Journal of Plant Growth Regulation https://doi.org/10.1007/s00344-017-9762-y

Transcriptome Profiling of Abiotic Stress-Responsive Genes During Cadmium Chloride-Mediated Stress in Two Indica Rice Varieties Saikat Paul1 · Aryadeep Roychoudhury1 Received: 3 August 2017 / Accepted: 16 October 2017 © Springer Science+Business Media, LLC, part of Springer Nature 2017

Abstract Rice growth and development is highly affected by the excessive concentration of the heavy metal cadmium (Cd). To elucidate the molecular basis of Cd tolerance, we carried out comprehensive transcriptome profiling of diverse groups of genes like those encoding antioxidative enzymes (APX, CAT, SOD, and GR), osmolyte-regulatory enzymes (P5CS, PDH, and BADH1), polyamine-regulatory enzymes (SAMDC, SPDS, SPMS, and DAO), transcription factors (TRAB-1 and WRKY-71), Osem and RbcS from leaf and root tissues under cadmium chloride (1.5 mM) stress at different time points (6, 12, and 24 h) of exposure in two indica rice varieties, that is, IR-64 (sensitive) and Nonabokra (tolerant). Analyses of massively parallel signature sequencing (MPSS) and publicly available microarray databases indicated the physiological role of the concerned genes and possible cross-talk between Cd and other abiotic stresses. Moreover, the pattern of expression, for a particular gene in leaves, varied in most cases from that in roots, suggesting a tissue-specific response. The higher accumulation of TRAB-1 transcript and protein in the tolerant cultivar Nonabokra in response to Cd stress suggested a role for TRAB-1 as a nodal component in Cd signaling pathway. Overall, the transcriptome analyses undertaken in the present study indicated that the genes, belonging to different functional classes and conferring Cd tolerance, were differentially regulated and mostly overrepresented under stress situations, thus providing novel insight into the functional basis of Cd tolerance in rice varieties and the importance of cross-talk of different signaling pathways. The present work will also pave the way in the future to select gene(s) for overexpression, so that rice can better tolerate Cd stress. Keywords  Cadmium chloride · Rice · Gene expression · Microarray · MPSS · Transcription factors · TRAB-1 · Osmolytes · Polyamines

Introduction Cadmium (Cd), which belongs to the group of non-essential transition metals, constitutes a highly toxic heavy metal contaminant to both humans and plants. Because of its long biological half-life and residence time in soil, Cd is a major threat to agricultural systems. The main sources of Cd contamination include effluents from industries, mining, burning of fossil fuels, leakage of municipal wastes, pesticides, Electronic supplementary material  The online version of this article (https://doi.org/10.1007/s00344-017-9762-y) contains supplementary material, which is available to authorized users. * Aryadeep Roychoudhury [email protected] 1



Post Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata, West Bengal 700016, India

discharges from paint and leather industries, phosphate fertilizers, and sewage sludge (Boyd 2010). Cd is readily taken up by plants, leading to toxic symptoms such as growth inhibition, damage to photosynthetic apparatus, lowering of chlorophyll content, inhibition of stomatal opening, ­NH4+ accumulation, oxidative stress, and DNA damage (Moussa and El-Gamal 2010; Kao 2014). Cd also inhibits several physiological processes like respiration, photosynthesis, cell elongation, plant-water relationships, nitrogen metabolism, and mineral nutrition, resulting in reduced plant biomass (Kuo and Kao 2004; Hsu and Kao 2005). Abscisic acid (ABA), the universal stress hormone, acts as a signaling molecule by promoting stomatal closure and triggering the expression of many stress-responsive genes under multiple abiotic stresses, that is, salinity (Gurmani and others 2013), drought (Yoshida and others 2010), cold, and heat (Xu and others 2014). Stress-tolerant cultivars were earlier reported to accumulate higher endogenous ABA level

