Patent Application
20190309316
The present disclosure relates to the field of plant genetics and plant transformation. There is provided in the instant disclosure a method of making abiotic stress tolerant transgenic plant, and methods thereof.
Plants, as sessile organisms, are endowed with highly sophisticated biological mechanisms to precisely regulate their growth and development in response to environmental risk factors. Among several abiotic stresses, water-deficit or dehydration is the fundamental factor that negatively affects plant productivity resulting in major yield loss of foremost crops worldwide (Altman et. al., Planta 218, 1-14 (2003); Bray, J. Exp. Bot. 55, 2331-2341 (2004), which are incorporated by reference). Dehydration response in plants is a complex phenomenon, and the exact structural and functional modifications caused by dehydration are poorly understood. Plants render dehydration tolerance either by escaping or by maintaining a favourable internal water balance (Oliveira et al., J. Exp. Bot. 55, 2365-2384 (2004); Bhushan et al., Mol. Cell. Proteomics 6, 1868-1884 (2007), which are incorporated by reference). The changes at molecular level are regulated by a number of different, and potentially overlapping, signal transduction pathways (Zhu et al., Plant Cell 14, S165-S183 (2002); Yamaguchi-Shinozaki et al., J. Exp. Bot. 58, 221-227 (2007), which are incorporated by reference). Interestingly, dehydration induces the expression of proteins not specifically related to this stress, but rather to reactions against cell damage (Zivy et al., Plant Physiol. 117, 1253-1263 (1998), which is incorporated by reference). The dehydration-responsive cellular cross-talk is thought to be modulated by a diverse population of secreted proteins, and thus, expression profiling of proteins has become an important tool to investigate various stress-responsive signaling networks.
The analysis of secretome appears to be a fundamental approach to map the quality and quantity of the extracellular proteins, which provides insight into possible biological pathways involved. The secretomes of plants submitted to stress condition usually contain significantly more leaderless secretory proteins (LSPs) than the secretomes of unstressed plants (Rakwal et al., Proteomics 10, 799-827 (2010), which is incorporated by reference). During the past few years, there have been rapid advances in plant secretomics (Djordjevic et al., J. Proteome Res. 7, 4508-4520 (2008); Sanchez et al., Mass Spectrom. Rev. 28, 844-867 (2009); Juegens et al., Biochim. Biophys. Acta 1834, 2429-2441 (2013), which are incorporated by reference). While most research interest in the secreted proteins has been restricted to the biotic stress responses, the study of the effect of abiotic stress remained highly limited. In the recent past, the studies on plant transcriptomics enhanced our understanding of the molecular basis of abiotic stress response (Yamaguchi-Shinozaki et al., J. Exp. Bot. 58, 221-227 (2007); Blum, Austral. J. Agr. Res. 56, 1159-1168 (2005), which are incorporated by reference).
Developmental control of protein expression in differentiated tissues is assumed to mask the identity of proteins that are differentially expressed as part of stress response. Therefore, investigation of stress-responsive proteins in undifferentiated cells, particularly in suspension-cultured cells (SCCs) has potential for: (i) identifying novel proteins related to stress which have not yet been characterized and (ii) recognizing already characterized proteins that have not been identified as stress-responsive proteins.
In an aspect of the present disclosure, there is provided a method of generating a stress tolerant transgenic plant, said method comprising: (a) obtaining plant cells; (b) obtaining a DNA construct comprising a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2, operably linked to a promoter and transforming a host cell with a DNA vector comprising said DNA construct to obtain a recombinant host cell comprising said DNA construct; (c) transforming said plant cells with said recombinant host cell to obtain transformed plant cells; and (d) selecting transformed plant cells and regenerating transgenic plants, wherein said transgenic plants are tolerant to stress compared to wild type non-transformed plants.
In an aspect of the present disclosure, there is provided a method of generating a stress tolerant transgenic plant, said method comprising: (a) obtaining plant cells; (b) obtaining a DNA construct comprising a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2, operably linked to a promoter; (c) transforming said plant cells with said DNA construct to obtain transformed plant cells; and (d) selecting transformed plant cells and regenerating transgenic plants, wherein said transgenic plants are tolerant to stress compared to wild type non-transformed plants.
In an aspect of the present disclosure, there is provided a stress tolerant transgenic plant, or parts thereof including seeds, capable of heterologous expression of a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2.
In an aspect of the present disclosure, there is provided a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2 for use in generating stress tolerant transgenic plants.
These and other features, aspects and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used for to limiting the scope of the claimed subject matter.
The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
For convenience, before further description of the present invention, certain terms employed in the specification, example and appended claims are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps. The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
“Primers” are synthesized nucleic acids that anneal to a complementary target DNA strand by hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by polymerase activity, e.g., a DNA polymerase. Primer pairs described in the present invention refer to their use for amplification of a target nucleic acid sequence, e.g., by polymerase chain reaction or other conventional nucleic-acid amplification methods.
