 80%, greater than 90% or greater than 95%.

In other embodiments, a PAD4, LSD1 , EDS1 polypeptides which are variants of SEQ ID NO: 1 to 3 (respectively) may share greater than 60% sequences similarity with the amino acid sequences of SEQ ID NO: 1 to 3 (respectively), preferably greater than 65%, greater than 70%, greater than 80%, greater than 90% or greater than 95%. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for

another or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Particular amino acid sequences variants may differ from a known PAD4, LSD1 , EDS1 polypeptides sequences as described herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, or more than 50 amino acids.

The Arabidopsis PAD4, LSD1 , EDS1 polypeptides sequences have the database accession numbers NP_19081 1 .1 , and NP_001 154257.1 and NP_190392.1 , respectively.

Nucleic acids as described herein may be wholly or partially synthetic. In particular, they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesized directly e.g. using an automated synthesizer.

A nucleic acid encoding a PAD4, LSD1 , EDS1 polypeptides may comprise or consist of the nucleotide sequences of SEQ ID NO: 4-6 (respectively) or may be a variant or fragment of the SEQ ID NO: 4-6 sequences (respectively).

A variant sequences may be a mutant, homologue, or allele of the SEQ ID NO: 4-6 sequences and may differ from the sequences of SEQ ID NO: 4-6 by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid that make no difference to the encoded amino acid sequences are included.

A nucleic acid encoding a PAD4, LSD1 , EDS1 polypeptides, which has a nucleotide sequences which is a variant of the SEQ ID NO: 4-6 sequences (respectively) may comprise a sequences having at least 60% sequences identity with the nucleic acid sequences of SEQ ID NO: 3 to 6, than 65%, greater than 70%, greater than 80%, greater than 90% or greater than 95%. Sequences identity is described above.

A fragment or variant may comprise a sequences which encodes a functional PAD4, LSD1 , EDS1 polypeptides i.e. a polypeptides which retains one or more functional characteristics of the polypeptide encoded by the wild-type PAD4, LSD1 , EDS1 genes, for example, the ability to stimulate biomass accumulation in a perennial plant (woody plant).

In other embodiments, a nucleic acid encoding a PAD4, LSD1 , EDS1 polypeptides, which have a nucleotide sequences which is a variant of the SEQ ID NO: 4-6 sequences (respectively) may selectively hybridize under stringent conditions with the nucleic acid sequences of SEQ ID NO: 4-6 (respectively) or the complement thereof.

In yet another embodiments, an isolated nucleic acid including a nucleotide sequence which encodes an amino acid sequence that has at least 80% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO. 1 is provided. The isolated nucleic acid encodes the amino acid sequence shown in SEQ ID NO: 1 to 3 and 10a to 12. The isolated nucleic acid further includes a sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 4 to 6 and 7 to 9

According to another aspect of the invention, a method for increasing the growth, photosynthesis, development, biomass production and water use efficiency of a plant, including the steps of genetically engineering said plant to contain and over-express or silence at least one preferably two or all three functional gene product of Phytoalexin Deficient 4 (PAD4), Enhanced Disease Susceptibilityl (EDS1 ) and Lesion Simulating Diseasel (LSD1 ) is provided. In some preferred embodiments, the method further provides PAD4, EDS1 and LSD1 polypeptides, which alone or in combinations includes an amino acid sequence that shares greater than 60% sequence identity with the amino acid sequence of SEQ ID NO: 1 to 3 and 10 to 12. The method further provides a nucleic acid molecule that includes a sequence having at least 60% sequence identity with the nucleic acid sequence of SEQ ID NO: 4-6 and 7 to 9.

According to an embodiment of the invention, the biomass of a plant is increased such that either wet biomass or dry biomass, or production of fruit, seeds, or all, of the plant is increased or may appear sooner. According to yet another aspect of the invention, the water use efficiency of the plant is increased such that the plant exhibits the ability to grow in a manner that is more successful than comparable control plants that are not genetically engineered, even under circumstances or conditions of stress for the plant with respect to growth rate, wet and dry biomass, color, maturation, fruit production.

