Plant Signaling & Behavior 7:4, 502–509; April 2012;
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Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance Moumita Ganguly,1 Karabi Datta,1 Aryadeep Roychoudhury,1 Dipak Gayen,1 Dibyendu N. Sengupta2 and Swapan K. Datta1,3,4,* 1
Plant Molecular Biology and Biotechnology Laboratory; Department of Botany; University of Calcutta; Kolkata, India; 2Division of Plant Biology; Bose Institute; Kolkata, India; 3 Indian Council of Agricultural Research; Krishi Bhawan; New Delhi, India; 4Indian Council of Agricultural Research; Krishi Bhawan, New Delhi India
Keywords: abiotic stress, responsive to abscisic acid, salt tolerance, transformation, transgenic rice Abbreviations: DAP, days after pollination; MS, Murashige and Skoog; LEA, late embryogenesis abundant; Rab, responsive to abscisic acid; Rab p, Rab promoter; WT, wild type
We report here the overexpression of Rab16A full length gene (promoter + ORF), from the salt-tolerant indica rice Pokkali, in the salt-susceptible indica rice variety Khitish, via particle bombardment. Molecular analysis of the transgenics revealed stable integration of the transgene upto T2 generation. High level of expression of the transgene (driven by its own stress-inducible promoter), as well as the protein, was detectable in the leaves under simulated salinity stress (250 mM NaCl, 24 h); the expression level being higher than wild type (WT) plants. The Rab16A transcript also increased gradually with seed maturity, with its maximal accumulation at 30 d after pollination (DAP) i.e., fully matured seeds, explaining the protective role of Rab16A gene during seed maturation. Enhanced tolerance to salinity was observed in the plants transformed with Rab16A. The superior physiological performances of the transgenics under salt treatment were also reflected in lesser shoot or root length inhibition, reduced chlorophyll damages, lesser accumulation of Na+ and reduced loss of K+, increased proline content as compared with the WT plants. All these results indicated that the overproduction of RAB16A protein in the transgenics enable them to display enhanced tolerance to salinity stress with improved physiological traits. Our work demonstrates the profound potential of Group 2 LEA proteins (to which RAB16A belongs to) in conferring stress tolerance in crop plants through their genetic manipulation.
Introduction Most crops growing under field conditions are often exposed to various abiotic stresses such as high or low temperature, drought and salinity, which influence plant metabolism directly or indirectly, thereby affecting plant growth, development and finally agricultural productivity and hence food security. The adverse effects of salinity stress include cell wall damages, plasmolysis, membrane damages, either that of cell membrane or chloroplast membrane, imbalance of Na+/K+ ratio due to excess Na+ accumulation or K+ efflux, reduction in photosynthesis and overall decline in germination and seedling growth,1 leading ultimately to diminished grain yield. One of the primary effects of salt stress is the dehydration effect, which is associated with the decrease in water potential in the extracellular solution.2 Producing crops tolerant to salinity stress is imperative to run into the growing food demand through sustainable agriculture. This could be achieved either through conventional selection and breeding or through modern molecular biology approaches. Moreover, the magnitude of genetic variation in the gene pools of most important crops is very low. Genetic engineering has certainly opened a new way to overcome crop losses due to salinity
stress prevalent in the agricultural ecosystems. Transcriptomic, proteomic and molecular genetics approaches have identified many stress related genes, which are generally classified into two major groups. One group is involved in signaling cascades, transcriptional control and the degradation of transcripts or proteins, whereas members of the other group function in membrane protection, osmoprotection, as antioxidants and as reactive oxygen species (ROS) scavengers. A systemic regulation of stress responsive genes is necessary for plant adjustment to salinity stress.3 Although molecular responses that are associated with the acclimatization to water and salinity stress are multigenic and often interrelated, there are instances where even the transfer of a single gene in the recipient plant improved stress tolerance. Overexpression of genes encoding biosynthesis of several osmolytes/antifreeze proteins/chaperones/ROS-detoxification enzymes/late embryogenesis abundant (LEA) proteins/transcription factors/protein kinases/ion transporters and channel proteins, either singly or in combination, are altogether considered as multiple strategies to generate salt tolerance in transgenic crops. In recent years, several stress-tolerant transgenic plants have been generated through overexpression of various groups of genes, viz., DREB1C,4 SOD2,5 TPS + TPP fusion,6 OsCDPK77 and
*Correspondence to: Swapan K. Datta; Email:
[email protected] Submitted: 12/19/11; Revised: 02/07/12; Accepted: 02/07/12 http://dx.doi.org/10.4161/psb.19646
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RESEARCH PAPER