The term “genetic transformation” refers to a process of introducing a DNA sequence or construct (e.g., a vector or expression cassette) into a cell in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.
The term “transgenic” refers to a cell contains a transgene, or whose genome has been altered by the introduction of a transgene. The term “transgenic” when used in reference to a tissue or to a plant refers to a tissue or plant, respectively, which comprises one or more cells that contain a transgene, or whose genome has been altered by the introduction of a transgene.
The term “transgene” refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations. A transgene may be an “endogenous DNA sequence,” or a “heterologous DNA sequence” (i.e., “foreign DNA”). A transgene is capable of causing the expression of one or more cellular products. Exemplary transgenes will provide the host cell, or plants regenerated therefrom, with a novel phenotype relative to the corresponding non-transformed cell or plant. Transgenes may be directly introduced into a plant by genetic transformation, or may be inherited from a plant of any previous generation which was transformed with the DNA segment.
The term “vector” refers to a DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operably linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.
The term “expression vector” refers to a vector comprising an expression cassette.
The term “polypeptide” and “peptide are used interchangeably for the purposes of the present disclosure.
The term “transformed cell” refers to a cell, the DNA complement of which has been altered by the introduction of an exogenous DNA molecule into that cell.
The term “transgenic plant” refers to a plant or progeny plant of any subsequent generation derived therefrom, wherein the DNA of the plant or progeny thereof contains an introduced exogenous DNA segment not originally present in a non-transgenic plant of the same strain. The transgenic plant may additionally contain sequences which are native to the plant being transformed, but wherein the “exogenous” gene has been altered in order to alter the level or pattern of expression of the gene.
The term “polynucleotide” used in the present invention refers to a DNA polymer composed of multiple nucleotides chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar in the adjacent nucleotide.
SEQ ID NO: 1 depicts the CaRRP1 cDNA sequence.
SEQ ID NO: 2 depicts the CaRRP1 protein sequence.
SEQ ID NO: 3 depicts the CaRRP1pGEM forward primer.
SEQ ID NO: 4 depicts the CaRRP1pGEM reverse primer.
SEQ ID NO: 5 depicts the CaRRP1pYES2 forward primer.
SEQ ID NO: 6 depicts the CaRRP1pYES2 reverse primer.
SEQ ID NO: 7 depicts the CaRRP1RT forward primer.
SEQ ID NO: 8 depicts the CaRRP1RT reverse primer.
SEQ ID NO: 9 depicts the CaEF1 forward primer.
SEQ ID NO: 10 depicts the CaEF1 reverse primer.
SEQ ID NO: 11 depicts the CaRRP1pENTR forward primer.
SEQ ID NO: 12 depicts the CaRRP1pENTR reverse primer.
In an embodiment of the present disclosure, there is provided a method of generating a stress tolerant transgenic plant, said method comprising: (a) obtaining plant cells; (b) obtaining a DNA construct comprising a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2, operably linked to a promoter and transforming a host cell with a DNA vector comprising said DNA construct to obtain a recombinant host cell comprising said DNA construct; (c) transforming said plant cells with said recombinant host cell to obtain transformed plant cells; and (d) selecting transformed plant cells and regenerating transgenic plants, wherein said transgenic plants are tolerant to stress compared to wild type non-transformed plants.
In an embodiment of the present disclosure, there is provided a method of generating a stress tolerant transgenic plant, said method comprising: (a) obtaining plant cells; (b) obtaining a DNA construct comprising a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2, operably linked to a promoter; (c) transforming said plant cells with said DNA construct to obtain transformed plant cells; and (d) selecting transformed plant cells and regenerating transgenic plants, wherein said transgenic plants are tolerant to stress compared to wild type non-transformed plants.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is monocot.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is rice.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is wheat.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is rye.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is millet.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is dicot.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said plant cells is selected from the group consisting of beans, peas, potato, eggplant, peppers, squash, melons, coffee, citrus, broccoli, turnips, legumes, yams, and apple.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said polynucleotide fragment sequence is as set forth in SEQ ID NO: 1.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said polynucleotide fragment is cDNA.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said transgenic plants tolerant to stress heterologously expresses a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said transformation is carried out by a process selected from the group consisting of Agrobacterium mediated transformation method, particle gun bombardment method, in-planta transformation method, liposome mediated transformation method, protoplast transformation method, microinjection, and macroinjection.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said transformation is carried out by Agrobacterium mediated transformation.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said transformation is carried out particle gun bombardment method.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said recombinant host cell is E. coli.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said method results in generation of transgenic plants tolerant to dehydration.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said method results in generation of transgenic plants tolerant to hypersalinity.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said method results in generation of transgenic plants tolerant to cold.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said method results in generation of transgenic plants tolerant to methyl viologen treatment.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said method results in generation of transgenic plants tolerant to jasmonic acid treatment.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said method results in generation of transgenic plants tolerant to salicylic acid treatment.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said polypeptide is present in extracellular space of said transgenic plants.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said polynucleotide fragment is nuclear genome encoded.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant, or parts thereof including seeds, capable of heterologous expression of a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 1.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is a monocot.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is a dicot.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is rice.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is wheat.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is rye.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is millet.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is selected from the group consisting of beans, peas, potato, eggplant, peppers, squash, melons, coffee, citrus, broccoli, turnips, legumes, yams, and apple.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is tolerant to dehydration.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is tolerant to hypersalinity.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is tolerant to cold.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is tolerant to methyl viologen treatment.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is tolerant to jasmonic acid treatment.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is tolerant to salicylic acid treatment.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is produced by a method comprising the steps: (a) obtaining plant cells; (b) obtaining a DNA construct comprising a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2, operably linked to a promoter and transforming a host cell with a DNA vector comprising said DNA construct to obtain a recombinant host cell comprising said DNA construct; (c) transforming said plant cells with said recombinant host cell to obtain transformed plant cells; and (d) selecting transformed plant cells and regenerating transgenic plants, wherein said transgenic plants are tolerant to stress compared to wild type non-transformed plants.
