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Expression of pigeonpea hybrid‐proline‐rich protein encoding gene (CcHyPRP) in yeast and Arabidopsis affords multiple abiotic stress tolerance

Expression of pigeonpea hybrid‐proline‐rich protein encoding gene (CcHyPRP) in yeast and... Introduction Abiotic stresses imposed by drought, salinity and extreme temperatures act as major impediments and pose serious threat to the growth and productivity of diverse crop plants with devastating socio-economic consequences. These stresses, in general, are characterized by dehydration resulting from the decreased availability of water to plant cells. Different plants, upon exposure to numerous abiotic stresses, were found to show a wide range of responses at molecular, cellular and whole plant levels ( Greenway and Munns, 1980 ; Zhu et al. , 1997 ; Yeo, 1998 ; Hasegawa et al. , 2000 ). Concomitant occurrence of multiple abiotic stresses, as compared with individual stresses, invariably proved detrimental to the plants grown under field conditions. However, not much is known about the molecular mechanisms underlying acclimation of plants to different combinations of more than one stress ( Rizhsky et al. , 2004 ). To tide over different environmental conditions, plant species have evolved specific adaptive mechanisms and exhibit wide variation in their ability to withstand abiotic stresses, owing to their inherent genetic plasticity ( Bartels and Ramanjulu, 2005 ; Yamaguchi-Shinozaki and Shinozaki, 2006 ). Predictions made from climate models point to the occurrence of abrupt fluctuations in weather conditions triggered by the long-term effects of global warming ( Salinger et al. , 2005 ; Cook et al. , 2007 ). To ensure sustainable crop productivity, it is imperative to design and evolve improved versions of crop plants that can endure the detrimental effects of changing environmental factors. In this context, it is mandatory to identify novel candidate genes with multiple stress tolerance by accessing the vast gene pools of diverse taxa. In model plants, significant information has been generated on biochemical and molecular aspects of gene regulation under different abiotic stress conditions. Drought, salinity, extreme temperatures and oxidative stress result in complex stimuli of varied nature that are often interconnected, and hence these stresses might induce similar damages at the cellular level ( Rodriguez et al. , 2005 ). Furthermore, stress responses of plants to various abiotic factors have been found difficult to dissect, as defense responses require regulatory changes for activation of multiple genes involved in different metabolic pathways ( Bohnert et al. , 2006 ). Functions of diverse plant genes that respond to abiotic stresses have been elucidated at physiological, biochemical and molecular levels ( Hasegawa et al. , 2000 ; Shinozaki and Yamaguchi-Shinozaki, 2000 ; Zhu, 2002 ). Plant genes involved in the signalling and regulatory pathways of stress response ( Saijo et al. , 2000 ; Shinozaki et al. , 2003 ; Kim et al. , 2007 ; Chen et al. , 2008 ), genes which encode proteins conferring stress tolerance ( Sunkar et al. , 2003 ; Nelson et al. , 2007 ), and genes coding for enzymes mediating metabolic pathways for synthesis of specific stress-responsive metabolites ( Kavi Kishore et al. , 1995 ; Park et al. , 2004 ), have been deployed for production of transgenic plants bestowed with abiotic stress tolerance. Plant hybrid-proline-rich proteins (HyPRPs) are putative cell wall proteins consisting of a repetitive proline-rich (PR) N-terminal domain and a conserved eight-cysteine-motif (8 CM) C-terminal domain ( Jose-Estanyol et al. , 2004 ; Dvorakova et al. , 2007 ). Proteins with 8 CM have been classified into different families, viz., proline-rich proteins, lipid transfer proteins (LTPs), 2S albumins and amylase/trypsin inhibitors, based on their functions during growth and development of plants ( Jose-Estanyol et al. , 2004 ). Also, based on the presence of a proline-rich domain and a secretory signal, HyPRPs were designated as a group of secreted structural cell wall proline-rich proteins ( Jose and Puigdomenech, 1993 ; Jose-Estanyol and Puigdomenech, 2000 ). In diverse plant species, HyPRPs were found associated with different abiotic and biotic stress conditions. In the salt tolerant Medicago sativa , a proline-rich protein encoding gene, MsPRP2 , was amply induced under drought conditions ( Deutch and Winicov, 1995 ). Cold tolerant alfalfa, as compared with cold sensitive plants, revealed higher levels of transcripts of a cold regulatory MSACIC gene ( Castonguay et al. , 1994 ). Transcripts of Brassica napus BNPRP gene were specifically induced when the plants were subjected to cold stress ( Goodwin et al. , 1996 ). In soybean, the expression profile of SbPRP gene was modulated by ABA, internal circadian rhythm, as well as abiotic and biotic stress factors ( He et al. , 2002 ). Arabidopsis plants with knocked down EARLI 1 genes, which code for HyPRPs, showed an increased freezing-induced cellular damage as compared with the wild-type plants ( Zhang and Schlappi, 2007 ). Pigeonpea ( C. cajan L) is a well known grain legume crop of the tropical and subtropical regions of the world, which grows well in hot and humid climates. It has an excellent deep root system with profuse laterals and is widely known as one of the most drought, salinity and alkalinity tolerant crops among legumes ( Nene and Sheila, 1990 ). To isolate stress-inducible genes, a highly drought tolerant pigeonpea variety has been used as a potential source of stress-tolerance genes. In this study, as a part of our ongoing programme, we have isolated a stress-responsive Cajanus cajan hybrid-proline-rich protein encoding gene ( CcHyPRP ) from the PCR-subtractive cDNA library. Over-expression of CcHyPRP in yeast cells conferred significant tolerance to multiple abiotic stresses. Similarly, transgenic Arabidopsis plants expressing CcHyPRP disclosed explicit tolerance against different abiotic stress conditions. Results Characterization of Cajanus cajan hybrid-proline-rich protein gene ( CcHyPRP ) A partial cDNA clone of 309 bp (Acc.No. CK394831 ) was obtained from the cDNA library of pigeonpea plants subjected to 20% PEG stress (−1.01 ± 0.02 Mpa), employing PCR-based cDNA subtraction. A full-length cDNA clone of 731 bp with a 5′- and 3′-untranslated region was recovered by 5′- and 3′-RACE PCR; it codes for a polypeptide of 131 amino acids including a signal peptide of 22 amino acids ( Figure 1 ). The CcHyPRP contained maximum number of leucines (21) followed by prolines (16), besides a proline-rich domain in the N-terminal region and a cysteine-rich hydrophobic domain in the C-terminal region. It was hence designated as the Cajanus cajan hybrid-proline-rich protein encoding gene ( CcHyPRP ). The CcHyPRP showed maximum similarity of ∼84% with that of Pvr5 of Phaseolus vulgaris ( AAC49369 ) followed by ∼68% with glycine-rich protein of Nicotiana tabacum ( BAA95941 ), ∼67% with SbPRP of Glycine max ( AAF78903 ), ∼58% with LTP of Capsicum annuum ( ABQ88334 ), ∼51% with CLCT protein of Arabidopsis thaliana ( AAM63191 ) and ∼47% with extensin-like protein of Glycine max ( AAM75351 ) ( Figure 1 ). To decipher the nature of CcHyPRP , the pigeonpea genomic DNA was digested independently with Bam HI, Eco RI, Hind III and Sal I enzymes, and was probed with CcHyPRP coding sequence. Southern analysis revealed single hybridization signals of varied size ranging from >3 Kb to ∼9 Kb ( Figure 2 ). Northern blot analysis of the RNA isolated from pigeonpea plants treated with NaCl (1 M)/PEG (20%)/heat (42°C)/cold (4°C)/ABA (20 & 50 ॖM) and untreated plants, revealed increased accumulation of CcHyPRP transcripts in the stressed plants as compared with the unstressed controls ( Figure 3a–e ). In various stress treatments, higher levels of CcHyPRP transcript accumulations were detected in the roots compared with the leaves of pigeonpea plants. Expression of CcHyPRP gene in yeast ( Saccharomyces cerevisiae ) confers abiotic stress tolerance Yeast system was employed to assess the effect of