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than sensitive cultivars (Moons and others 1995; Yang and others 2014). The higher concentration of ABA plays a significant role in Cd tolerance in rice seedlings, the degree of tolerance being genotype dependent (Hsu and Kao 2003, 2005). During Cd stress, the ABA level increased rapidly in roots and leaves of the Cd-tolerant cultivar (Tainung 67), but not in the Cd-sensitive cultivar (Taichung Native 1). Salinity tolerance is also accounted for by higher endogenous ABA levels in rice. The salt-tolerant cultivars (Pokkali and Nonabokra) were shown to exhibit constitutive and higher expression levels of ABA-inducible genes than the salt-sensitive varieties (IR-64, IR-29, M-1-48), the latter showing lower and inducible expression (Roychoudhury and others 2008; Cotsaftis and others 2011; Basu and Roychoudhury 2014). Nonabokra, the salt-tolerant variety, also exhibits better Cd tolerance than IR-29, the salt-sensitive variety, in terms of higher activity of antioxidative enzymes, increased accumulation of higher polyamines (spermidine, Spd and spermine, Spm), lower lipoxygenase activity, protein oxidation, and much lower chlorophyll loss in the former (Roychoudhury and others 2012). Earlier studies have shown that on exposure to Cd, the transcription factors (TFs) belonging to different families, such as WRKY, basic leucine zipper (bZIP), and myeloblastosis (MYB) proteins play a significant role in tolerance by controlling the expression of their downstream genes (DalCorso and others 2010). In Cdtreated Thlaspi caerulescens, MYB28 and WRKY53 were strongly expressed, thereby regulating the activity of other TFs (Van De Mortel and others 2008; Wei and others 2008). Upon induction by Cd, the bZIP TF, OBF5, regulates the expression of glutathione S-transferase 6 (GST6) by recognizing its promoter region (Suzuki and others 2001). The same group has observed the transcriptional induction of rd29A with Cd treatment, following the binding of the TF, DREB2A, to the dehydration responsive element (DRE) of rd29A. The ABA-inducible, cold-responsive/late embryogenesis abundant (COR/LEA) genes, that is, WRAB15 and WRAB18, showed positive correlation with Cd stress in terms of expression and tolerance of wheat seedlings (Talanova and others 2013). Likewise, the LEA gene, TaLEA1 from Tamarix androssowii also provides tolerance to Cd stress by increasing reactive oxygen species (ROS)scavenging ability and decreasing lipid peroxidation (Gao and others 2012). Polyamines (PAs) play a significant role in Cd tolerance by preventing ­H2O2 generation and reducing Cd uptake (Hsu and Kao 2007). The severe growth inhibition with higher lipid peroxidation and reduction in functioning of antioxidant systems during Cd stress due to antisense inhibition of spermidine synthase (SPDS) gene in European pear (Pyrus communis L.) shoot, suggested the protective role of Spd in Cd tolerance by influencing the antioxidative systems (Wen and others 2011). The enhanced polyamine oxidase (PAO) activity in roots of Malus hupehensis

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(Rehd.) under Cd stress initiated programmed cell death by dramatically decreasing free Spd and Spm and generating ­H2O2 (Jiang and others 2012). To decipher further the Cd tolerance mechanism, we have, in the present investigation, performed comparative transcriptome profiling of diverse genes encoding stress-inducible TFs, ROS-scavenging antioxidative enzymes, osmolytes (proline, Pro, and glycinebetaine, GB)-, and PA (Spd and Spm)-metabolizing enzymes, between salt-sensitive (IR-64) and salt-tolerant (Nonabokra) rice under varying durations of Cd stress exposure, so as to correlate the varietal response of Cd with gene expression patterns.

Materials and Methods Expression Analysis of Stress‑Responsive Genes Using Massively Parallel Signature Sequencing (MPSS) Database For the Oryza sativa (rice) genome, 20-nucleotide long signature sequences were used for determining the tissuespecific expression of stress-responsive genes by using the MPSS database (http://mpss.udel.edu). The descriptions of the MPSS library of O. sativa are provided in Table S1 (electronic supplementary material). The normalized abundance of the signature sequences was log-transformed and heat maps were generated with the help of Multiple Experiment Viewer (MeV) software (Saeed and others 2003).

Expression Analysis Using Microarrays To compare the gene expression for Cd with other abiotic stresses, the Affymetrix CEL files for rice were obtained from the National Center for Biotechnology Information Gene Expression Omnibus database (GSE6901, GSE37557, and GSE25206) (Barrett and others 2013). The rice microarray slides were processed in R with ricecdf (as defined by Affymetrix CDF) as described earlier (Movahedi and others 2011). Briefly, the array quality of the experiment was assessed by MA plot for individual arrays. The robust multi-array average (RMA) algorithm along with ricecdf was implemented for normalizing the raw data. These steps include background correction, log transformation, and quantile normalization and summarization. The average intensity values for the replicated slides were calculated and used for analysis. The normalized signal intensities were transformed into log10 scale, heat maps were produced, and hierarchical clustering was done by the average linkage method using MeV software package (Saeed and others 2003).