In an embodiment of the present disclosure, there is provided a stress tolerant transgenic plant as described herein, wherein said plant is produced by a method comprising the steps: (a) obtaining plant cells; (b) obtaining a DNA construct comprising a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2, operably linked to a promoter; (c) transforming said plant cells with said DNA construct to obtain transformed plant cells; and (d) selecting transformed plant cells and regenerating transgenic plants, wherein said transgenic plants are tolerant to stress compared to wild type non-transformed plants.
In an embodiment of the present disclosure, there is provided a polynucleotide fragment encoding a polypeptide having amino acid sequence as set forth in SEQ ID NO: 2 for use in generating stress tolerant transgenic plants.
In an embodiment of the present disclosure, there is provided a polynucleotide fragment as described herein, wherein said polynucleotide fragment is as set forth in SEQ ID NO: 1.
In an embodiment of the present disclosure, there is provided a polynucleotide fragment as described herein, wherein said polynucleotide fragment is cDNA.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said DNA construct promoter is a constitutive promoter.
In an embodiment of the present disclosure, there is provided a method as described herein, wherein said DNA construct promoter is 35S promoter.
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skills in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. The example is provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention.
There has been much interest in the ability of eukaryotic cells to regulate their internal water potentials (Epstein et al., Science 210, 399-404 (1980)). Although considerable knowledge of this process in certain algae is available (Macrobbie et al., University of California Press, Berkeley 676-713 (1974), which is incorporated by reference), our understanding of dehydration regulation in higher plants is much more limited. While suspension cultures may be considered as over simplification of the complex mechanisms involved in dehydration response, they represent a highly controllable and homogeneous experimental system. However, there is little information on the degree of dehydration and physio-biochemical characteristics of suspension culture. Sugar alcohols such as mannitol and sorbitol act as common source of carbon and energy and also, as osmotica in response to environmental stress including dehydration (Liu et al., Bot. Bull. Acad. Sci. 43, 107-113 (2002), which is incorporated by reference). On the contrary, PEG is known to mimic dehydration by reducing water availability and modifying the osmotic potential of nutrient solution (Rains, Cambridge University Press, Cambridge, UK 181-196 (1989), which is incorporated by reference). We therefore used PEG as an ideal osmoticum to model dehydration conditions in chickpea suspension culture.