OsSDIR1.8 All of them showed markedly enhanced tolerance (responsive to abscisic acid) belongs to the Group 2 LEA protein. to salt stress (and to water deprivation) compared with non The gene Rab16A is ABA-inducible; it is highly expressed in seeds transgenics. Expression of stress-inducible promoters such as as well as in vegetative tissues during salinity, dehydration and rab16A, 4X ABRE, 2X ABRC were also studied in rice.9 cold stress or during exogenous stress or ABA treatments.24 The Although plant species differ in their thresholds for stress promoter of Rab16A possesses both ACGT-containing typical tolerance, most cultivated crop plants are highly sensitive when abscisic acid responsive element (ABRE) called Motif I, as well as they are exposed to long periods of stress. In general, rice shows coupling element (CE)-like sequences called Motif II, which are differences in sensitivity toward excess salinity at various develop- duplicated (Motif IIa and Motif IIb). The salt-tolerant rice mental stages during its life cycle. It is considered relatively varieties have constitutive and higher level of Rab gene/protein tolerant to salinity at the germination stage while the young expression compared with the salt-sensitive varieties, where seedling stage and early reproductive stages, i.e., panicle initiation the expression is only inducible by stress.13 This proves the and pollination are the most salinity-sensitive growth stages, involvement of Rab16A gene in generating salt tolerance in saltdirectly affecting the crop yield.10-12 Moreover, rice exhibits sensitive rice varieties. In the present study, we overexpressed the full length Rab16A genetic diversity in their sensitivity to salt stress. The high yielding indica rice varieties like IR-64, IR-72 and IR-29, along with some gene, from the salt-tolerant rice variety Pokkali, in a salt-sensitive local ones like Khitish, Satabdi, etc. are mostly salt susceptible, and high yielding, local indica rice variety named Khitish, with while the low yielding varieties like Pokkali and Nonabokra are the aim of improving salt tolerance level. The molecular analyses salt tolerant.13 Hence to feed the world, there have been of the transgenics for the transcript or protein accumulation were considerable efforts to develop genetically engineered stress performed, followed by the salt tolerance assay of the transgenics tolerant rice through overexpression of genes conferring salt against the WT, in artificial hydroponic culture. The agronomic tolerance. Availability of the genome sequence of rice has proved performances and several physiological parameters, like shoot and to be an additional benefit to look for the suitable target genes root length, endogenous Na+ and K+ ion chlorophyll content and that can be employed for selected gene manipulation to achieve proline content were compared between the transgenics and WT abiotic stress tolerance in rice. plants, to highlight the overall superior performance of the The acquisition of desiccation tolerance during late stages of transgenics under the imposed salinity stress. seed or embryo development (when desiccation tolerance is acquired) is correlated with the induction of a set of small, highly Results hydrophilic, heat-resistant proteins called late embryogenesis Abundant (LEA) proteins, which constitute around 4% of Confirmation of the T2 transgenics through DNA gel blot the total cellular proteins. During seed maturation, lea gene hybridization. The construct used for bombardment is shown expression is induced by plant stress hormone, abscisic acid schematically in Figure 1A. The results of DNA gel blot hybridiza(ABA).14 However, there are certain lea genes that are induced tion (Fig. 1B) with five PCR-positive plants i.e., T2/3/11, T2/7/3, also in vegetative and reproductive tissues in response to drought, T2/12/1, T2/15/9 and T2/22/3 showed stable introgression of salinity, extreme temperatures or by exogenous ABA,15,16 leading 0.9 Kbp Rab F (Lanes 1–5), as compared against positive control to the hypothesis that these proteins play a role in the adaptation (PC) (Lane 6). of plants to such stress condition. The expression of several groups of lea genes during environmental stress and their role in stress tolerance has been recently reported in several species.2 Based on sequence similarity and conserved domains, LEA proteins have been classified into nine groups.16,17 Among the nine groups of LEA proteins, role of only Group 1, 2 and 3 has been investigated in imparting abiotic stress tolerance. Wheat LEA proteins, PMA1959 (Group I) and PMA80 (Group 2) conferred dehydration tolerance in transgenic rice.18 The Group 3 LEA, HVA1 was successfully introduced in rice19-21 which rendered tolerance against both salt and water deficit conditions via maintaining cell membrane stability. The Group 2 LEA proteins also referred to as dehydrins, include 15-amino acid conservative structure (EKKGIMDKIKEKLPG) at the C-terminus and play an important role in metabolism as molecular chaperones Figure 1. (A) Physical map of the plasmid vector pCAMBIA1200 containing and defending protein structures. Localization of specific Rab F (0.3 Kbp Rab p + 0.6 Kbp ORF) at HindIII and BamHI site. (B) DNA-blot hybridization analyses showing the integration of 0.9 Kbp Rab16A gene, dehydrins at the plasma membrane22 and also on the M represents DNA molecular weight marker, PC represents positive control and endomembranes is consistent with their role in stabilizing NC negative control. 23 cellular structures during dehydration stress. The rice RAB