CcHyPRP protein against heat, salinity and osmotic stress. Yeast cells containing CcHyPRP, under the regulation of GAL promoter, expressed a polypeptide of ∼14 kDa which was absent in the cells transformed with the vector (pYES2/NTC) alone (Figure S1). Yeast cells harbouring pYES2/NTC- CcHyPRP along with the control (pYES2/NTC) were subjected to different stresses caused by PEG, NaCl, LiCl, cold (4°C) and heat (42°C). LiCl is commonly 100 mM LiCl could induce both salt and osmotic stresses. Under normal (stress-free) conditions, the growth pattern of pYES2/NTC- CcHyPRP yeast was found similar to that of control yeast containing the vector alone ( Figure 4 ). Yeast cells expressing CcHyPRP showed normal growth under 20% PEG/1.0 mM NaCl/100 mM LiCl/heat (42°C) stress as compared with the negligible growth observed in the control yeast ( Figure 4 ). On the other hand, both control and CcHyPRP -yeast cells, subjected to cold stress (4°C) failed to show any growth even after 48 h of culture. Overexpression of CcHyPRP gene in Arabidopsis imparts multiple stress tolerance To evaluate the functional role of CcHyPRP, Arabidopsis was transformed with CcHyPRP driven by CaMV 35S/rd 29A promoters ( Figure 5a,b ). PCR analysis of the DNA isolated from kanamycin tolerant Arabidopsis plants, using CcHyPRP primers, revealed a ∼400 bp amplified fragment, while no such band was observed in the wild-type plants (data not shown). RT-PCR analysis, using the total RNA from stressed rd29A- CcHyPRP lines and unstressed CaMV35S- CcHyPRP lines as well as wild-type plants, employing CcHyPRP- specific primers, disclosed the presence of a ∼400 bp amplicon in the transgenic lines ( Figure 5c ). Tolerance exhibited by the homozygous (T3) transgenic lines, viz., CS1 and CS2 (CaMV35S- CcHyPRP ) and CR1 and CR2 (rd29A- CcHyPRP ), to different abiotic stresses was analysed by subjecting them to mannitol, PEG, NaCl, LiCl, heat (42°C) and cold (4°C) stress conditions. Under unstressed conditions, CR1 and CR2 lines showed similar growth pattern as that of wild-type plants. Whereas, CS1 and CS2 lines revealed noticeable decreases in plant growth as compared with the wild-type plants ( Figure 6 ). Transgenic lines grown under 300 mM mannitol/15% PEG/200 mM NaCl/15 mM LiCl/heat (42°C) stress, in comparison with the wild-type plants, showed observable increases in plant growth ( Figure 6 ). However, both CR1 and CR2 lines, similar to wild-type plants, failed to withstand the heat stress ( Figure 6 ). Effect of CcHyPRP on plant survival, root growth and biomass under stress conditions Transgenic lines, grown under different stress conditions, exhibited higher plant survival rate, increased root length and enhanced plant biomass as compared with the wild-type plants. CcHyPRP -transgenics subjected to mannitol (300 mM) showed higher (85.00%–88.33%) survival rate compared with the wild-type plants (43.33%) ( Figure 7a ). Transgenic lines also exhibited enhanced (96.00%–144.50%) biomass with substantial increases (184.62%–215.30%) in root length when compared with wild-type plants ( Figure 7b,c ). Similarly, transgenic lines subjected to PEG (15%) revealed higher (85.00%–88.30%) survival rates compared with 43.30% survival observed in wild-type plants ( Figure 7a ). Furthermore, compared with control plants, transgenics disclosed enhanced (111.30%–200.00%) plant biomass with significant increases (138.40%–166.90%) in root length ( Figure 7b,c ). Transgenic plants grown under salt (200 mM) stress exhibited higher (85.00%–88.33%) plant survival while wild-type plants showed 13.30% survival ( Figure 7a ). Transgenics also revealed marked increases (524.60%–671.20%) in plant biomass and root length (286.60%–420.00%) as compared with the wild-type plants ( Figure 7b,c ). Likewise, transgenic lines subjected to LiCl (15 mM) showed higher (85.00%–91.60%) survival rate compared with 30.00% survival observed in the wild-type plants ( Figure 7a ). Transgenic lines also exhibited increased (124.60%–251.30%) biomass along with significant increases (178.10%–206.80%) in the root length when compared with the control plants ( Figure 7b,c ). Under heat (42°C) stress, CS1 and CS2 transgenics showed higher (93.33%–95.00%) plant survival when compared with CR-transgenics (34.00%) and wild-type (33.33%) plants ( Figure 7a ). CS-transgenics also revealed significant increases (156.50%–164.00%) in total biomass and root length (212.50%–225.00%) compared with that of CR-transgenics and wild-type plants ( Figure 7b,c ). However, both CS- and CR-transgenics and wild-type plants, when subjected to cold stress (4°C), failed to show observable differences among them for plant survival, root growth and total biomass (data not shown). Discussion Extensive damage caused to the global agriculture by various abiotic stresses warrants evolvement of crop plants with enhanced tolerance to different environmental factors. To develop customized crop plants with inbuilt tolerance for multiple stresses, efforts should be made to clone novel stress-tolerance genes from diverse species acclimated to various adverse conditions. Pigeonpea is a major pulse crop and is endowed with the innate ability to resist adverse environmental conditions. In this study, a stress-responsive CcHyPRP gene has been isolated from the cDNA library of pigeonpea plants subjected to drought stress. The functional role of CcHyPRP in imparting multiple abiotic stress tolerance has been established by its overexpression in yeast and Arabidopsis systems. The deduced amino acid sequence of CcHyPRP showed similarity to LTP/alpha amylase inhibitor/seed storage family proteins ( Figure 1 ). The proteins belonging to this family were characterized by the presence of an eight-cysteine-motif (8CM) at the C-terminal end ( Jose-Estanyol et al. , 2004 ). The CcHyPRP contained a proline-rich domain and a hydrophobic cysteine-rich domain besides a signal peptide at the N-terminal end. The conserved nature of spacing between leucine residues noticed in CcHyPRP indicates their plausible involvement in the formation of leucine-zipper motifs as was observed in various other plant HyPRPs ( Subramaniam et al. , 1994 ; He et al. , 2002 ). Different plant proteins, containing 8CM as well as signal peptide, have been found to differ in their functionality ( Jose-Estanyol et al. , 2004 ). In diverse plant species, involvement of HyPRP proteins has been implicated under both abiotic and biotic stress conditions ( Ryan, 1990 ; Molina and Garcı′a-Olmedo, 1993 ; Showalter, 1993 ; Yubero-Serrano et al. , 2003 ; Jang et al. , 2004 ; Zhang and Schlappi, 2007 ). Different proline-rich proteins (PRPs) of plant-cell walls were found to act in various processes of plant growth and development in a tissue- and cell-specific manner ( Showalter and Rumeau, 1990 ; Jose-Estanyol et al. , 1992 ). Other classes of proline-rich cell wall structural proteins containing 8CM sequences (HyPRPs), were found to express in a tissue-specific manner, and were induced by specific stresses or hormones, but their functions were not clearly determined ( Jose-Estanyol and Puigdomenech, 2000 ). Southern blot analysis of the genomic DNA digested with different restriction enzymes, using CcHyPRP as a probe, disclosed single hybridizable bands of varied size, implying single-copy nature of CcHyPRP gene and the absence of paralogs in the pigeonpea genome ( Figure 2 ). Northern blot analysis revealed intense hybridizable signals in PEG-/salt-/heat-stressed plants and moderate signals in cold-stressed plants compared with the weak signals in unstressed plants, thus suggesting the stress-responsive nature of CcHyPRP gene ( Figure 3 ). Accumulation of significantly higher levels of CcHyPRP transcripts under PEG, NaCl, heat and cold stress, denotes that CcHyPRP is regulated by a stress-inducible promoter in the pigeonpea. Presence of increased levels of CcHyPRP transcripts in ABA-treated plants indicates that CcHyPRP is induced by ABA, thereby affirming its crucial role in the abiotic stress tolerance. In different stress treatments, higher levels of transcript accumulations were observed in the roots as compared with