To quantitate the influence of dehydration on the physiology of SCCs, the culture was subjected to various degree of dehydration at varying PEG concentration (0 to 20%). The culture was initially examined for change in fresh weight. No obvious deleterious changes could be observed up to 10% PEG in the media. Subsequent increase in the PEG concentration resulted in sharp decrease in fresh weight (
To compute the temporal effect of dehydration on the physiology of culture, 10% PEG treated suspension culture was examined up to 192 h. The morphometric analysis did not show any visible change in the SCCs until 72 h of dehydration (
The purity of the secretome was examined by catalase assay as an intracellular marker. The secreted proteins from dehydration treated suspension culture did not show any significant catalase activity, while proteins prepared from the calli showed high activity (
It has been frequently observed that dehydration causes simultaneous activation of specific protein synthesis and inhibition of the synthesis of some constitutive cellular proteins (Scolnik et al., Plant Physiol. 96, 291-296 (1991), which is incorporated by reference). The expression of dehydration-responsive proteins in the suspension culture was thus monitored using 1- and 2-DE analyses. We attempted to resolve the proteins initially onto a basic pH range (pH 6-11) (
The 202 dehydration-responsive secreted proteins varied in molecular weight with significant increase in the secretion of high molecular weight proteins. In stressed as well as unstressed conditions, maximum number of the identified secreted proteins has relatively high molecular weights in the range >100 kDa (
Differentially regulated secreted proteins were examined to predict their locations using TargetP program (www.cbs.dtu.dk/services/TargetP). Of the identified proteins, 43 were predicted to be secreted with signal peptide sequences (
Glycosylphosphatidylinositol (GPI) linked proteins are secreted via the ER and Golgi apparatus to the extracellular space. In this study, no proteins were predicted to contain GPI anchors when examined with big-PI Plant Predictor program. There have been many reports of the existence of non-classical proteins in the extracellular space. Protein sequences were thus analyzed on the neural network server SecretomeP. The analysis predicted 63 LSPs (NN-scores >0.5;
It is increasingly clear that most environmental signals utilize the extra cellular space (ECS) as a conference point for their communication, albeit the role of secreted proteins in such cross-talk remains largely uninvestigated. To gain an insight into protein secretion during stress induced signal transduction, the identified proteins were grouped into 4 functional classes (
The communication between the cytoskeleton and the apoplast is one of the most characteristic features of cellular mechanism that allows cells to respond effectively to various extracellular signals, possibly through regulation of ROS (Menzel et al., Plant Physiol. 133, 482-491 (2003), which is incorporated by reference). Induced actin polymer formation is reported during disturbance of ROS homeostasis (Apostolakos et al., Cytoskeleton 69, 1-21 (2012), which is incorporated by reference). Protein kinase is a ubiquitous enzyme in eukaryotes and prokaryotes that catalyzes the transfer of the y-phosphate from ATP to substrate auto-phosphorylation, thus contributing to downstream signaling by producing GTP for the activation of GTP-binding proteins. We observed dehydration induced regulation of protein kinase. Overexpression of protein kinase was previously reported to reduce the accumulation of ROS and provide tolerance against different abiotic stresses (Moon et al., Proc. Natl. Acad. Sci. U.S.A. 100, 358-363 (2003), which is incorporated by reference). Probable serine/threonine-specific protein kinase, xylulose kinase-like, inactive purple acid phosphatase and protein phosphatase are likely candidates that might be involved in the signal transduction network that operates in the apoplast. Subtilisin-like protease, aspartic proteinase, cysteine proteinase and protease inhibitor were previously reported to be regulated differentially under stress conditions (Peng et al., J. Plant Res. 120, 465-469 (2007), which is incorporated by reference). The enzymes related to secondary metabolism were also notable among the list of dehydration-responsive proteins like GMP synthase, an enzyme from the strictosidine biosynthesis pathway. The alkaloids have multiple functions, such as structural support, pigmentation, defense and signaling (Goldberg et al., Annu. Rev. Biochem. 65, 801-847 (1996), which is incorporated by reference). Most of the secondary metabolism related differentially expressed proteins, identified in this study, have been reported in the cell walls of different organisms. Cysteine synthase was earlier reported in cell wall of Medicago (Sumner et al., Phytochemistry 65, 1709-1720 (2004), which is incorporated by reference). Spermidine synthase, serine hydroxymethyltransferase and aldolase were reported in the secondary cell wall of developing xylem tracheary elements (Carol et al., Int. J. Plant Sci. 165, 243-256 (2004), which is incorporated by reference). Hexosaminidase, hydroquinone glucosyltransferase and dolichyl-diphosphooligosaccharide catalyzes an important trafficking step in cell wall modification. Tankyrase-2-like protein is required for synthesis of a variety of cellular constituents including cell wall polymers and glycoproteins. These results suggest that the differentially expressed secreted proteins may utilize the cell wall polysaccharides as a reservoir to produce sugar monomers, which maintain osmotic balance in plants under dehydration conditions.
During stress adaptation, protein degradation is necessary for the removal of abnormal or damaged proteins, and for altering the balance of proteins (Goldberg et al., Annu. Rev. Biochem. 65, 801-847 (1996)). In this study, proteins involved in the degradation pathway were also identified. The 26S proteasome has been reported to be involved in ubiquitin-mediated turnover of misfolded proteins (DiDonato et al., Mol. Cell. Biol. 16, 1295-1304 (1996), which is incorporated by reference). Since intact proteins are less sensitive to oxidation than misfolded proteins, protein chaperones are usually upregulated in response to various stresses. Different chaperones have been documented to play complementary and sometimes overlapping roles in protection of proteins. Many proteins from this class were also identified in the chickpea secretome viz., peptidyl-prolyl cis-trans isomerase, Hsp70 and 10 kDa chaperonin. Furthermore, nodal modulator, which is involved in nodal signaling and subsequent organization of axial structures, was also identified as differentially expressed protein.