Journal of Plant Growth Regulation

Plant Materials, Growth Conditions, and Stress Treatments

Immunoblot Analysis to Detect TRAB‑1 Protein Accumulation

The rice seeds of IR-64 were obtained from Chinsurah Rice Research Station (Hooghly, West Bengal, India) and Nonabokra seeds from Central Soil Salinity Research Institute (Canning, West Bengal, India). The seeds were surface sterilized with 0.1% (w/v) H ­ gCl 2 for 20  min, washed extensively, imbibed in deionized water for 6–8 h, and allowed to germinate in Petri dishes at 37 °C in the dark for three days. The germinated seedlings were grown in the presence of deionized water at 32 °C under 16 h light and 8 h dark photoperiodic cycle with 50% relative humidity and 700 µmol photons ­m − 2s − 1 for the desired period in a plant growth chamber. The 10-day-old seedlings were either maintained under control conditions (0 mM ­CdCl2) or treated with 1.5 mM ­CdCl2 for varying durations (6, 12, and 24 h). The selection of ­CdCl2 concentration for stress imposition was made on the basis of our earlier report (Roychoudhury and others 2012). All the test samples were washed thoroughly, roots and leaves harvested, immediately frozen in liquid nitrogen, and kept at − 80 °C until initiation of experiment.

TRAB-1 cDNA was amplified by PCR from Nonabokra using gene-specific primers and sub-cloned into the pET28a+ (Novagen) expression vector. The sequence was submitted to the NCBI genome database with accession number KT037117. The TRAB-1 protein was purified, and immunoblot analysis was performed as described by Paul and Roychoudhury (2017a, b). Briefly, the TRAB-1 protein was overexpressed in E. coli BL21 (DE3) cells and the 44 kDa polypeptide was produced after 1 mM IPTGmediated induction as a fusion protein with the 6× His tag at the N-terminus. The fusion protein was purified with nickel–nitrilotriacetic acid–agarose affinity resin (Qiagen) and concentrated using centrifugal filters (Amicon, Millipore) and quantified spectrophotometrically. Total protein was isolated from young leaf tissues, blotted onto nitrocellulose membrane, and immunoblot analysis was carried out using antibody generated against the purified protein (BioBharati LifeScience, India). The detection of TRAB-1 was done by developing the blot in substrate solution containing NBT-BCIP (Sambrook and Russell 2001).

Semi‑quantitative RT‑PCR Analyses

Results and Discussion

Total RNA was extracted from leaf and root tissues of control and C ­ dCl 2-treated seedlings of IR-64 and Nonabokra, using TRIzol reagent (Invitrogen). Before first strand cDNA synthesis, the RNA was treated with DNase I (Thermo Scientific) to remove DNA contamination. About 5 µg of total RNA (DNase I treated) was reversetranscribed using a Maxima First Strand cDNA synthesis kit (Thermo Scientific). About 100 ng of cDNA was used as a template for semi-quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) using gene-specific primers, with actin as the standard (Chen and others 2007). The primer sequences and corresponding gene accession ID are shown in Table S2 (electronic supplementary material). Following densitometric scanning of band intensity of each gene, the derived value for gene expression level at the respective time point was normalized by dividing the value with that of the internal standard (actin). The fold change (increase or decrease) in gene expression was calculated as log2 of the ratio of expression of genes under stress conditions to expression under the control (untreated) condition. The average data from semi-quantitative RT-PCR analysis were imported into the TM4 microarray software suite for heat map analysis (Saeed and others 2003).