the leaves. Similar results were reported in P. vulgaris, where pvr 5 was found to show preferential expression in roots with maximum accumulation of transcripts in the cortical root meristem cells ( Choi et al. , 1996 ). An overview of northern profiles authenticates that CcHyPRP expression is highly modulated by various environmental conditions. In diverse plant species, expression of HyPRPs was induced by different abiotic and biotic stress conditions. In alfalfa, expression of MsPRP2 gene was induced by water-deficit in the salt tolerant plants ( Deutch and Winicov, 1995 ), while MsACIC was expressed in the cold tolerant plants ( Castonguay et al. , 1994 ). The BNPRP gene of B. napus ––encoding a putative cell wall-plasma membrane linker-protein––was highly expressed under low temperature conditions ( Goodwin et al. , 1996 ). Similarly, expression of soybean SbPRP gene was up-regulated by salicylic acid, viral infection, salt and drought stress conditions ( He et al. , 2002 ). The CcHyPRP gene was transformed into the yeast system to analyse its functionality, owing to the ability of yeast to perform various post-translational modifications ( Cereghino and Cregg, 2000 ). CcHyPRP -yeast transformants revealed marked superiority in their growth rate in comparison with the control yeast as evidenced by the colony size ( Figure 4b ), confirming that the stress-responsive CcHyPRP imparts substantial tolerance at the cellular level for multiple abiotic stresses. Over-expression of Arabidopsis DBF2 kinase ( Lee et al. , 1999 ), trehalose-6P synthase of yeast ( Soto et al. , 1999 ), Arabidopsis AtGSK ( Piao et al. , 1999 ) and rice rHsp 90 ( Liu et al. , 2006 ) genes in yeast cells conveyed significant tolerance against different abiotic stresses. Yeast cells expressing Arabidopsis EARLI1 genes coding for HyPRP exhibited higher rates of freezing survival than the control cells ( Zhang and Schlappi, 2007 ). Arabidopsis plants were transformed with CcHyPRP gene, driven by constitutive CaMV35S or by stress-inducible rd29A promoter, to validate its role in bestowing abiotic stress tolerance. Four transgenic Arabidopsis lines expressing CcHyPRP , when subjected to multiple stresses imposed by PEG/mannitol/NaCl/LiCl/heat, disclosed healthy seedlings with profuse root system in contrast to stunted and debilitated seedlings observed in the wild-type plants ( Figure 5 ). However, the vulnerability of CR1 and CR2 transgenics to heat stress may be attributed to the absence of heat-shock-inducible element(s) in the rd29A promoter. Furthermore, CcHyPRP -transgenics exhibited conspicuous tolerance against multiple abiotic stresses as evidenced by increased seedling survival, profuse root growth and enhanced plant biomass ( Figure 6 & Figure 7 ). These results amply demonstrate the unequivocal involvement of CcHyPRP in affording abiotic stress tolerance at the whole plant level. Deploying various candidate genes accessed from diverse gene pools, crop plants have been genetically engineered against different abiotic stresses. Ectopic expression of DREB1a transcription factor in Arabidopsis furnished increased tolerance to drought, salinity and freezing stress ( Kasuga et al. , 1999 ). Similarly, expression of Arabidopsis CBF1 ( DREB1b ) in transgenic tomato provided ample tolerance against drought, chilling and oxidative stress ( Hsieh et al. , 2002 ). Transgenic tobacco, expressing rice zinc-finger protein gene (OSISAP1) , conferred distinct tolerance against cold, dehydration and salt stress ( Mukhopadhyay et al. , 2004 ). Likewise, expression of calcium-dependent protein kinase ( OsCDPK7 ) gene in rice conveyed increased tolerance to cold, salt and drought stresses ( Saijo et al. , 2000 ). Tobacco plants expressing AtMDAR1 gene exhibited tolerance against ozone, salt and PEG stresses ( Eltayeb et al. , 2007 ). Similarly, expression of pea MnSOD gene in transgenic rice accorded enhanced tolerance against drought and oxidative stresses ( Wang et al. , 2005 ). Transgenic Arabidopsis , expressing CcHyPRP under CaMV35S promoter, exhibited diminished characteristics such as stunted plants with tiny leaves and undersized siliques, presumably owing to the constitutive expression of CcHyPRP protein. Use of strong constitutive promoters like CaMV35S and rice actin were shown to induce undesirable phenotypic effects upon over-expression of stress-responsive genes ( Kasuga et al. , 1999 ; Mahalakshmi et al. , 2006 ). The various negative effects observed in the transformants obtained with CaMV35S promoter, under unstressed conditions, are attributable to the energy-drain caused by the constitutive expression of transgenes. Conversely, CcHyPRP -transgenics driven by the stress-inducible rd29A revealed similar stress-tolerance as that of CaMV35S-lines without any sort of negative effects on the plant morphology, implying that stress-inducible promoters are preferable for development of stress tolerant transgenic plants. The pigeonpea CcHyPRP contained a highly conserved 8CM domain and resembled proline-rich proteins, LTPs, 2S albumins, extensins and trypsin inhibitors, involved in the storage of proteins and minerals, protection against pathogens, enzyme inhibition and lipid transfer mechanisms ( Jose-Estanyol et al. , 2004 ). Earlier, it was proposed that different 8CM proteins have evolved by the insertion of different sets of amino acid residues within the 8CM domain to assume varied functions. For instance, variations such as the presence of an internal hydrophobic cavity in LTPs; addition of a proline-rich domain to the hydrophobic 8CM of HyPRPs; proline-/glutamine-rich domain in prolamins; and sequences that promote loop proteolytic cleavage in 2S-albumins, have been observed in different 8CM proteins ( Jose-Estanyol et al. , 2004 ). As Arabidopsis transgenic lines expressing CcHyPRP exhibited marked tolerance against different abiotic stresses, we envisage a broad mechanism of action analogous to that of 8CM proteins operating at the cellular level for protection of plants against multiple stresses. However, additional investigations are needed for an in-depth analysis of CcHyPRP’s function in abiotic stress tolerance. To sum up, a multifunctional CcHyPRP gene has been isolated from the subtracted cDNA library constructed from the PEG-stressed pigeonpea plants. The CcHyPRP disclosed single-copy nature and was induced in pigeonpea by different abiotic stress conditions. Expression of CcHyPRP in yeast and Arabidopsis imparted substantial tolerance to PEG, mannitol, NaCl, LiCl and high temperature. The overall results affirm that the Cajanus stress-responsive gene confers high-level multiple stress tolerance in the heterologous systems. The pigeonpea CcHyPRP, by virtue of its surpassing tolerance against drought, salinity and heat stress, seems promising as a prime candidate gene for stress tolerance. As such, CcHyPRP may be deployed in the genetic enhancement of diverse crop plants for multiple abiotic stress tolerance. Experimental procedures Plant material and growth conditions Drought tolerant pigeonpea accession ICP 8744, procured from the ICRISAT, Hyderabad (India), was multiplied and used in this investigation. Seeds were surface sterilized with 0.1% mercuric chloride for 5 min followed by three washes, each for 10 min, in sterile distilled water under aseptic conditions. The sterilized seeds were germinated in pots and plants were maintained in the green house at 28 ± 2.0 °C. Seeds of Arabidopsis thaliana ecotype Columbia, obtained from the CCMB, Hyderabad, were treated with ethanol for 10 min followed by 0.05% mercuric chloride for 3 min. Later, these seeds were washed thoroughly six times with sterile water. Sterilized seeds, after stratification for 64 h at 4°C, were grown on MS medium (MS salts + 2% sucrose + 0.8% agar) or synthetic soil mix at 200 °C with 16 h photoperiod (long day) under fluorescent light (7000 lux at 20 cm) in Conviron growth chamber (Model TC16). Measurement of water potential Water potential of the 4-week old control (unstressed) and Polyethylene glycol (PEG 6000)-stressed pigeonpea plants were measured using the pressure chamber