The differential regulation of signaling molecules viz., GTP-binding protein and Ran GTPases, in the extracellular space suggests a complex signal transduction network. There are several reports that suggest extensive crosstalk among various environmental stress-responsive pathways (Shinozaki et al., Plant Cell 6, 251-264 (1994); Cheong et al., Plant Physiol. 129, 661-677 (2002), which are incorporated by reference). The PR proteins form a novel class of proteins that play pleiotropic roles, both in abiotic and biotic stresses (Hotta et al., Appl. Biochem. Biotechnol. 120, 169-174 (2005), which is incorporated by reference). Induction of different isoforms of glucanases and thaumatin like-protein, among others, could be attributed to their multiple roles. In unstressed condition, the translation elongation factor (EF-Tu) catalyzes the GTP-dependent binding of the aminoacyl-tRNA to the ribosome during the elongation phase of protein synthesis. However, EF-Tu can act as a molecular chaperone during stress and might be involved in protein folding and protection (Richarme et al., J. Biol. Chem. 273, 11478-11482 (1998), which is incorporated by reference).
The significance of non-classical secretion in plants, and the possible functions of leaderless secretory proteins (LSPs) are largely unknown. Most of the LSPs are related to stress response, and to explain their secretion, a number of alternate secretion mechanisms have been anticipated (Seedorf et al., Annu. Rev. Cell Dev. Biol. 24, 287-308 (2008); Bissell, et al., Nat. Rev. Mol. Cell Biol. 10, 228-234 (2009), which are incorporated by reference). Leaderless secretion facilitates rapid release of stress-responsive proteins via Golgi/ER-independent pathway. It also allows a normally cytoplasmic protein to relocate to the ECS where it can perform alternate functions. One of the dehydration-responsive LSPs identified, in this study, is nucleolin, a highly conserved and ubiquitously expressed protein in eukaryotes (Bouvet et al., Trends Cell Biol. 17, 80-86 (2007), which is incorporated by reference). It contains RGG repeats which participate in interactions with other proteins. Although it is highly abundant in the nucleus but often found in the plasma membrane and cytoplasm, where it is involved in numerous cellular processes. Lipid transfer protein is known to bind calcium intracellularly, but its role as an extracellular polypeptide signal has also been proposed (Schechter et al., Eur. J. Biochem. 212, 589-596 (1993), which is incorporated by reference). It is likely that these proteins serve dual roles, both intracellular and extracellular, depending on environmental cues. A recent secretome study in mammalian cells revealed an intracellular cysteine protease as a regulator of non-classical protein secretion (Beer et al., Cell 132, 818-831 (2008), which is incorporated by reference). The quantitative study on Arabidopsis suspension culture confirmed that a relatively high number of LSPs are secreted into the ECS when treated with salicylic acid (Williamson et al., J. Proteome Res. 8, 82-93 (2009), which is incorporated by reference). There are an increasing number of reports of individual proteins or enzyme activities that localize to the apoplast in response to stress despite the fact that such proteins lack signal peptide. We identified different isoforms of ubiquitin, proteasome and endopeptidases that lacked the signal peptide (
Screening of the secretome led to the identification of a non-classical secreted protein, henceforth designated CaRRP1 (ripening related protein). The domain analysis of CaRRP1 using InterProScan revealed the presence of Bet v 1 family domain (
Genomic sequence comparison revealed the transcript size of CaRRP1 to be 737 bp with coding region of 459 bp, and 43 bp 5′-UTR and 235 bp 3′-UTR. Further, the CaRRP1 coding sequence is interrupted by a single intron (
While in silico analysis using SignalP did not identify any clear targeting sequence, SecretomeP neural network programme could recognize the secretion of CaRRP1 via non-classical secretion. To validate the location, the coding region of CaRRP1 was introduced into plant expression vector, harboring YFP reporter gene. The YFP fluorescence of the fusion protein in tobacco leaves was visualized following Agrobacterium-mediated transient expression. A time-dependent expression assay detected the YFP-CaRRP1 in the ECS (
Regulation of gene expression at the transcriptional level plays a crucial role in the development and physiological status of plant. To investigate stress-responsive expression patterns of CaRRP1 in chickpea, we carried out Northern blot analysis. The CaRRP1 transcripts were highly upregulated under dehydration, displaying maximum accumulation at 48 h but dropped thereafter and reached the background level at 72 h (
Over the years, complementation analysis in yeast led to the identification of more than 50 proteins involved in protein trafficking in the secretory pathway (Bonifacino, Trends Cell Biol. 24, 3-5 (2014), which is incorporated by reference). YJL036w is a sorting nexin protein involved in proteasome function (Dixon et al., Nat. Rev. Mol. Cell Biol. 3, 919-931 (2002), which is incorporated by reference) and also functions in vesicular transport in yeast. YJL036w lacking yeast strains are defective in protein transport and exhibit aberrations in growth and appearance. To investigate the role of CaRRP1 in protein trafficking, we tested whether it could complement the YJL036w mutant. We used the YJL036w-deficient mutant for the complementation assay and monitored the growth of BY4741 (wild-type), YJL036w mutant and complemented (YJL036w:CaRRP1) strains in presence of various stressors that include 2.5 mM H2O2 for oxidative stress, 0.5 M mannitol for osmotic stress, 0.8 M NaCl and 0.2 M LiCl for hypersalinity. No significant difference in growth pattern of transformants, wild-type and mutants was observed either on SD-URA or YPD plates (
Maintenance of SSCs and dehydration treatment: Seeds of chickpea (Cicer arietinum L. cv. JG-62) were surface-sterilized using 70% ethanol followed by 0.1% HgCl2 for 10 min and then rinsed 5-7 times with sterile distilled water. Surface-sterilized seeds were allowed to imbibe water for at least 16 h and kept aseptically in dark. The explants were prepared from root-shoot-cut embryo axes and inoculated on MS media (pH 5.8). The nascent calli were maintained at 25±2° C., under 16 h photoperiod (300 μmol m−2 s−1 light intensity). The friable embryogenic calli were used to initiate suspension cultures in liquid MS media as described earlier (Gupta et al., J. Proteome Res. 10, 5006-5015 (2011), which is incorporated by reference). Dehydration stress was imposed after second sub-culturing by adding PEG 6000. The unstressed and the stressed cultures were maintained in parallel under same conditions. Secretory fractions of the SCCs, harvested at different time intervals, were used for downstream experiments.
