Plant Mol Biol Rep DOI 10.1007/s11105-014-0827-9
ORIGINAL PAPER
OsBAT1 Augments Salinity Stress Tolerance by Enhancing Detoxification of ROS and Expression of Stress-Responsive Genes in Transgenic Rice Narendra Tuteja & Ranjan Kumar Sahoo & Kazi Md. Kamrul Huda & Suresh Tula & Renu Tuteja
# Springer Science+Business Media New York 2014
Abstract Coping with salinity-induced reduction in crop yield is essential for food security. Human leukocyte antigen-B associated transcript 1 (BAT1) also called as UAP56 is a DExD/H-box protein involved in messenger RNA (mRNA) splicing. Function of plant homologue of BAT1, especially its involvement in stress tolerance, has not been reported so far. Here, we demonstrate the localization of rice BAT1 (OsBAT1) in the nucleus and in the plasma membrane and its novel function in salinity stress tolerance in rice (Oryza sativa L. cv. IR64). Rice overexpressing OsBAT1 (T1 and T2 generations) show tolerance to high salinity (200 mM NaCl) stress. The T1 transgenics exhibited higher levels of biochemical parameters such as water and chlorophyll contents, net photosynthetic rate, stomatal conductance and intercellular CO2 content as compared to null-segregant (control) plants. The activities of ascorbate peroxidase, guaiacol peroxidase, malondialdehyde and glutathione reductase were significantly higher in transgenics indicating the presence of an efficient antioxidant defence system which helps to cope with salinity-induced oxidative damages. Agronomic parameters were also higher in transgenics as compared to control. Microarray analysis of OsBAT1 overexpressing transgenic lines revealed up-regulation of stress-responsive genes of different pathways including the spliceosome. Our results provide the first direct evidence for a promising function of OsBAT1 in mediating salinity stress response/tolerance in rice.
N. Tuteja (*) : R. K. Sahoo : K. M. K. Huda : S. Tula : R. Tuteja International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India e-mail:
[email protected] N. Tuteja e-mail:
[email protected]
Keywords Antioxidant enzymes . BAT1/UAP56 . DNA and RNA helicases . HLA-B associated transcript 1 . Oryza sativa . Photosynthesis . Reactive oxygen species (ROS) . Salinity stress
Introduction Abiotic stresses decrease both the growth and productivity of crops by reducing photosynthesis, decreasing seedling fresh weight, germination percentage and biomass as well as increasing the generation of reactive oxygen species (ROS) (Hadiarto and Tran 2011). The regulation of plant growth and survival in response to stress is a complex process which is dependent on the severity of stress, developmental stage at the time of stress and organ or cell identity (Claeys and Inze 2013). Salinity is a widespread soil problem limiting crop productivity worldwide, especially in the tropical countries and irrigated fields where salinization has caused deterioration of agricultural lands (Mahajan and Tuteja 2005; Munns and Tester 2008). Several studies have demonstrated that the introduction of specific foreign genes into crop plants provides resistance against biotic and abiotic stresses (Xiong et al. 2006; Mazzucotelli et al. 2008; Chen et al. 2013). Earlier studies had revealed that some RNA/DNA helicases also play an important role in the abiotic stress management/ resistance (Liu et al. 2002; Vashisht and Tuteja 2006; Kant et al. 2007; Li et al. 2008). DNA and RNA helicases are highly conserved proteins that catalyse the unwinding of duplex nucleic acids. Kant et al. (2007) demonstrated that two DEAD-box RNA helicases (STRS1; At1g31970, STRS2; At5g08620) attenuate the sensitivity to multiple abiotic stresses,
Plant Mol Biol Rep
functioning in ABA-dependent and ABA-independent abiotic stress signalling pathways in Arabidopsis thaliana. In addition, two RNA helicases, namely AtRH9 and AtRH25, have been reported to be involved in seed germination under salt stress (Kim et al. 2008). In rice, the OsBIRH1 encodes a functional DEAD-box RNA helicase which when overexpressed, it functions in defence responses against biotic and abiotic stresses (Li et al. 2008). Furthermore, a functional involvement of a putative alfalfa helicase in the antioxidative responses of plants has also been reported (Luo et al. 2009). RNA helicases have been reported to perform defined roles during environmental stresses, and their expression and/ or activity is frequently altered during cellular response to abiotic stress (Owttrim 2013). Recently, the overexpression of a mitochondrial helicase OsSUV3 has been reported to impart salinity stress tolerance in rice plants without yield loss (Tuteja et al. 2013). Human leukocyte antigen-B associated transcript 1 (BAT1), also termed HEL in fruitfly (Drosophila melanogaster), Sub2p in yeast and UAP56 in mammals, is a bonafide member of DExD/H-box family of helicases, which mediates pre-messenger RNA (mRNA) splicing (Pause and Sonenberg 1992; Kammel et al. 2013). Previously, rice UAP56 (termed AIP1/2) has been identified, and its role in degeneration of tapetum during anther development was reported (Li et al. 2011). However, the role of BAT1 rice homologue (here termed as OsBAT1) in abiotic stress tolerance has not been investigated yet. In this study, we have developed transgenic rice plants (Oryza sativa L., cv. IR64) by overexpressing OsBAT1 gene, which leads to the enhancement of salinity stress tolerance by interacting with mechanisms involved in coping with stress-induced oxidative damage.
Materials and Methods Subcellular Localization of OsBAT1 Onion epidermal cells were used to study the localization of OsBAT1 protein. The complete ORF of OsBAT1 was fused with GFP and cloned into Xba1 site of the pMBPII-GFP expression vector (Sharma et al. 2012) under the control of 2XCaMV35S (approximately six times higher expression than that of the CaMV 35S1x promoter) promoter to create a 35S:OsBAT1–GFP fusion construct. As a control, 35S:GFP construct was used. These constructs were bombarded to onion epidermal cells using a particle gun-mediated method (Genepulser Excell System, Bio-Rad, Hercules, CA, USA), and the expression of GFP was observed using confocal
microscopy (Cell observer SD, Apotome 2, LSM 710, Carl Zeiss Iberia, SL, ESPANA). Cloning of Rice BAT1 Gene and Agrobacterium-Mediated Transformation of IR64 The coding region of BAT1 gene was PCR-amplified from rice (O. sativa L. cv. Swarna) complementary DNA (cDNA) using primers 5′-CCATGGATGGCCGAAGCCGAGGTTAAG-3′ and 5′-CCATGGTTAAGAAGGCATATAT-3′ and subsequently cloned into pRT-100 at the NcoI site to obtain the 35S promoter:BAT1:poly A signal cassette. The complete cassette containing the BAT1 gene was cloned into PstI site of plant transformation vector pCAMBIA1301 (GenBank accession number: GQ 478227; Locus id: LOC_Os01g36890.2), keeping the GUS (β-glucuronidase) gene present in the vector. For rice plant transformation, the plasmid was introduced into Agrobacterium tumefaciens LBA4404 and subsequently transformed into embryogenic calli from mature rice (O. sativa L. cv. IR64) seeds as described by Sahoo and Tuteja (2012). The transgenic rice plants were compared to the ideal control (null-segregant) plants derived from tissue culture method, which are genetically similar to the transgenic plants but do not contain any inserted genetic materials. In the further experiments, 10 plants from each transgenic line were taken for one biological repeat. Therefore, a total of 30 plants from each transgenic line were used in three biological repeats. The sample numbers of T1 seedlings obtained from stress tests were analysed. Analysis of T1 Plants: PCR, Southern Blot and Histochemical Assay for GUS Activity and Tolerance Index The integration of OsBAT1 gene was confirmed through PCR, using genomic DNA extracted from healthy leaves (0.5 g) of transgenic IR64 rice plants. PCR amplification was performed with 0.15–0.20 μg genomic DNA, using gene-specific forward and reverse primers as well as promoter-specific forward and gene-specific reverse primers. For Southern analysis, 20 μg of DNA was digested with XbaI, and samples were resolved on 0.8 % agarose gels. DNA was transferred to negatively charged nylon membrane (Hybond-N + , Amersham, Inc.) as described by Sambrook et al. (1989). The radioactive probe was prepared by using promoter (CaMV35S)-specific forward and reverse primers using α-[32P] dCTP. Hybridization with the probe at 53 °C and washing at 65 °C was conducted according to the method described by Sambrook et al. (1989). GUS activity was assayed histochemically using the indigogenic X-gluc (5-bromo-4-chloro-3-indolyl beta-D-glucuronide) substrate, as described by Jefferson et al. (1987). The tolerance index of BAT1 T1 transgenic (L5, L8, L12 and L17) and control plants in 200 mM NaCl were calculated
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by the following formula: TI ð%Þ ¼ ðplant dry weight with 200 mM NaClÞ
.