method of Scholander et al. (1964) . Synthesis of cDNA library Total RNA was isolated from the four-week old unstressed (−0.49 ± 0.02 Mpa) and 20% PEG 6000-stressed (−1.01 ± 0.02 Mpa) pigeonpea plants by guanidinium thiocyanate method ( Sambrook and Russell, 2001 ). Poly (A + ) RNA was purified from the total RNA through oligo (dT) cellulose chromatography using the mRNA isolation kit (Amersham Pharmacia Biotech, Asia pacific Ltd, Quarry Bay, Hong Kong). cDNA library was constructed using PCR select cDNA subtractive hybridization kit (Clontech, Mountain View, CA, USA) employing 2 ॖg mRNA each of control and PEG-stressed plants. PCR products were ligated into pGEM-T Easy vector (Promega Corporation, Madison, WI, USA) using rapid ligation kit (Fermentas) and were transformed into E. coli (Top10) cells ( Sambrook and Russell, 2001 ). Cloned cDNA fragments were sequenced using automated DNA sequencer and homology search of sequences at nucleotide and protein levels were analyzed using NCBI BLAST and ExPASy tools. Multiple sequence alignment was performed employing CLUSTALW ( http://align.genome.jp/ ). RACE PCR Total RNA was isolated from root and leaf tissues of four-week old 20% PEG-stressed pigeonpea plants (−1.01 ± 0.02 Mpa) as described earlier, and cDNA was synthesized using Clontech Marathon cDNA amplification kit. RACE PCR was carried out employing primers, 5′-CACCCACCAGTCCCAAAGCCACC-3′ (gene specific) and 5′-CCATCCTAATACGACTCACTATAGGGC-3′ (adaptor specific); 5′-CTCAATGA GAGAGCAGCATGGGG-3′ (gene specific) and 5′-CCATCCTAATACGACTCACTAT AGGGC-3′ (adaptor specific), to extend the 5′ and 3′-regions of the CcHyPRP partial clone (Acc. No. CK394831 ). The amplified fragments were eluted and ligated in pGEM-T Easy vector (Promega) and transformed into E. coli cells, and the recombinant clones were subjected to DNA sequencing. Southern blot analysis Genomic DNA (15 ॖg), isolated from pigeonpea plants by CTAB method ( Saghai-Maroof et al. , 1984 ), was digested independently with Bam HI, Eco RI, Hind III and Sal I restriction enzymes. The digested DNA was resolved on 0.8% agarose gel along with lambda Hind III marker, and was transferred to the positively charged nylon membrane as per the manufacturer’s instructions (Amersham Pharmacia Biotech Asia pacific Ltd, Quarry Bay, Hong Kong). Southern blot analysis was performed according to Sambrook and Russell (2001) . The CcHyPRP coding region was radiolabelled with ॅ-32P dCTP using Ready to Go DNA labelling beads (Amersham Pharmacia Biotech) and was used as a probe. Northern blot analysis Total RNAs (15 ॖg), isolated from pigeonpea plants treated independently with ABA (20 and 50 ॖM) for 12 h; PEG (20%) and NaCl (1 m ) for 1, 3 and 6 h; Cold (4°C) for 1 and 3 h; and heat (42°C) for 1 and 2 h, were resolved on denaturing agarose gel (1.5% Agarose, 2.2 M formaldehyde and 1× MOPS) along with the RNA from control (unstressed) plants. The separated RNAs were transferred to the positively charged nylon membrane and northern blot was performed as described ( Sambrook and Russell, 2001 ). The CcHyPRP coding region was radiolabelled as described above and was used as a probe. Transformation of yeast ( Saccharomyces cerevisiae ) Saccharomyces cerevisiae strain INVSc 1 was used as a host to test the function of CcHyPRP at cellular level. The coding region of CcHyPRP was ligated at Bam HI and Eco RI sites of PYES2/NTC shuttle vector containing GAL promoter and transformed into the INVSc1 by lithium acetate method (Invitrogen Corporation, Carlsbad, CA, USA). An empty vector (PYES2/NTC) was introduced into INVSc1 to serve as the control. Transformants were selected on SC medium lacking uracil (SC-U). Yeast cells expressing CcHyPRP along with the control cells were grown on YPD solid medium (1% yeast extract + 2% peptone + 2% dextrose). Stress experiments in yeast Stress tolerance of yeast cells, harbouring pYES2NTC- CcHyPRP /pYES2NTC, were analysed independently under different stress conditions of PEG, NaCl, LiCl and temperature. Recombinant yeast cells were propagated in the induction medium (SC–U medium containing 2% galactose) for 12 h, density was adjusted to OD 2.0 followed by serial dilutions, and were cultured on YPD solid medium containing PEG (20%)/LiCl (100 mM)/NaCl (1 M) at 30°C. Cultures were also subjected to 4°C and 42°C to assess cold and heat tolerance, respectively. All the cultures were incubated for 48 h for recording the data. Construction of plant expression cassettes and transformation of Arabidopsis Arabidopsis rd29A promoter ( AY973635 ) was amplified by employing the primers 5′-GCAAGCTTCGACTCAAAACAAACTTACG-3′ and 5′-GCGGATCCAA TCAAACCCTTATTCCTG-3′. PCR amplified product of the promoter was cloned at Hind III and Bam HI sites in pBI121 vector by replacing CaMV35S promoter. Coding region of CcHyPRP gene was cloned into pBI121 at Bam HI and Sac I, driven by either CaMV 35S or rd29A promoter. The pBI121 vector containing CcHyPRP and npt II expression units were mobilized into EHA105 strain of Agrobacterium through triparental mating. Transformation of A. thaliana was carried out using the modified vacuum infiltration method ( Bechtold and Pelletier, 1998 ). Transformed seedlings were selected on MS medium supplemented with kanamycin (50 ॖg/mL). Molecular analysis of transgenic plants PCR analysis PCR was carried out using the genomic DNA isolated from the kanamycin tolerant plants. DNA from the untransformed plants was used as a negative control and plasmid DNA of pBI 35S npt II- 35S CcHyPRP /pBI 35S npt II- rd29A CcHyPRP were used as positive controls. For PCR, plasmid DNA (10 ng) and genomic DNA (50 ng) were used as templates. PCR analyses were performed employing CcHyPRP gene specific primers, 5′-CGCGCCATGGCTTCCAAGGCTGCACTC-3′ and 5′-CCGCGCGGATCCTTAAGC GCAGATGAAATC-3′. The reaction mixture, containing template, primers, buffer, dNTPS and Taq DNA polymerase, was subjected to initial denaturation (94°C) for 5 min; followed by repeated denaturation (94°C) for 45 s, annealing (60°C) for 45 s, and elongation (72°C) for 1 min for a total of 35 cycles. Final elongation step was carried out at 72°C for 10 min. Amplified PCR products were analysed by gel electrophoresis on 1.0% agarose gel. RT-PCR analysis Total RNA was isolated independently from transgenic and untransformed plants using TRIZOL method (Invitrogen). RT PCR analysis was carried out employing CcHyPRP gene specific primers, and the reaction was set to 50 ॖL containing Tris-HCl (10 mM), KCl (50 mM), MgCl 2 (1.5 mM), dNTPs (200 ॖM each), Taq DNA Polymerase (1.5 units), MMLV reverse transcriptase (2 units), total RNA (∼1.0 ॖg), forward primer (10 pM), and reverse primer (10 pM). The reaction mixture was incubated at 50°C for 30 min and PCR was carried out as described above. Amplified PCR products were analysed by the gel electrophoresis on 1.0% agarose gel. Stress tolerance studies in Arabidopsis thaliana Homozygous T3 seedlings of transgenic Arabidopsis were used in various stress tolerance experiments. Ten-day old transgenic and wild-type seedlings were transferred to MS medium supplemented independently with PEG (15%)/NaCl (200 mM)/mannitol (300 mM)/LiCl (15 mM), and were allowed to grow for 7 day. For heat stress, seedlings were grown in incubator initially at 37°C for 1½ h followed by 42°C for 2 h. To test the effect of cold, seedlings were transferred to incubator set at 4°C for 4 day. Culture plates were placed vertically in the growth chamber and root length was measured from all the seedlings (15) before and after stress treatments. Stressed seedlings were allowed to recover for 15 day in the growth chamber under normal conditions, and data were recorded on plant survival rate, root length and total biomass. Plant biomass was estimated based on fresh weight. In each treatment, 15 seedlings were used and experiments were replicated three times. Statistical analysis Mean value, standard error and t -test were computed with the help of pre-loaded software in excel, programmed for statistical calculations. http://www.Physics.csbsju.edu/stats/t-test.html . http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Biotechnology Journal Wiley