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Additionally, the fold induction in Rab F transcript was also measured at different stages of seed development. The maximum expression was noted for the transgenic line T2/22/3 at 30 DAP, i.e., in the fully matured seed. Here, the transcript level was induced 16.98-fold compared with 10 DAP. On the other hand, the WT seeds at 30 DAP showed only 3.76-fold inductions in Rab F mRNA with respect to 10 DAP (Fig. 2D). Analysis of Rab F protein accumulation. A protein band of 21 KDa, was detected in the transgenic plants upon salt treatment (250 mM NaCl, 24 h) in all the transgenic lines. Even the untreated transgenics showed high protein expression. The band was hardly detectable in the WT plants even after stress imposition (Fig. 3A), proving that only the transgenic plants were the expressors of RAB16A protein. Performance of T2 transgenics under salinity stress. Monitoring of plant growth continuously for 21 d in 250 mM NaCl showed better performance of the T2 transgenics as compared with the WT plants, which either showed retarded growth wilting as well as chlorosis (Fig. 3B). No significant agronomic differences were observed between WT plants (without stress) and T2 transgenic plants after salinity stress under greenhouse conditions. The T2 transgenic (with stress) plants behaved identical to the WT plants (without stress) in several vital growth parameters tested (Table 1). No detrimental effects on overall physiological performance, growth and development, flowering pattern or seed production was observed due to introgression of Rab16A. Estimation of endogenous Na+ and K+ ions. Salt treated WT plants showed very high accumulation of Na+ and loss of K+ ions was also evident from both shoot and root after salinity stress. The salt-treated WT plants showed 1.5-fold higher accumulation of Na+ in the shoot than the untreated plants. The stressed transgenic plants showed only 1.0–1.3 times accumulation of Na+ as compared with the untreated condition, i.e., lesser Na+ accumulation (Fig. 4A). While the loss of K+ from the shoots of the salt-treated WT plants was 1.8-fold compared with untreated condition, the salt-treated transgenics showed 1–1.2 times loss compared with non stressed plants (Fig. 4B). Similarly in the roots of salttreated WT plants, the accumulation of Na+ ion was 2.3-fold over untreated plants. In the salttreated transgenics, the increase was 1.2–1.4 times than the untreated condition (Fig. 4C). While the loss of K+ in salt-treated transgenics was Figure 2. (A) RT-PCR analyses showing the expression of 0.6 Kbp Rab F in the leaves of T2 transgenics in response to salinity stress (250 mM NaCl, 24 h). “C” represents transgenics 1.1–1.5-fold than untreated plants, the loss was without stress and “S” represents transgenics with 250 mM NaCl stress. (B) qRT-PCR 2.1-fold in stressed WT plants compared with the analysis, showing the fold induction of Rab F expression with salt stress (250 mM NaCl, untreated condition (Fig. 4D). 24 h) treatment and (C) without stress in the leaves of WT and T2 transgenics, (D) fold Chlorophyll content estimation. Salinity stress induction of Rab F expression at different developmental stages of seed maturation in (250 mM NaCl, 24 h) considerably reduced the the T2 transgenics. The expression of genes was determined using tubulin as an internal control. The values were means from three independent series, and the results are chlorophyll content of the WT plants (40%) represented as means ± SE, based on three replicates. The statistical differences at p # 0.05 whereas the transgenic plants suffered chlorophyll have been calculated. loss to a much lower extent, viz., 25%, 21%, 31%,
Rab F expression analysis of T2 transgenic lines. RT-PCR analysis with five T2 transgenic plants (Fig. 2A) showed the accumulation of 0.6 Kbp transcript of Rab F cDNA, particularly in response to NaCl treatment. Although the transcript was also detected in WT plants (which may be because of the fact that the gene is endogenous in rice), its level was much lower even upon NaCl application, as compared with the salt-treated transgenic plants. It is also noteworthy that the inducibility of Rab F in the transgenics was more pronounced upon stress application. In addition, qRT-PCR analyses showed 8.6–18.2-fold induction in Rab F mRNA levels in the transgenic lines compared with the WT plants under salt treated condition (Fig. 2B). Under control condition, the transgenic plants showed 5.05–13.5-fold induction in the transcript level with respect to WT plants (Fig. 2C). The fold-induction levels varied widely in the transgenics, the maximum induction over WT plants being recorded in T2/22/3, which was 18.2-fold than WT upon NaCl treatment and 13.46-fold over WT under untreated condition, whereas the least induction was observed in line T2/12/1, which were 8.6-fold over WT on stress treatment and 5.03-fold over WT under non stressed condition.