ðplant dry weight with waterÞ 100:
conditions (Garg et al. 2012). The estimation of lipid peroxidation, electrolytic leakage and relative water content (RWC) was performed by following the method described by Tuteja et al. (2013). Measurement of Photosynthetic Characteristics
RNA Isolation and qRT-PCR Three-week-old IR 64 T1OsBAT1 and control rice seedlings grown in a hydroponic system were treated with 200 mM NaCl for 24 h, and total RNA was isolated using the TRIzol reagent (Invitrogen Life Technologies, USA) as per the manufacturer’s instructions. The total RNA obtained was used as template for cDNA synthesis. The first-strand cDNA was synthesized from 5 mg of total RNA using Superscript II Reverse Transcriptase (Invitrogen Life Technologies USA) using oligo(dT)18 primer according to the manufacturer’s instructions. The quantitative real-time PCR (qRT-PCR) was performed with gene-specific primers (forward, 5′-CTATGA CATGCCCGATTCTGC-3′ and reverse, 5′-CCATGGAGA AGGCATATATGTCGAAGT-3′), and the cycle was as follows: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The procedure was according to the manufacturer’s instructions (StepOne Real-Time PCR system Applied Biosystems). The quantitative variation between different samples was evaluated by the ΔΔCT method. To validate the qRT-PCR results, the experiments were repeated three times. The mean values for the expression levels of the genes were calculated from three independent experiments. The relative expression level was calculated as 2−ΔΔCT (Jayaraman et al. 2008). Leaf Disc Senescence Assay for Salinity Tolerance Healthy and fully expanded rice leaf squares of 1 cm×1 cm dimension were taken from similar age transgenic lines (L5, L8, L12 and L17) of T1 and T2 generation and control plants. The discs were floated in 100- and 200-mM solution of NaCl for 72 h. The experiment was performed at room temperature with three biological replicates as previously described (Tuteja et al. 2013). Antioxidant Assays in OsBAT1 Transgenic Lines For this experiment, 21-day old seedlings of control and OsBAT1 transgenic plants were grown in 200 mM NaCl for 24 h and then used for biochemical analysis. Activities of ascorbate peroxidase (APX), catalase (CAT), guaiacol peroxidase (GPX) and glutathione reductase (GR) enzymes were measured as described by Garg et al. 2012. In addition, the amount of both hydrogen peroxide (H2O2) and proline was measured due to their active roles played during stress
Mature OsBAT1 transgenic and control IR64 rice plants were treated with 200 mM NaCl and 0 mM NaCl for 30 days. An infra-red gas analyser (IRGA, LiCor, Lincoln, NE, USA) was used on a sunny day between 1000 and 1200 hours to estimate net photosynthetic rate (Pn), stomatal conductance (gs) and intercellular CO2 concentration (Ci) on the fourth and fifth fully expanded leaves of transgenic lines (L5, L8, L12 and L17) and control plants. The atmospheric conditions during the measurement were photosynthetically active radiation (PAR), 1,050±7 μmol m−2 s−1, relative humidity 66±4 %, atmospheric temperature 24±2 °C and atmospheric CO2, 350 μmol mol−1. Agronomic Characteristics of T1 Transgenic Plants Agronomic characteristics of mature OsBAT1 transgenic and control plants were measured after treatment with 200 and 0 mM NaCl for 30 days. Growth and yield performance of transgenic and control lines was assessed after salinity stress. Several agronomic parameters like plant height, number of tillers/plant, number of panicle/plant, number of filled grain/ panicle, number of chaffy grains/panicle, straw dry weight, 100 grain weight, root length, root dry weight, leaf area and plant dry weight were measured at 3 weeks after initiating the 200-mM NaCl treatment in T1 transgenic and water-grown control plants. The shoot and root