Expression of pigeonpea hybrid‐proline‐rich protein encoding gene (CcHyPRP) in yeast and Arabidopsis affords multiple abiotic stress tolerance

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Abstract

Introduction Abiotic stresses imposed by drought, salinity and extreme temperatures act as major impediments and pose serious threat to the growth and productivity of diverse crop plants with devastating socio-economic consequences. These stresses, in general, are characterized by dehydration resulting from the decreased availability of water to plant cells. Different plants, upon exposure to numerous abiotic stresses, were found to show a wide range of responses at molecular, cellular and whole plant levels ( Greenway and Munns, 1980 ; Zhu et al. , 1997 ; Yeo, 1998 ; Hasegawa et al. , 2000 ). Concomitant occurrence of multiple abiotic stresses, as compared with individual stresses, invariably proved detrimental to the plants grown under field conditions. However, not much is known about the molecular mechanisms underlying acclimation of plants to different combinations of more than one stress ( Rizhsky et al. , 2004 ). To tide over different environmental conditions, plant species have evolved specific adaptive mechanisms and exhibit wide variation in their ability to withstand abiotic stresses, owing to their inherent genetic plasticity ( Bartels and Ramanjulu, 2005 ; Yamaguchi-Shinozaki and Shinozaki, 2006 ). Predictions made from climate models point to the occurrence of abrupt fluctuations in weather conditions triggered by the long-term effects of global warming ( Salinger et al. , 2005 ; Cook et al. , 2007 ). To ensure sustainable crop productivity, it is imperative to design and evolve improved versions of crop plants that can endure the detrimental effects of changing environmental factors. In this context, it is mandatory to identify novel candidate genes with multiple stress tolerance by accessing the vast gene pools of diverse taxa. In model plants, significant information has been generated on biochemical and molecular aspects of gene regulation under different abiotic stress conditions. Drought, salinity, extreme temperatures and oxidative stress result in complex stimuli of varied nature that are often interconnected, and hence these stresses might induce similar damages at the cellular level ( Rodriguez et al. , 2005 ). Furthermore, stress responses of plants to various abiotic factors have been found difficult to dissect, as defense responses require regulatory changes for activation of multiple genes involved in different metabolic pathways ( Bohnert et al. , 2006 ). Functions of diverse plant genes that respond to abiotic stresses have been elucidated at physiological, biochemical and molecular levels ( Hasegawa et al. , 2000 ; Shinozaki and Yamaguchi-Shinozaki, 2000 ; Zhu, 2002 ). Plant genes involved in the signalling and regulatory pathways of stress response ( Saijo et al. , 2000 ; Shinozaki et al. , 2003 ; Kim et al. , 2007 ; Chen et al. , 2008 ), genes which encode proteins conferring stress tolerance ( Sunkar et al. , 2003 ; Nelson et al. , 2007 ), and genes coding for enzymes mediating metabolic pathways for synthesis of specific stress-responsive metabolites ( Kavi Kishore et al. , 1995 ; Park et al. , 2004 ), have been deployed for production of transgenic plants bestowed with abiotic stress tolerance. Plant hybrid-proline-rich proteins (HyPRPs) are putative cell wall proteins consisting of a repetitive proline-rich (PR) N-terminal domain and a conserved eight-cysteine-motif (8 CM) C-terminal domain ( Jose-Estanyol et al. , 2004 ; Dvorakova et al. , 2007 ). Proteins with 8 CM have been classified into different families, viz., proline-rich proteins, lipid transfer proteins (LTPs), 2S albumins and amylase/trypsin inhibitors, based on their functions during growth and development of plants ( Jose-Estanyol et al. , 2004 ). Also, based on the presence of a proline-rich domain and a secretory signal, HyPRPs were designated as a group of secreted structural cell wall proline-rich proteins ( Jose and Puigdomenech, 1993 ; Jose-Estanyol and Puigdomenech, 2000 ). In diverse plant species, HyPRPs were found associated with different abiotic and biotic stress conditions. In the salt tolerant Medicago sativa , a proline-rich protein encoding gene, MsPRP2 , was amply induced under drought conditions ( Deutch and Winicov, 1995 ). Cold tolerant alfalfa, as compared with cold sensitive plants, revealed higher levels of transcripts of a cold regulatory MSACIC gene ( Castonguay et al. , 1994 ). Transcripts of Brassica napus BNPRP gene were specifically induced when the plants were subjected to cold stress ( Goodwin et al. , 1996 ). In soybean, the expression profile of SbPRP gene was modulated by ABA, internal circadian rhythm, as well as abiotic and biotic stress factors ( He et al. , 2002 ). Arabidopsis plants with knocked down EARLI 1 genes, which code for HyPRPs, showed an increased freezing-induced cellular damage as compared with the wild-type plants ( Zhang and Schlappi, 2007 ). Pigeonpea ( C. cajan L) is a well known grain legume crop of the tropical and subtropical regions of the world, which grows well in hot and humid climates. It has an excellent deep root system with profuse laterals and is widely known as one of the most drought, salinity and alkalinity tolerant crops among legumes ( Nene and Sheila, 1990 ). To isolate stress-inducible genes, a highly drought tolerant pigeonpea variety has been used as a potential source of stress-tolerance genes. In this study, as a part of our ongoing programme, we have isolated a stress-responsive Cajanus cajan hybrid-proline-rich protein encoding gene ( CcHyPRP ) from the PCR-subtractive cDNA library. Over-expression of CcHyPRP in yeast cells conferred significant tolerance to multiple abiotic stresses. Similarly, transgenic Arabidopsis plants expressing CcHyPRP disclosed explicit tolerance against different abiotic stress conditions. Results Characterization of Cajanus cajan hybrid-proline-rich protein gene ( CcHyPRP ) A partial cDNA clone of 309 bp (Acc.No. CK394831 ) was obtained from the cDNA library of pigeonpea plants subjected to 20% PEG stress (−1.01 ± 0.02 Mpa), employing PCR-based cDNA subtraction. A full-length cDNA clone of 731 bp with a 5′- and 3′-untranslated region was recovered by 5′- and 3′-RACE PCR; it codes for a polypeptide of 131 amino acids including a signal peptide of 22 amino acids ( Figure 1 ). The CcHyPRP contained maximum number of leucines (21) followed by prolines (16), besides a proline-rich domain in the N-terminal region and a cysteine-rich hydrophobic domain in the C-terminal region. It was hence designated as the Cajanus cajan hybrid-proline-rich protein encoding gene ( CcHyPRP ). The CcHyPRP showed maximum similarity of ∼84% with that of Pvr5 of Phaseolus vulgaris ( AAC49369 ) followed by ∼68% with glycine-rich protein of Nicotiana tabacum ( BAA95941 ), ∼67% with SbPRP of Glycine max ( AAF78903 ), ∼58% with LTP of Capsicum annuum ( ABQ88334 ), ∼51% with CLCT protein of Arabidopsis thaliana ( AAM63191 ) and ∼47% with extensin-like protein of Glycine max ( AAM75351 ) ( Figure 1 ). To decipher the nature of CcHyPRP , the pigeonpea genomic DNA was digested independently with Bam HI, Eco RI, Hind III and Sal I enzymes, and was probed with CcHyPRP coding sequence. Southern analysis revealed single hybridization signals of varied size ranging from >3 Kb to ∼9 Kb ( Figure 2 ). Northern blot analysis of the RNA isolated from pigeonpea plants treated with NaCl (1 M)/PEG (20%)/heat (42°C)/cold (4°C)/ABA (20 & 50 ॖM) and untreated plants, revealed increased accumulation of CcHyPRP transcripts in the stressed plants as compared with the unstressed controls ( Figure 3a–e ). In various stress treatments, higher levels of CcHyPRP transcript accumulations were detected in the roots compared with the leaves of pigeonpea plants. Expression of CcHyPRP gene in yeast ( Saccharomyces cerevisiae ) confers abiotic stress tolerance Yeast system was employed to assess the effect of CcHyPRP protein against heat, salinity and osmotic stress. Yeast cells