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Levels of proline in T2 transgenic plants during salt stress. In all the transgenic lines, proline content was remarkably higher compared after imposing stress with WT plants under 250 mM NaCl for 24 h. The proline level increased considerably in transgenic lines, reaching a maximum up to seven times over their untreated counterparts as in the case of T2/22/3 (Fig. 5D). In WT plants, the increase after salt treatment was only 1.5 times over untreated plants. Discussion Plants respond to diverse environmental stresses at molecular level by altering the expression of different sets of genes that are involved in signaling and regulatory pathways or genes that encode proteins conferring stress tolerance or enzymes present in pathways leading to the synthesis of structural metabolites and osmolytes.25-29 Plant genetic engineering strategies for abiotic stress Figure 3. (A) Protein immunoblot analysis showing the expression of 21 kDa RAB16A protein in tolerance depend upon the expression of such several T2 transgenics in response to control (0 mM NaCl, 24 h) and salt stress (250 mM NaCl, genes. Most of these genes respond to ABA 24 h) compared against WT plants. (B) Salt tolerant assay of one week old plants of T2 through specific transcription factors which transgenic plant (T2/22/3), in response to 250 mM NaCl treatments continuously for 21 d. bind to the cis regulatory elements present in The salt tolerance of the transgenics was compared with WT type plants. their promoters. One such group of genes encodes the LEA proteins that were initially 20% and 18% in T2/3/11, T2/7/3, T2/12/1, T2/15/9 and identified in plant seeds.30 The hydrophilic, random coil structure T2/22/3 respectively (Fig. 5C). of LEA proteins possibly serve as water-binding domains, act as Percentage of shoot length and root length inhibition. Both hydration buffer to regulate water status, or may also interact the shoot and root growth was significantly affected in WT plants with the surface of macromolecules as a water matrix to oppose after imposing stress with 250 mM NaCl, as compared with the protein denaturation in the dehydrating tissues, thereby solvating different transgenic lines. In WT plants, the shoot and root the cytosolic structures. The Group 2 LEA proteins or dehydrins, growth were significantly inhibited by 63% and 46% respectively. to which RAB16A protein belongs, are water deficit-inducible The least inhibition was recorded in the transgenic line T2/22/3, proteins, customarily expressed in the embryos during the late which showed only 20% and 19% of shoot and root growth stages of embryogenesis and also induced in vegetative tissues in inhibition respectively. The percentage inhibition of shoot length response to those stresses causing ultimate cellular dehydration ranged from 26–39% (Fig. 5A), while that of root length ranged (e.g., drought, low temperature and salinity). During seed from 19–39% (Fig. 5B) in the transgenic plants. maturation, LEA expression is induced by ABA.15,16 The Group 2 LEA proteins contain a lysine-rich, 15 amino acid motif (K-segment; EKKGIMDKIKEKLPG), which is predicted Table 1. Yield performance of the transgenic lines survived after salinity to form an amphipathic a-helix, a tract of contiguous serine stress of different transgenic lines compared with WT Khitish residues and a conserved motif, containing the consensus Plant no. Plant No. of Spikelet Grain sequence DEYGNP in the N-terminal section of the protein. Height Fertile fertility yield per They have detergent and chaperone-like properties; stabilize (cm) tillers (%) plant (g) membranes, proteins and cellular compartments.31 The lea2 genes T2/3/11 96.55 7 50.65 8.43 have been manipulated in many plants in order to increase T2/7/3 96.25 9 55.32 9.32 drought resistance. For example, a wheat dehydrin, DHN-5, was T2/12/1 95.13 7 50.23 7.10 ectopically overexpressed in Arabidopsis and the transgenic plants displayed superior growth, seed germination rate, water retention, T2/15/9 100.21 11 57.21 12.01 ion accumulation, more negative water potential and higher T2/22/3 97.14 14 61.46 14.75 proline contents than WT plants under salt and/or drought Khitish (without stress) 99.76 17 61.21 16.00 stress.32 The Rab16A transcript and protein level were earlier Khitish (stressed) 59.13 3 11.21 3.34 shown to be expressed at a much higher level in salt-tolerant rice