Determination of RWC and cell viability: The SCCs were weighed (fresh weight, FW) after filtration through pre-weighed 0.2 micron membrane. The cells were rehydrated until fully turgid, surface dried and weighed (turgid weight, TW), followed by oven drying at 80° C. for 48 h and reweighed (dry weight, DW). The RWC was calculated by the following formula (Bhushan et al., Mol. Cell. Proteomics 6, 1868-1884 (2007)): RWC (%)=[(FW−DW/TW−FW)×100]. Cell viability of suspension culture was determined and quantified as described earlier20 using Evans blue and FDA.
Proline estimation, evaluation of electrolyte leakage and lipid peroxidation: Free proline content was measured as described earlier. Proline content was determined as per the formula: proline (μg/g FW)=36.6×A520×volume/2×FW. Electrolyte leakage was assayed by estimating the ions leaching from the SCCs. Aliquots of 20 ml SCCs in two sets were examined. The first set was kept at 25±2° C. for 4 h and the conductivity (C1) was recorded. The second set was autoclaved followed by recording the conductivity (C2), and electrolyte leakage [1−(C1/C2)×100] was calculated. In a separate experiment, lipid peroxidation was determined in terms of malondialdehyde (MDA) production.
Purification of secreted proteins from suspension culture: The suspension cultures were left untreated or treated with 10% PEG 6000 for 72 h and the secreted proteins were isolated (Gupta et al., J. Proteome Res. 10, 5006-5015 (2011)). In brief, the suspension culture was centrifuged at 3000×g for 5 min followed by filtration using 0.45 μm Durapore membrane (Millipore). The resulting callus culture filtrate (CCF) was concentrated using Amicon Ultra-15 centrifugal filter unit (10 kDa cut-off each; Millipore) until the final protein concentration was >1 mg/ml. Further, the retentate was extracted as described earlier (Peck et al., Mol. Cell. Proteomics 8, 145-156 (2009), which is incorporated by reference). Also, proteins from calli were isolated (Schiltz et al., Plant Physiol. 135, 2241-2260 (2004), which is incorporated by reference) with few modifications. Approximately, 300 mg calli were ground to powder in liquid nitrogen with 0.3% (w/w) polyvinylpolypyrollidone (PVPP) and powdered tissue was homogenized in homogenizing buffer [50 mM Tris-HCl (pH 8.2), 2 mM EDTA, 20% glycerol, 5 mM DTT and 2 mM PMSF]. The proteins were recovered as supernatant by centrifugation at 6000×g for 10 min at4 ° C. (Bhushan et al., J. Proteome Res. 5, 1711-1720 (2006)). Protein samples were allowed to cool at 25±2° C., precipitated with 9 volumes of 100% chilled acetone overnight at −20° C. The precipitates were recovered at 10,000×g for 10 min at 4° C. Protein pellets were washed twice with 80% acetone to remove excess SDS, air-dried and protein concentration was measured using the 2-D Quant Kit (GE Healthcare).
Enzymatic assay of catalase: The catalase activity was determined calorimetrically as described earlier ° C. (Bhushan et al., J. Proteome Res. 5, 1711-1720 (2006)). The reaction mixture was prepared by adding 10 μg protein, in 50 μl, to 940 μl of 70 mM potassium phosphate buffer (pH 7.5). Reaction was initiated by adding 10 μl of H2O2 (3% v/v), and decrease in absorbance at 240 nm was monitored for 5 min.