containing CcHyPRP, under the regulation of GAL promoter, expressed a polypeptide of ∼14 kDa which was absent in the cells transformed with the vector (pYES2/NTC) alone (Figure S1). Yeast cells harbouring pYES2/NTC- CcHyPRP along with the control (pYES2/NTC) were subjected to different stresses caused by PEG, NaCl, LiCl, cold (4°C) and heat (42°C). LiCl is commonly 100 mM LiCl could induce both salt and osmotic stresses. Under normal (stress-free) conditions, the growth pattern of pYES2/NTC- CcHyPRP yeast was found similar to that of control yeast containing the vector alone ( Figure 4 ). Yeast cells expressing CcHyPRP showed normal growth under 20% PEG/1.0 mM NaCl/100 mM LiCl/heat (42°C) stress as compared with the negligible growth observed in the control yeast ( Figure 4 ). On the other hand, both control and CcHyPRP -yeast cells, subjected to cold stress (4°C) failed to show any growth even after 48 h of culture. Overexpression of CcHyPRP gene in Arabidopsis imparts multiple stress tolerance To evaluate the functional role of CcHyPRP, Arabidopsis was transformed with CcHyPRP driven by CaMV 35S/rd 29A promoters ( Figure 5a,b ). PCR analysis of the DNA isolated from kanamycin tolerant Arabidopsis plants, using CcHyPRP primers, revealed a ∼400 bp amplified fragment, while no such band was observed in the wild-type plants (data not shown). RT-PCR analysis, using the total RNA from stressed rd29A- CcHyPRP lines and unstressed CaMV35S- CcHyPRP lines as well as wild-type plants, employing CcHyPRP- specific primers, disclosed the presence of a ∼400 bp amplicon in the transgenic lines ( Figure 5c ). Tolerance exhibited by the homozygous (T3) transgenic lines, viz., CS1 and CS2 (CaMV35S- CcHyPRP ) and CR1 and CR2 (rd29A- CcHyPRP ), to different abiotic stresses was analysed by subjecting them to mannitol, PEG, NaCl, LiCl, heat (42°C) and cold (4°C) stress conditions. Under unstressed conditions, CR1 and CR2 lines showed similar growth pattern as that of wild-type plants. Whereas, CS1 and CS2 lines revealed noticeable decreases in plant growth as compared with the wild-type plants ( Figure 6 ). Transgenic lines grown under 300 mM mannitol/15% PEG/200 mM NaCl/15 mM LiCl/heat (42°C) stress, in comparison with the wild-type plants, showed observable increases in plant growth ( Figure 6 ). However, both CR1 and CR2 lines, similar to wild-type plants, failed to withstand the heat stress ( Figure 6 ). Effect of CcHyPRP on plant survival, root growth and biomass under stress conditions Transgenic lines, grown under different stress conditions, exhibited higher plant survival rate, increased root length and enhanced plant biomass as compared with the wild-type plants. CcHyPRP -transgenics subjected to mannitol (300 mM) showed higher (85.00%–88.33%) survival rate compared with the wild-type plants (43.33%) ( Figure 7a ). Transgenic lines also exhibited enhanced (96.00%–144.50%) biomass with substantial increases (184.62%–215.30%) in root length when compared with wild-type plants ( Figure 7b,c ). Similarly, transgenic lines subjected to PEG (15%) revealed higher (85.00%–88.30%) survival rates compared with 43.30% survival observed in wild-type plants ( Figure 7a ). Furthermore, compared with control plants, transgenics disclosed enhanced (111.30%–200.00%) plant biomass with significant increases (138.40%–166.90%) in root length ( Figure 7b,c ). Transgenic plants grown under salt (200 mM) stress exhibited higher (85.00%–88.33%) plant survival while wild-type plants showed 13.30% survival ( Figure 7a ). Transgenics also revealed marked increases (524.60%–671.20%) in plant biomass and root length (286.60%–420.00%) as compared with the wild-type plants ( Figure 7b,c ). Likewise, transgenic lines subjected to LiCl (15 mM) showed higher (85.00%–91.60%) survival rate compared with 30.00% survival observed in the wild-type plants ( Figure 7a ). Transgenic lines also exhibited increased (124.60%–251.30%) biomass along with significant increases (178.10%–206.80%) in the root length when compared with the control plants ( Figure 7b,c ). Under heat (42°C) stress, CS1 and CS2 transgenics showed higher (93.33%–95.00%) plant survival when compared with CR-transgenics (34.00%) and wild-type (33.33%) plants ( Figure 7a ). CS-transgenics also revealed significant increases (156.50%–164.00%) in total biomass and root length (212.50%–225.00%) compared with that of CR-transgenics and wild-type plants ( Figure 7b,c ). However, both CS- and CR-transgenics and wild-type plants, when subjected to cold stress (4°C), failed to show observable differences among them for plant survival, root growth and total biomass (data not shown). Discussion Extensive damage caused to the global agriculture by various abiotic stresses warrants evolvement of crop plants with enhanced tolerance to different environmental factors. To develop customized crop plants with inbuilt tolerance for multiple stresses, efforts should be made to clone novel stress-tolerance genes from diverse species acclimated to various adverse conditions. Pigeonpea is a major pulse crop and is endowed with the innate ability to resist adverse environmental conditions. In this study, a stress-responsive CcHyPRP gene has been isolated from the cDNA library of pigeonpea plants subjected to drought stress. The functional role of CcHyPRP in imparting multiple abiotic stress tolerance has been established by its overexpression in yeast and Arabidopsis systems. The deduced amino acid sequence of CcHyPRP showed similarity to LTP/alpha amylase inhibitor/seed storage family proteins ( Figure 1 ). The proteins belonging to this family were characterized by the presence of an eight-cysteine-motif (8CM) at the C-terminal end ( Jose-Estanyol et al. , 2004 ). The CcHyPRP contained a proline-rich domain and a hydrophobic cysteine-rich domain besides a signal peptide at the N-terminal end. The conserved nature of spacing between leucine residues noticed in CcHyPRP indicates their plausible involvement in the formation of leucine-zipper motifs as was observed in various other plant HyPRPs ( Subramaniam et al. , 1994 ; He et al. , 2002 ). Different plant proteins, containing 8CM as well as signal peptide, have been found to differ in their functionality ( Jose-Estanyol et al. , 2004 ). In diverse plant species, involvement of HyPRP proteins has been implicated under both abiotic and biotic stress conditions ( Ryan, 1990 ; Molina and Garcı′a-Olmedo, 1993 ; Showalter, 1993 ; Yubero-Serrano et al. , 2003 ; Jang et al. , 2004 ; Zhang and Schlappi, 2007 ). Different proline-rich proteins (PRPs) of plant-cell walls were found to act in various processes of plant growth and development in a tissue- and cell-specific manner ( Showalter and Rumeau, 1990 ; Jose-Estanyol et al. , 1992 ). Other classes of proline-rich cell wall structural proteins containing 8CM sequences (HyPRPs), were found to express in a tissue-specific manner, and were induced by specific stresses or hormones, but their functions were not clearly determined ( Jose-Estanyol and Puigdomenech, 2000 ). Southern blot analysis of the genomic DNA digested with different restriction enzymes, using CcHyPRP as a probe, disclosed single hybridizable bands of varied size, implying single-copy nature of CcHyPRP gene and the absence of paralogs in the pigeonpea genome ( Figure 2 ). Northern blot analysis revealed intense hybridizable signals in PEG-/salt-/heat-stressed plants and moderate signals in cold-stressed plants compared with the weak signals in unstressed plants, thus suggesting the stress-responsive nature of CcHyPRP gene ( Figure 3 ). Accumulation of significantly higher levels of CcHyPRP transcripts under PEG, NaCl, heat and cold stress, denotes that CcHyPRP is regulated by a stress-inducible promoter in the pigeonpea. Presence of increased levels of CcHyPRP transcripts in ABA-treated plants indicates that CcHyPRP is induced by ABA, thereby affirming its crucial role in the abiotic stress tolerance. In different stress treatments, higher levels of transcript accumulations were observed in the roots as compared with the leaves. Similar results were reported in P. vulgaris, where pvr 5 