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Figure 4. Effect of salinity stress (250 mM NaCl, 24 h) on the accumulation of inorganic ions (Na+ and K+) in the shoots (A and B) and roots (C and D) of WT and T2 transgenic plants. The level of inorganic ions, determined from equal amount of tissues (in terms of fresh weight), was expressed in terms of percent dry weight of the tissue (either shoot or root). The untreated (0 mM NaCl) seedlings served as experimental control. Values are the mean ± SE (n = 3), calculated from the standard solutions of metal ions. The statistical significance at p # 0.05 has been calculated.
Figure 5. (A) Effect of salt stress (250 mM NaCl, 24 h) on the percentage inhibition of shoot length (B) root length (C) total chlorophyll (D) estimation of proline content of WT and T2 transgenic lines; the untreated (0 mM NaCl) seedlings served as experimental control. Total chlorophyll, were represented as mg g21 fresh weight (FW) whereas proline was represented in mg g21 FW. Data are the means ± SE, and statistical significance at p # 0.05 has been calculated.
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varieties as compared with the sensitive ones,33 proving their evident role in salt tolerance. In this study, we therefore developed a salt tolerant rice variety by overexpressing Rab16A gene governed by the endogenous stressinducible Rab16A promoter and all the transgenics showed sufficiently enhanced salt tolerance up to 250 mM NaCl. The appearance and development of symptoms of damages caused by salinity were delayed in the transgenic lines. Both the transcript and protein accumulation were induced upon salt treatment, with their level being sufficiently higher in the transgenic plants as compared with WT. The morphological features of the transgenic plants were comparable to the non transgenic plants under greenhouse conditions, without any significant phenotypic changes in growth, flowering and seed setting. Previously, high levels of Rab16A mRNA were found in embryos between 20 DAP and maturity (30 DAP), i.e., the level of expression was increased as maturation proceeded. The long lived transcript was found in mature dry grains.24 Our observation also supported the earlier results in that the seeds of the
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transgenic plants at 20 DAP and 30 DAP showed higher Rab16A transcript accumulation compared with the WT plants. This appears to be a very significant observation as it will probably account for greater tolerance and hence higher productivity or grain yield of the transgenics, if they are challenged with salinity stress at the vulnerable stages of panicle formation or seed development. The inhibition of seed germination and retarded plant growth under salt stress is primarily due to osmotic effect, whereas toxic effect produced by excessive salt accumulation in plants also becomes evident at the later stages of growth.34 The decrease in leaf chlorophyll content or increased leaching of chlorophyll under high NaCl concentration is due to increased membrane permeability to salt.35 For most species, Na+ appears to reach a toxic concentration before other ions do, and thus the control of Na+ homeostasis is vital.36 Maintenance of appropriate intracellular K+/Na+ balance is critical for metabolic function, as Na+ cytotoxicity is largely due to competition with K+ for binding sites in enzymes essential for cellular functions.37 Often, higher K+/Na+ ratios are regarded as determinants of salt tolerance itself without considering any other agronomical or physiological traits. From our results, it is evident that the transgenic plants were able to tolerate both the initial osmotic stress as well as the toxic effect of salt in the later stages of growth and development as they produced similar fruit yield under NaCl treatment as WT plants grown under non stress conditions. The WT plants died due to prolonged exposure to salinity stress conditions. Inhibition of shoot length or root length due to salt stress was considerably reduced in the transgenics, accompanied with lesser chlorophyll damages and increased proline content. The accumulation of Na+ in the transgenic plants in response to salinity was significantly less both in leaves and roots and the loss of K+ was also much lesser, especially from the leaves, compared