length was measured on meter scale. Plant dry weight was measured by drying fresh plant samples in a hot-air oven (Memmert, Model 500, Germany) at 80 °C for 4 days until constant weight and incubated in a desiccator for dry weight measurement. The leaf area was measured by a leaf area meter (Systronics, Hyderabad, India). Estimation of Endogenous Ion and Soluble Sugar Content Leaves from T1 transgenic and control plants grown for 8 weeks on either 200 mM NaCl or water, respectively, were used for estimation of endogenous ion (nitrogen, phosphorus, potassium and sodium) content. Total nitrogen content was determined according to Jackson (1973). The phosphorus content was calculated using a spectrophotometer as described by Gupta (2004). Potassium content was estimated through flame ionization photometer following standard protocol (Chapman and Pratt 1982). Sodium content was measured following the method described by Munns et al. (2010). Glucose and fructose contents in the roots and shoots of both transgenic as well as control plants were measured after salt
Plant Mol Biol Rep
stress for 24 h using the method described by Karkacier et al. (2003).
Results OsBAT1 Is Localized in Nucleus and Plasma Membrane
Segregation, Molecular and Phenotypic Expression of T2 Transgenic Plants The functionality and stability of the OsBAT1 transgene in the T1 and T2 generation were analysed. For this experiment, T1 and T2 seeds were plated onto hygromycin (50 mg l−1) containing MS medium. The survival of progeny was evaluated for resistance to hygromycin in four independent transformants. The molecular and phenotypic expression of OsBAT1 gene was studied in T2 generation. The T2 transgenic lines (L5, L8, L12 and L17) were grown up to maturity, and the integration of transgene was checked by PCR and phenotypic expression. T2 transgenic plants along with control plants were kept together in one big tank filled with 200 mM NaCl instead of water. The response of these plants was recorded in 30-day intervals. Microarray Analysis Control and transgenic plants were grown in greenhouse for 14 days and subjected to stress for 6 h by using 200 mM NaCl. The total RNA was extracted from seedlings and sent to Genotypic Technology Pvt Ltd (Bangalore, India) for microarray analysis. Using 10 μg of total RNA, double-stranded cDNA was synthesized with a T7 promoter-containing oligo (dT) primer followed by in vitro transcription using Agilent’s Quick-Amp labelling Kit. Resulting cRNA was hybridized using Agilent’s In situ Hybridization kit. Microarray analysis was performed using an Agilent (http://www.agilent.co.in/) microarray chip for rice [4X44K (CAT: G2519F_015241)] which was then scanned using an Agilent gene array scanner. The data generated was normalized with Gene Spring GX11.5 software using percentile shift normalization. Genes exhibiting more than a 2-fold enhanced or reduced transcription level are considered as significant alterations in expression. Chi-squared test was performed for statistical analyses. The genes for which P0.05 level as determined by DMRT ND no data
plants survived efficiently up to maturity and formed viable seeds (Fig. 5f, g), whereas control plants died completely. Stress-Responsive Genes Are Up-regulated in OsBAT1 Overexpressing Transgenic Rice To compare gene expression profiles between the OsBAT1 overexpressing line and control plants, we performed microarray analysis using the Affymetrix rice gene chip. A total of 6197 genes (3788 genes up-regulated and 2409 genes downregulated) presented alterations in their expression in the OsBAT1 (line L5) transgenic rice in response to salinity stress. The genes which showed a >2-fold change in transcript level in the OsBAT1 overexpressing line compared with control are shown in Table 5. Important selected