was found to show preferential expression in roots with maximum accumulation of transcripts in the cortical root meristem cells ( Choi et al. , 1996 ). An overview of northern profiles authenticates that CcHyPRP expression is highly modulated by various environmental conditions. In diverse plant species, expression of HyPRPs was induced by different abiotic and biotic stress conditions. In alfalfa, expression of MsPRP2 gene was induced by water-deficit in the salt tolerant plants ( Deutch and Winicov, 1995 ), while MsACIC was expressed in the cold tolerant plants ( Castonguay et al. , 1994 ). The BNPRP gene of B. napus ––encoding a putative cell wall-plasma membrane linker-protein––was highly expressed under low temperature conditions ( Goodwin et al. , 1996 ). Similarly, expression of soybean SbPRP gene was up-regulated by salicylic acid, viral infection, salt and drought stress conditions ( He et al. , 2002 ). The CcHyPRP gene was transformed into the yeast system to analyse its functionality, owing to the ability of yeast to perform various post-translational modifications ( Cereghino and Cregg, 2000 ). CcHyPRP -yeast transformants revealed marked superiority in their growth rate in comparison with the control yeast as evidenced by the colony size ( Figure 4b ), confirming that the stress-responsive CcHyPRP imparts substantial tolerance at the cellular level for multiple abiotic stresses. Over-expression of Arabidopsis DBF2 kinase ( Lee et al. , 1999 ), trehalose-6P synthase of yeast ( Soto et al. , 1999 ), Arabidopsis AtGSK ( Piao et al. , 1999 ) and rice rHsp 90 ( Liu et al. , 2006 ) genes in yeast cells conveyed significant tolerance against different abiotic stresses. Yeast cells expressing Arabidopsis EARLI1 genes coding for HyPRP exhibited higher rates of freezing survival than the control cells ( Zhang and Schlappi, 2007 ). Arabidopsis plants were transformed with CcHyPRP gene, driven by constitutive CaMV35S or by stress-inducible rd29A promoter, to validate its role in bestowing abiotic stress tolerance. Four transgenic Arabidopsis lines expressing CcHyPRP , when subjected to multiple stresses imposed by PEG/mannitol/NaCl/LiCl/heat, disclosed healthy seedlings with profuse root system in contrast to stunted and debilitated seedlings observed in the wild-type plants ( Figure 5 ). However, the vulnerability of CR1 and CR2 transgenics to heat stress may be attributed to the absence of heat-shock-inducible element(s) in the rd29A promoter. Furthermore, CcHyPRP -transgenics exhibited conspicuous tolerance against multiple abiotic stresses as evidenced by increased seedling survival, profuse root growth and enhanced plant biomass ( Figure 6 & Figure 7 ). These results amply demonstrate the unequivocal involvement of CcHyPRP in affording abiotic stress tolerance at the whole plant level. Deploying various candidate genes accessed from diverse gene pools, crop plants have been genetically engineered against different abiotic stresses. Ectopic expression of DREB1a transcription factor in Arabidopsis furnished increased tolerance to drought, salinity and freezing stress ( Kasuga et al. , 1999 ). Similarly, expression of Arabidopsis CBF1 ( DREB1b ) in transgenic tomato provided ample tolerance against drought, chilling and oxidative stress ( Hsieh et al. , 2002 ). Transgenic tobacco, expressing rice zinc-finger protein gene (OSISAP1) , conferred distinct tolerance against cold, dehydration and salt stress ( Mukhopadhyay et al. , 2004 ). Likewise, expression of calcium-dependent protein kinase ( OsCDPK7 ) gene in rice conveyed increased tolerance to cold, salt and drought stresses ( Saijo et al. , 2000 ). Tobacco plants expressing AtMDAR1 gene exhibited tolerance against ozone, salt and PEG stresses ( Eltayeb et al. , 2007 ). Similarly, expression of pea MnSOD gene in transgenic rice accorded enhanced tolerance against drought and oxidative stresses ( Wang et al. , 2005 ). Transgenic Arabidopsis , expressing CcHyPRP under CaMV35S promoter, exhibited diminished characteristics such as stunted plants with tiny leaves and undersized siliques, presumably owing to the constitutive expression of CcHyPRP protein. Use of strong constitutive promoters like CaMV35S and rice actin were shown to induce undesirable phenotypic effects upon over-expression of stress-responsive genes ( Kasuga et al. , 1999 ; Mahalakshmi et al. , 2006 ). The various negative effects observed in the transformants obtained with CaMV35S promoter, under unstressed conditions, are attributable to the energy-drain caused by the constitutive expression of transgenes. Conversely, CcHyPRP -transgenics driven by the stress-inducible rd29A revealed similar stress-tolerance as that of CaMV35S-lines without any sort of negative effects on the plant morphology, implying that stress-inducible promoters are preferable for development of stress tolerant transgenic plants. The pigeonpea CcHyPRP contained a highly conserved 8CM domain and resembled proline-rich proteins, LTPs, 2S albumins, extensins and trypsin inhibitors, involved in the storage of proteins and minerals, protection against pathogens, enzyme inhibition and lipid transfer mechanisms ( Jose-Estanyol et al. , 2004 ). Earlier, it was proposed that different 8CM proteins have evolved by the insertion of different sets of amino acid residues within the 8CM domain to assume varied functions. For instance, variations such as the presence of an internal hydrophobic cavity in LTPs; addition of a proline-rich domain to the hydrophobic 8CM of HyPRPs; proline-/glutamine-rich domain in prolamins; and sequences that promote loop proteolytic cleavage in 2S-albumins, have been observed in different 8CM proteins ( Jose-Estanyol et al. , 2004 ). As Arabidopsis transgenic lines expressing CcHyPRP exhibited marked tolerance against different abiotic stresses, we envisage a broad mechanism of action analogous to that of 8CM proteins operating at the cellular level for protection of plants against multiple stresses. However, additional investigations are needed for an in-depth analysis of CcHyPRP’s function in abiotic stress tolerance. To sum up, a multifunctional CcHyPRP gene has been isolated from the subtracted cDNA library constructed from the PEG-stressed pigeonpea plants. The CcHyPRP disclosed single-copy nature and was induced in pigeonpea by different abiotic stress conditions. Expression of CcHyPRP in yeast and Arabidopsis imparted substantial tolerance to PEG, mannitol, NaCl, LiCl and high temperature. The overall results affirm that the Cajanus stress-responsive gene confers high-level multiple stress tolerance in the heterologous systems. The pigeonpea CcHyPRP, by virtue of its surpassing tolerance against drought, salinity and heat stress, seems promising as a prime candidate gene for stress tolerance. As such, CcHyPRP may be deployed in the genetic enhancement of diverse crop plants for multiple abiotic stress tolerance. Experimental procedures Plant material and growth conditions Drought tolerant pigeonpea accession ICP 8744, procured from the ICRISAT, Hyderabad (India), was multiplied and used in this investigation. Seeds were surface sterilized with 0.1% mercuric chloride for 5 min followed by three washes, each for 10 min, in sterile distilled water under aseptic conditions. The sterilized seeds were germinated in pots and plants were maintained in the green house at 28 ± 2.0 °C. Seeds of Arabidopsis thaliana ecotype Columbia, obtained from the CCMB, Hyderabad, were treated with ethanol for 10 min followed by 0.05% mercuric chloride for 3 min. Later, these seeds were washed thoroughly six times with sterile water. Sterilized seeds, after stratification for 64 h at 4°C, were grown on MS medium (MS salts + 2% sucrose + 0.8% agar) or synthetic soil mix at 200 °C with 16 h photoperiod (long day) under fluorescent light (7000 lux at 20 cm) in Conviron growth chamber (Model TC16). Measurement of water potential Water potential of the 4-week old control (unstressed) and Polyethylene glycol (PEG 6000)-stressed pigeonpea plants were measured using the pressure chamber method of Scholander et al. (1964) . Synthesis of cDNA library Total