with the WT plants. This clearly reveals the capability of the transgenic plants to eliminate the deleterious effects of salinity stress to the endogenous membranes and protecting the photosynthetically active parts of the leaf by restricting the entry and distribution of Na+ and loss of high level of K+ from tissues. This is in accordance with the earlier results where dehydrins were found to undergo function-related conformational changes at the water/membrane interface, related to the stabilization of vesicles or other endomembrane structures under stress condition.38 Proline content was comparatively higher in all the transgenics compared with that of their WT plants and this result was in accordance with those described by Gracia et al.39 This result in membrane stability, preventing increases in RNase, reducing ionic toxicity and solute leakage, and protease activity, chlorophyll degradation and other processes culminating in senescence.40 Thus, the Rab16A transgenic plants showed overall superior physiological traits under high salt regimes. To our knowledge, this is the first report on the generation of a transgenic indica rice variety using Rab16A full length gene. Our studies reveal that Rab16A has the potentiality in conferring relatively high degree of tolerance against salinity conditions. In future, we will assess the growth performance of these transgenic plants under drought conditions, as well as when subjected to a combination of drought and salinity stress.
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Materials and Methods Preparation of gene construct. The amplification of the full length Rab16A gene (Rab p + ORF, interrupted by a short intron), designated as Rab F, using the primer pairs, Rab21–5 (5'-CGACAAGCTTCACG CGCACAGTACACACACAT-3') and Rab21–3B (5'-ATAGAGCTCTGGATCCTCAGTGCTG GCCGGGCAGCTT-3') Roychoudhury et al.41 The 0.9 Kbp Rab F was subcloned in pCAMBIA1200 at HindIII and BamHI sites, followed by nopaline synthase (nos) terminator. Plant transformation. Biolistic transformation was performed following the protocol described by Datta et al.42 The immature embryos (10–12 d after pollination, DAP) of an elite indica rice variety Khitish were procured from Chinsurah Rice Research Station, Hooghly, West Bengal. They were bombarded with pCAMBIA: Rab F construct using Biolistic PDS-1000/He (Biorad), involving nine bombardments. Southern hybridization. Genomic DNA was extracted from the leaves of 21 d-old seedlings of both transgenic and WT plants.43 About 30 mg of DNA was digested with Hind III and BamHI. The probe was labeled using Biotin labeling kit (Fermentas) according to the manufacturer’s instructions and hybridization was done according to Sambrook and Russell.44 RT-PCR and qRT-PCR. Total RNA was extracted using Trizol reagent from the leaves of 21 d-old WT and T2 transgenic plants, either untreated or after exposing stress (250 mM NaCl) for 24 h. The primer pairs used for RT-PCR were Rab16–5A (5'-GCATGGATCCAAATGGAGCACCAGGGGCAGCA-3') and Rab21–3B (5'-ATAGAGCTCTGGATCCTCAGTGCTGGCC GGGCAGCTT-3'). The expression of the tubulin gene, was checked to normalize the data. Apart from this, the total RNA was also prepared from the immature embryos/seeds of different growth stages, i.e., 10 d-old, 20 d-old and 30 d-old seedlings, in order to study the expression profile at different stages of seed development. The qRTPCR was performed with gene specific primers and the cycle was as follows: 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The procedure was according to the manufacturer’s instructions (CFX 96 Real time system, Biorad). The quantitative variation between different samples was evaluated by the DDCt method, and the amplification of tubulin gene was used as internal control to normalize all data. Protein immunoblot analysis. Total protein was extracted from the leaves of two-week old WT and T2 transgenic plants either, control or salt treated (250 mM NaCl, 24 h), following the method of Richard et al.45 with some modifications. Salt tolerance and agronomic performances of the transgenics. WT and T2 transgenic seeds were germinated in the dark at 30°C on MS medium alone and MS medium containing hygromycin (70 mg l–1) respectively. The 10 d-old surviving seedlings were then transferred to Yoshida solution (hydroponic solution) containing 250 mM NaCl. The seedlings were maintained in salinized nutrient solution. The response of the transgenic plants to salt stress was monitored for 21 d against WT. Yield performance of the transgenic lines survived after salinity stress was compared with wild type plant Khitish. Several agronomic parameters like plant height (cm), number of fertile tillers, spikelet