genes encoding stressrelated proteins, signalling and transcription factors were upregulated in OsBAT1 transgenics. Transcription factor genes like MYB, NAC and WRKY (Zhang et al. 2012), DREB (Reddy et al. 2011) and MADS-box protein (Tardif et al. 2007) that are known to improve stress adaptation were found to be up-regulated in OsBAT1 overexpressing lines (Table 5). Genes encoding proteins involved in protein phosphorylation including protein phosphatase, serine/threonine-protein kinase and leucine rich receptor-like kinase were also up-regulated. Transporter proteins play a positive regulatory role in stress tolerance, and a number of these proteins such as potassium transporter, Na+/H+ antiporter, plasma membrane Ca2+ ATPase, ABC transporter and nitrate transporter were also found to be up-regulated in OsBAT1 expressing transgenic plants. The up-regulation of heat shock protein, peroxidase protein and calmodulin-binding protein indicates the
involvement of OsBAT1 in modulating ROS pathways (Table 5). Gene ontology (GO) analysis of up-regulated genes revealed that overexpression of OsBAT1 regulates several biological processes (Table 6). The genes of different pathways including metabolic pathways (130 genes), biosynthesis of plant hormones (31 genes), glycolysis I (22 genes) and spliceosome (20 genes) were up-regulated in OsBAT1 overexpressing transgenic plants. In addition, the genes involved in peroxisome, oxidative phosphorylation, amino sugar metabolism, nitrogen metabolism, jasmonic acid biosynthesis, brassinosteroid biosynthesis II, glutathione metabolism and proline biosynthesis were also significantly up-regulated (Table 6). These findings indicate that different stress-responsive pathways are elevated in OsBAT1 transgenics upon exposure to salinity stress leading to salinity stress tolerance. The GO analysis of down-regulated genes also revealed that overexpression of OsBAT1 regulates biological processes. Under salt stress, several degradation-associated (e.g. lysine degradation, methionine degradation, RNA degradation, isoleucine degradation II and glutamine degradation III) and metabolic pathway-related (213 genes) genes were down-regulated (Table 7).
Discussion Salinity is a multigenic trait which controls the whole plant machinery, and rice productivity is severely affected due to this stress. The new role of helicases in plant abiotic stress tolerance such as salinity and drought has been reported in
Plant Mol Biol Rep
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Fig. 4 Soluble sugar content in roots and shoots of OsBAT1 overexpressing T1-transgenic lines (L5, L8, L12 and L17) compared to control rice plants exposed to 24-h salinity stress (200 mM NaCl). a Glucose content in roots. b Fructose content in roots. c Glucose content in shoots. d Fructose content in shoots. Each value represents the mean of
three replicates±SE. Means were compared using ANOVA. Data followed by the same letters are not significantly different at P0.05 level as determined by Duncan’s multiple range test (DMRT) ND no data
Plant Mol Biol Rep
Fig. 5 Analysis of T2OsBAT1 overexpressing transgenic lines. a Germination test of T2 seeds on solid MS medium with 200 mM NaCl. b PCR conformation of transgenic lines. c Visualization of GUS activity from transgenic lines. d Leaf disc senescence assay under 100 and 200 mM NaCl. e Chlorophyll content (mg g−1 fw) in T2OsBAT1 transgenic lines after salt stress. f Salt tolerance response of T2OsBAT1 transgenic plants (L5, L8 and L12) and control in 0 day of 200-mM NaCl
stress. g Salt tolerance of the same set of mature plants after 30 days of NaCl stress. Each value represents the mean of three replicates±SE. Means were compared using ANOVA. Data followed by the same letters are not significantly different at P