Immunoblot screening: Immunoblotting was carried out by resolving secreted and calli proteins on 12.5% SDS-PAGE followed by electrotransfer onto nitrocellulose membrane (GE Healthcare). The blot was probed with anti-RbcL antibody (AS03037; Agrisera AB) at a dilution of 1:5000 in TBS. Immunoreactive protein was detected by incubation with alkaline phosphatase conjugated anti-rabbit IgG as secondary antibody (Sigma).
Electrophoresis of secreted proteins: The proteins were resuspended in 2-D rehydration buffer [8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) pharmalyte (pH 4-7) and 0.05% (w/v) bromophenol blue]. Isoelectric focusing was carried out with 150 μg protein in 250 μl buffer using 13 cm IPG strips (GE Healthcare) by in-gel rehydration method. Electrofocusing was performed using IPGphor system (GE Healthcare) at 20° C. for 30,000 Vh. In a separate experiment, the proteins were fractionated on 13 cm 1-DE. The electrophoresed proteins were visualized with MS-compatible silver staining (Bio-Rad Laboratories).
Image acquisition and data analysis: Image acquisition was achieved by digitization of gel images with a Bio-Rad FluorS system. Quantitative and qualitative differences between the replicate 2-DE gels were analyzed using PDQuest version 7.2.0 (Bio-Rad Laboratories) followed by generation of reference image (Bhushan et al., Mol. Cell. Proteomics 6, 1868-1884 (2007)). The replicate gels used for making the first level matchset had, at least, a correlation coefficient value of 0.8. In order to compare gels from individual time points, a second level matchset was created. A data matrix of high quality spots was constructed from unstressed and stressed samples for further analysis.
In-gel digestion, mass spectrometry and bioinformatics analysis: The protein spots or lane (sliced into 29 gel pieces each of ˜1.5 mm) were excised and subjected to trypsinolysis (Bhushan et al., J. Proteome Res. 5, 1711-1720 (2006)). The peptides were analyzed using QSTAR Elite mass spectrophotometer (Applied Biosystem) coupled with an on-line Tempo nano-MDLC system. The acquired mass spectra were searched against the chickpea protein sequence available in chickpea genome annotation v.1.0 (Varshney et al., Nat. Biotechnol. 31, 240-246 (2013), which is incorporated by reference) (22893 sequences; 9330989 residues) using Mascot search engine (www.matrixscience.com). Proteins were assigned as identified if the MOWSE score was above the significance level. The function of proteins was assigned using protein function database Pfam (www.sanger.ac.uk/software/Pfam/) or Inter-Pro (www.ebi.ac.uk/interpro/). Further, the criteria used to assign the function of the proteins were based on other reports besides the predicted biochemical and biological functions by GO classification.
The presence and location of signal peptide cleavage sites were predicted by the SignalP 3.0 program (www.cbs.dtu.dk/services/SignalP). Proteins identified by SignalP were re-examined using ScanProsite (au.expasy.org/prosite/). To identify non-classical secreted proteins, proteins lacking N-terminal signal sequences were analyzed by SecretomeP (www.cbs.dtu.dk/services/SecretomeP/). The big-PI Plant Predictor program GPI (mendel.imp.ac.at/gpi/plants/gpi_plants.html) was used to identify potential GPI lipid anchors. The domain analysis was done by NCBI CDD (www.ncbi.nlm.nih.gov).
Isolation of CaRRP1, sequence analyses and construction of phylogram: The cDNA fragment of CaRRP1 was cloned into the pGEM-T vector (Promega) and the sequence identity was determined. The amino acid sequence, molecular weight and isoelectric points of CaRRP1 were obtained from ExPASy. Secondary structures, including the locations of the Bet v 1 domain were determined by InterProScan (www.ebi.ac.uk/interpro/). The SPIDEY (www.ncbi.nlm.nih.gov/spidey) program was used to determine the genomic organization of CaRRP1. The phylogram was constructed from amino acid alignment by neighbor-joining method (www.ebi.uk/Tool/clustalw/) using MEGA software version 5.11, with a bootstrap value of 10060.
Quantitative real-time PCR: The chickpea seedlings were grown in green house, and maintained at 25±2° C. and 50±5% relative humidity under 16 h photoperiod (300 μM m−2 s−1 light intensity) as described previously. Three-week-old seedlings were independently subjected to dehydration and hypersalinity treatment (50, 100 and 200 mM NaCl). The unstressed seedlings were also maintained in the same green house and tissues were collected every day during the course of the dehydration experiment, and finally pooled to normalize the growth and development effects, if any. ABA (25, 50 and 100 μM), MV (50, 100 and 200 μM), SA (5 mM) and JA (100 μM) treatment were accomplished by spraying solution on the leaflets. The low temperature treatment was given by keeping the seedlings at 4° C. The harvested tissues were instantly frozen in liquid nitrogen and stored at −80° C. Total RNA was isolated from 3-week-old seedlings using the TriPure reagent (Invitrogen). cDNA was prepared using SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). The qRT-PCR assays were performed with the ABI PRISM 7700 sequence detection system (Applied Biosystems) using SYBR Green PCR Master mix in a final volume of 20 μl including cDNA template and appropriate primers (Table 2).