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percentage (%) and grain yield per plant (g) were performed with both WT and different transgenic plants at different stages of their life cycle.46 Estimation of endogenous ion content. The roots and leaves were harvested separately from untreated and salt treated (250 mM NaCl, 24 h) WT and T2 transgenic plants. The samples were kept at 80 ± 5°C for 48 h and the dry weight of each sample was recorded in Atomic Absorption Spectrophotometer.47 Chlorophyll content. The chlorophyll content of healthy and fully expanded leaves of three week-old WT and transgenic plants, both untreated and salt (250 mM NaCl, 24 h) treated, was estimated following the method of Arnon.48 Shoot length and root length. Seeds from T2 transgenics were sown on 0.5 ¾ MS basal medium with hygromycin (70 mg l21) and kept in light for five days at 30°C. The hygromycin resistant transgenic seedlings were salt-treated (250 mM) along with WT seedling (of the same age) and incubated at 37°C for one week; the lengths of the shoot and primary root were measured for five different transgenic plants. The percentage of relative growth inhibition of shoot and root were measured for three independent experiments. References 1.
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Sahi C, Singh A, Kumar K, Blumwald E, Grover A. Salt stress response in rice: genetics, molecular biology, and comparative genomics. Funct Integr Genomics 2006; 6:263-84; PMID:16819623; http://dx.doi.org/10.1007/ s10142-006-0032-5 Lenka SK, Katiyar A, Chinnusamy V, Bansal KC. Comparative analysis of drought-responsive transcriptome in Indica rice genotypes with contrasting drought tolerance. Plant Biotechnol J 2011; 9:315-27; PMID: 20809928; http://dx.doi.org/10.1111/j.1467-7652.2010. 00560.x Shinozaki K, Yamaguchi-Shinozaki K. Gene networks involved in drought stress response and tolerance. J Exp Bot 2007; 58:221-7; PMID:17075077; http://dx.doi. org/10.1093/jxb/erl164 Zarka DG, Vogel JT, Cook D, Thomashow MF. Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol 2003; 133:910-8; PMID: 14500791; http://dx.doi.org/10.1104/pp.103.027169 Zhao F, Guo S, Zhang H, Zhao Y. Expression of yeast SOD2 in transgenic rice results in increased salt tolerance. Plant Sci 2006; 170:216-24; http://dx.doi. org/10.1016/j.plantsci.2005.08.017 Jang IC, Oh SJ, Seo JS, Choi WB, Song SI, Kim CH, et al. Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth. Plant Physiol 2003; 131:516-24; PMID:12586876; http://dx.doi. org/10.1104/pp.007237 Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A, Hirochika H, et al. Function, intracellular localization and the importance in salt tolerance of a vacuolar Na(+)/H(+) antiporter from rice. Plant Cell Physiol 2004; 45:146-59; PMID:14988485; http://dx.doi.org/ 10.1093/pcp/pch014 Gao T, Wu Y, Zhang Y, Liu L, Ning Y, Wang D, et al. OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice. Plant Mol Biol 2011; 76: 145-56; PMID:21499841; http://dx.doi.org/10.1007/ s11103-011-9775-z
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Estimation of proline content. T2 transgenics seeds were sown on 0.5 ¾ MS basal medium with hygromycin (70 mg l21) and kept in light for five days at 30°C. The hygromycin resistant transgenic seedlings were salt-treated (250 mM) along with the wild type (WT) seedling (of the same age) for 24 h and incubated at 37°C condition for two weeks. Proline was extracted and determined according to Bates et al.49 Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
The financial support from the Department of Biotechnology, Government of India (Grant No: BT/01/COE/06/05) in the form of DBT Program Support is thankfully acknowledged. A partial grant from UGC is also acknowledged. Authors are extremely thankful to Prof Timothy J. Close, Department of Botany and Plant Sciences, University of California, Riverside, for providing the anti-dehydrin antiserum as a kind gift. The authors thank Ms Sayani Majumder for laboratory assistance and Mr Pratap Ghosh for field work.
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