RNA was isolated from the four-week old unstressed (−0.49 ± 0.02 Mpa) and 20% PEG 6000-stressed (−1.01 ± 0.02 Mpa) pigeonpea plants by guanidinium thiocyanate method ( Sambrook and Russell, 2001 ). Poly (A + ) RNA was purified from the total RNA through oligo (dT) cellulose chromatography using the mRNA isolation kit (Amersham Pharmacia Biotech, Asia pacific Ltd, Quarry Bay, Hong Kong). cDNA library was constructed using PCR select cDNA subtractive hybridization kit (Clontech, Mountain View, CA, USA) employing 2 ॖg mRNA each of control and PEG-stressed plants. PCR products were ligated into pGEM-T Easy vector (Promega Corporation, Madison, WI, USA) using rapid ligation kit (Fermentas) and were transformed into E. coli (Top10) cells ( Sambrook and Russell, 2001 ). Cloned cDNA fragments were sequenced using automated DNA sequencer and homology search of sequences at nucleotide and protein levels were analyzed using NCBI BLAST and ExPASy tools. Multiple sequence alignment was performed employing CLUSTALW ( http://align.genome.jp/ ). RACE PCR Total RNA was isolated from root and leaf tissues of four-week old 20% PEG-stressed pigeonpea plants (−1.01 ± 0.02 Mpa) as described earlier, and cDNA was synthesized using Clontech Marathon cDNA amplification kit. RACE PCR was carried out employing primers, 5′-CACCCACCAGTCCCAAAGCCACC-3′ (gene specific) and 5′-CCATCCTAATACGACTCACTATAGGGC-3′ (adaptor specific); 5′-CTCAATGA GAGAGCAGCATGGGG-3′ (gene specific) and 5′-CCATCCTAATACGACTCACTAT AGGGC-3′ (adaptor specific), to extend the 5′ and 3′-regions of the CcHyPRP partial clone (Acc. No. CK394831 ). The amplified fragments were eluted and ligated in pGEM-T Easy vector (Promega) and transformed into E. coli cells, and the recombinant clones were subjected to DNA sequencing. Southern blot analysis Genomic DNA (15 ॖg), isolated from pigeonpea plants by CTAB method ( Saghai-Maroof et al. , 1984 ), was digested independently with Bam HI, Eco RI, Hind III and Sal I restriction enzymes. The digested DNA was resolved on 0.8% agarose gel along with lambda Hind III marker, and was transferred to the positively charged nylon membrane as per the manufacturer’s instructions (Amersham Pharmacia Biotech Asia pacific Ltd, Quarry Bay, Hong Kong). Southern blot analysis was performed according to Sambrook and Russell (2001) . The CcHyPRP coding region was radiolabelled with ॅ-32P dCTP using Ready to Go DNA labelling beads (Amersham Pharmacia Biotech) and was used as a probe. Northern blot analysis Total RNAs (15 ॖg), isolated from pigeonpea plants treated independently with ABA (20 and 50 ॖM) for 12 h; PEG (20%) and NaCl (1 m ) for 1, 3 and 6 h; Cold (4°C) for 1 and 3 h; and heat (42°C) for 1 and 2 h, were resolved on denaturing agarose gel (1.5% Agarose, 2.2 M formaldehyde and 1× MOPS) along with the RNA from control (unstressed) plants. The separated RNAs were transferred to the positively charged nylon membrane and northern blot was performed as described ( Sambrook and Russell, 2001 ). The CcHyPRP coding region was radiolabelled as described above and was used as a probe. Transformation of yeast ( Saccharomyces cerevisiae ) Saccharomyces cerevisiae strain INVSc 1 was used as a host to test the function of CcHyPRP at cellular level. The coding region of CcHyPRP was ligated at Bam HI and Eco RI sites of PYES2/NTC shuttle vector containing GAL promoter and transformed into the INVSc1 by lithium acetate method (Invitrogen Corporation, Carlsbad, CA, USA). An empty vector (PYES2/NTC) was introduced into INVSc1 to serve as the control. Transformants were selected on SC medium lacking uracil (SC-U). Yeast cells expressing CcHyPRP along with the control cells were grown on YPD solid medium (1% yeast extract + 2% peptone + 2% dextrose). Stress experiments in yeast Stress tolerance of yeast cells, harbouring pYES2NTC- CcHyPRP /pYES2NTC, were analysed independently under different stress conditions of PEG, NaCl, LiCl and temperature. Recombinant yeast cells were propagated in the induction medium (SC–U medium containing 2% galactose) for 12 h, density was adjusted to OD 2.0 followed by serial dilutions, and were cultured on YPD solid medium containing PEG (20%)/LiCl (100 mM)/NaCl (1 M) at 30°C. Cultures were also subjected to 4°C and 42°C to assess cold and heat tolerance, respectively. All the cultures were incubated for 48 h for recording the data. Construction of plant expression cassettes and transformation of Arabidopsis Arabidopsis rd29A promoter ( AY973635 ) was amplified by employing the primers 5′-GCAAGCTTCGACTCAAAACAAACTTACG-3′ and 5′-GCGGATCCAA TCAAACCCTTATTCCTG-3′. PCR amplified product of the promoter was cloned at Hind III and Bam HI sites in pBI121 vector by replacing CaMV35S promoter. Coding region of CcHyPRP gene was cloned into pBI121 at Bam HI and Sac I, driven by either CaMV 35S or rd29A promoter. The pBI121 vector containing CcHyPRP and npt II expression units were mobilized into EHA105 strain of Agrobacterium through triparental mating. Transformation of A. thaliana was carried out using the modified vacuum infiltration method ( Bechtold and Pelletier, 1998 ). Transformed seedlings were selected on MS medium supplemented with kanamycin (50 ॖg/mL). Molecular analysis of transgenic plants PCR analysis PCR was carried out using the genomic DNA isolated from the kanamycin tolerant plants. DNA from the untransformed plants was used as a negative control and plasmid DNA of pBI 35S npt II- 35S CcHyPRP /pBI 35S npt II- rd29A CcHyPRP were used as positive controls. For PCR, plasmid DNA (10 ng) and genomic DNA (50 ng) were used as templates. PCR analyses were performed employing CcHyPRP gene specific primers, 5′-CGCGCCATGGCTTCCAAGGCTGCACTC-3′ and 5′-CCGCGCGGATCCTTAAGC GCAGATGAAATC-3′. The reaction mixture, containing template, primers, buffer, dNTPS and Taq DNA polymerase, was subjected to initial denaturation (94°C) for 5 min; followed by repeated denaturation (94°C) for 45 s, annealing (60°C) for 45 s, and elongation (72°C) for 1 min for a total of 35 cycles. Final elongation step was carried out at 72°C for 10 min. Amplified PCR products were analysed by gel electrophoresis on 1.0% agarose gel. RT-PCR analysis Total RNA was isolated independently from transgenic and untransformed plants using TRIZOL method (Invitrogen). RT PCR analysis was carried out employing CcHyPRP gene specific primers, and the reaction was set to 50 ॖL containing Tris-HCl (10 mM), KCl (50 mM), MgCl 2 (1.5 mM), dNTPs (200 ॖM each), Taq DNA Polymerase (1.5 units), MMLV reverse transcriptase (2 units), total RNA (∼1.0 ॖg), forward primer (10 pM), and reverse primer (10 pM). The reaction mixture was incubated at 50°C for 30 min and PCR was carried out as described above. Amplified PCR products were analysed by the gel electrophoresis on 1.0% agarose gel. Stress tolerance studies in Arabidopsis thaliana Homozygous T3 seedlings of transgenic Arabidopsis were used in various stress tolerance experiments. Ten-day old transgenic and wild-type seedlings were transferred to MS medium supplemented independently with PEG (15%)/NaCl (200 mM)/mannitol (300 mM)/LiCl (15 mM), and were allowed to grow for 7 day. For heat stress, seedlings were grown in incubator initially at 37°C for 1½ h followed by 42°C for 2 h. To test the effect of cold, seedlings were transferred to incubator set at 4°C for 4 day. Culture plates were placed vertically in the growth chamber and root length was measured from all the seedlings (15) before and after stress treatments. Stressed seedlings were allowed to recover for 15 day in the growth chamber under normal conditions, and data were recorded on plant survival rate, root length and total biomass. Plant biomass was estimated based on fresh weight. In each treatment, 15 seedlings were used and experiments were replicated three times. Statistical analysis Mean value, standard error and t -test were computed with the help of pre-loaded software in excel, programmed for statistical calculations. http://www.Physics.csbsju.edu/stats/t-test.html .

Journal

Plant Biotechnology Journal – Wiley

Published: Jan 1, 2010

Keywords: ; ; ; ; ;

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