Localization of CaRRP1: The coding region of CaRRP1 was amplified by PCR using gene-specific primers (Table 2) and cloned into pENTR-D/TOPO followed by recombination into pGWB441, and introduced into Agrobacterium strain GV3101. The cells were infiltrated into tobacco leaves for the transient expression of CaRRP1-YFP fusion protein. The YFP fluorescence was detected with TCS SP2 confocal system (Leica, Germany) and the images were captured 2-3 days after infiltration.
Functional complementation in yeast: The yeast mutant for vesicular transport YJL036w [BY4741; MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YJL036w:: kanMX4] and the background strain BY4741 [MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0] were transformed with pYES2 and the recombinant pYES2:CaRRP1 construct independently. Overnight cultures were grown in SD-URA and serial dilutions [OD600 ˜0.1] were plated. The plates were incubated at 30° C. for 2 days. Growth assays were performed by using serial dilution of cultures in the respective media containing various stress-inducing chemicals (2.5 mM H2O2, 0.5 M mannitol, 0.8 M NaCl and 0.2 M LiCl).
Table 1 depicts the reproducibility of 2-DE gels.
aAverage number of spots present in three replicate gels of each time point.
bSpots having quality score more than 30 assigned by PDQuest (Ver.7.2.0).
cSE represents standard error for the replicate gels.
Table 2 depicts list of primers.
Genetic transformation of plant: The coding region of CaRRP1 was amplified by PCR using gene-specific primers and cloned into pENTR-D/TOPO followed by recombination into pGWB441. The CaRRP1-EYFP construct in pGWB441 gateway vector under the control of CaMV 35S promoter was used to transform Agrobacterium strain GV3101. The bacteria were grown to stationary phase in liquid culture at 25-28° C., 250 rpm in sterilized YEP (10 g yeast extract, 10 g peptone, and 5 g NaCl per liter) supplemented with spectinomycin (100 mg/ml). Cultures were initiated with 1:100 dilution of primary overnight cultures and grown for roughly 18-20 h. Cells were harvested by centrifugation (5,500×g) for 20 min at room temperature. The harvested cells were re-suspended in inoculation medium [½ strength MS Medium (M-5519; Sigma Chemicals), 5.0% sucrose, 44 nM benzylamino purine (10 ml l−1 of a 1 mg ml−1 stock in DMSO) and 0.008% Silwet L-77] pH adjusted to 5.7 to a final OD600 of approximately 2.0. The seedlings of Arabidopsis were transformed by floral-dip method (Clough and Bent (1998) Plant J. 16(6):735-43, which is incorporated by reference).
The coding region of CaRRP1 was amplified by PCR using gene-specific primers (SEQ ID NO: 11 and SEQ ID NO: 12) and cloned into pENTR-D/TOPO followed by recombination into pGWB441. The CaRRP1-EYFP construct in pGWB441 gateway vector under the control of CaMV 35S promoter was used to transform Agrobacterium strain GV3101. The bacteria were grown to stationary phase in liquid culture at 25-28° C., 250 rpm in sterilized YEP (10 g yeast extract, 10 g peptone, and 5 g NaCl per liter) supplemented with spectinomycin (100 mg ml−1). Cultures were initiated with 1:100 dilution of primary overnight cultures and grown for roughly 18-20 h. Cells were harvested by centrifugation (5,500×g) for 20 min at room temperature. The harvested cells were resuspended in inoculation medium [½ strength MS Medium (M-5519; Sigma Chemicals), 5.0% sucrose, 44 nM benzylamino purine (10 ml l−1 of a 1 mg ml−1 stock in DMSO) and 0.008% Silwet L-77] pH adjusted to 5.7 to a final OD600 of approximately 2.0. The seedlings of Arabidopsis were transformed by floral-dip method [Clough and Bent (1998) Plant J. 16(6):735-43.].
Overall, the present disclosure provides a method of producing transgenic plants which are tolerant to stressors such as dehydration, hypersalinity, cold, and chemical stressors such as salicylic acid. These plants are better suited to survive environmental stress conditions, which otherwise may be detrimental to the life of non-transgenic plants. Such plants are of economic significance as environmental stress factor can wreak havoc on plants, which are otherwise agronomically important. Also provided are constructs, vectors, and recombinant cells for facilitation of generating said stress tolerant transgenic plants.
| Number | Date | Country | Kind |
|---|---|---|---|
| 3983/DEL/15 | Dec 2015 | IN | national |
This application is the U.S. National Phase Application of PCT/IN2016/050437, filed Dec. 7, 2016, which claims priority to Indian Patent Application No. 3983/DEL/15, filed Dec. 7, 2015, the contents of such applications being incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2016/050437 | 12/7/2016 | WO | 00 |