In a preferred embodiment, the plant may be selected from the group consisting of annual plant Arabidopsis, lettuce, tobacco, soybeans, potato, tomato, canola, rice, corn, wheat and perennial plant, wherein the perennial plant may be a woody plant such as a hardwood plant, conifer or a fruit bearing plant or selected from the group consisting of cotton, bamboo and rubber plants. The hardwood plant may be selected from the group consisting of acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple and sycamore. The hardwood plant may be a plant of the Populus or Salicaceae groups. The conifer may be selected from the group consisting of cypress, Douglas fir, fir, sequoia, hemlock, cedar, juniper, larch, pine, redwood, spruce and yew. The fruit bearing plant may be selected from the group consisting of apple, plum, pear, banana, orange, kiwi, lemon, cherry, grapevine and fig.

According to yet another aspect of the invention, the genetically engineered plant may cause an increase in a level of hydrogen peroxide and/or superoxide ion and/or salicylic acid and/or auxins and/or ethylene and changes the stomata density in the plant. The genetically engineered plant may not cause an increase in a level of hydrogen peroxide and/or superoxide ion and/or salicylic acid and/or auxins and/or ethylene and changes the stomata density in said plant. The genetically engineered plant may cause a change tissue density and compaction via modification of cell division/programmed cell death processes in the plant, and/or a change in chemical composition, physical, mechanical and technological properties of cell walls in the plant.

According to an embodiment of the invention, a transgenic plant is provided, wherein the transgenic plant is genetically engineered to contain and over-express or silence at least one, preferably two or three functional gene product of Phytoalexin Deficient 4 (PAD4), Enhanced Disease Susceptibilityl (EDS1 ) and Lesion Simulating Diseasel (LSD1 ). In some preferred embodiment of, the transgenic plant of claim 8, at least one, preferably two or three functional gene product is selected from the group consisting of Phytoalexin Deficient 4 (PAD4), Enhanced Disease Susceptibilityl (EDS1 ) and Lesion Simulating Diseasel (LSD1 ).

In a preferred embodiment, the transgenic plant may exhibit increased biomass, wherein either wet biomass or dry biomass, either wet biomass or dry biomass, or production of fruit, seeds, or all, of said plant is increased or may appear sooner, or both is increased. The transgenic plant may wherein exhibit increased stress tolerance and increased water use efficiency. The transgenic plant may also exhibit the ability to grow in a manner that is more successful than comparable

control plants that are not genetically engineered, even under circumstances or conditions of stress for the plant with respect to growth rate, wet and dry biomass, color, maturation, fruit production.

According to yet another preferred embodiment, the transgenic plant may be of a type selected from the group consisting of annual plant Arabidopsis, lettuce, tobacco, soybeans, potato, tomato, canola, rice, corn, wheat and perennial plant, wherein the perennial plant is a woody plant. The woody plant may be a hardwood plant, a conifer, a fruit bearing plant, or may be selected from a group consisting of cotton, bamboo and rubber plants. The hardwood plant may be selected from the group consisting of acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple and sycamore. The hardwood plant may be a plant of the Populus or Salicaceae groups. The conifer may be selected from the group consisting of cypress, Douglas fir, fir, sequoia, hemlock, cedar, juniper, larch, pine, redwood, spruce and yew. The fruit bearing plant may be selected from the group consisting of apple, plum, pear, banana, orange, kiwi, lemon, cherry, grapevine and fig.

It is also an aspect of the invention that the transgenic plant exhibits an increase in a level of hydrogen peroxide and/or superoxide ion and/or salicylic acid and/or auxins and/or ethylene acid and changes the stomata density or that it does not exhibit an increase in a level of hydrogen peroxide and/or superoxide ion and/or salicylic acid and/or auxins and/or ethylene and changes the stomata density. The transgenic plant may include changed tissue density and modified cell division/programmed cell death ratio processes and the transgenic plant may include a changed the chemical composition, physical, mechanical and technological properties of cell wall.

While the invention has been explained in relation to various embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading this specification. Therefore, it is to be understood that the invention provided herein is intended to cover such modifications as may fall within the scope of the appended claims.