The role of ABA in plant growth and development along with its role in abiotic stress tolerance is well-established (Skubacz and others 2016). Nonabokra has considerably higher endogenous ABA levels than the sensitive varieties (Moons and others 1995). The Cd tolerance in rice seedlings was earlier shown to be ABA-dependent (Hsu and Kao 2005) and the salt-tolerant Nonabokra, with higher ABA accumulation, has also been shown to behave as a Cd-tolerant variety (Roychoudhury and others 2012). Because the molecular regulation of the concerned genes in Cd stress tolerance is not fully understood, in the present study, we have carried out extensive transcriptome profiling by using the publicly available MPSS and microarray databases, along with timekinetic analysis of the expression of stress-responsive genes in the leaf and root of IR-64 and Nonabokra rice varieties under Cd stress.

Massively Parallel Signature Sequencing (MPSS)‑Based Expression Profiling of Rice Stress‑Responsive Genes MPSS is a very sensitive method used in gene expression analysis of various plant systems including rice, either at the control level or under stress conditions (Kushwaha and others 2009; Singh and others 2015a, b). In the MPSS database, all the libraries, except few as mentioned in Table S1

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Previous studies have shown that ABA plays an important role in Cd stress tolerance in rice along with tolerance to various other abiotic stresses such as salt, drought, and cold. We carried out transcriptome analysis by using a publicly available microarray database to compare the expression pattern of a group of stress-inducible genes between Cd

and other abiotic stresses (salt, drought, cold, and ABA) (Fig. 1b). The microarray data GSE6901 (expression data for salt, drought, and cold stress) and GSE37557 (for ABA treatment) involved 7-day-old seedlings and GSE25206 (for Cd stress) involved 10-day-old seedlings of the rice variety IR-64. Because of the limited reports on gene expression during Cd stress, the publicly available microarray database has been used to get a preliminary idea about the inducibility and expression behavior of the examined genes under multiple stresses (salt, drought, ABA) along with ­CdCl2 stress, prior to the onset of our work, as well as to analyze the cross-talk and overlapping gene expression among various abiotic stresses. To confirm beforehand whether at all such genes would be induced by Cd stress as well analyze crosstalk between multiple stresses with respect to gene expression, the microarray data seemed useful, informative, and hence significant. As only the root transcriptomic data of rice in response to 100 µM Cd are available in the database, we used such data for our analysis. Moreover, although the available microarray data are based on Cd concentrations different from that used in our semi-quantitative RT-PCR analysis, the prime knowledge gained through such data is the inducibility in the expression of all such genes under Cd stress, irrespective of Cd concentration. Our results showed that the CAT, SOD, GR, and APX transcripts were highly induced under all the abiotic stress, except APX which showed lower transcript levels in roots under Cd stress (Fig. 1b). The P5CS transcript was more accumulated under salt, drought, ABA, and Cd stresses (Igarashi and others 1997), whereas BADH1 (Hasthanasombut and

Fig. 1  The MPSS-based expression profile of stress-responsive genes in different tissues and organs of Nipponbare rice; the heat map shows the gene expression profile based on hierarchical clustering of various stress-responsive genes. The names of the MPSS libraries are mentioned below each heat map (a); microarray-based analysis of stress-responsive expression of genes in IR-64 rice under various

abiotic stresses, that is, salt, drought, cold, and cadmium. The differential expression profile of stress-responsive genes was represented as a heat map and hierarchical clustering was done by average linkage method. The relative signal values in log10 scale were represented by the color bar at the bottom (b). The heat maps were prepared with the help MeV software. (Color figure online)

were constructed using the Nipponbare (japonica) rice background. The MPSS database was primarily used only to extract information about the relative abundance of transcripts of stress-responsive genes in various tissues/organs of rice, thereby giving us preliminary knowledge or an idea regarding the location and tissue-specific expression pattern, as well as the concerned physiological functions of such genes under stress conditions, so as to validate the semiquantitative RT-PCR data from the leaf and root samples in our experiment. Expression analysis of genes using the rice MPSS database (Fig. 1a) showed that the CAT, P5CS, BADH1, SAMDC, SPDS, TRAB-1, WRKY-71, and RbcS transcripts were largely accumulated in all the tissues including leaves, roots, and reproductive stages, whereas the expression of Osem was more up regulated in seed, suggesting that these genes are involved in rice vegetative and reproductive growth and development. The SPMS transcript was more accumulated in root tissue. This study indicated the possible function of key representative genes of diverse metabolic pathways in different tissues and developmental stages.

Expression Profile of Rice Stress‑Responsive Genes Using Microarray

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Journal of Plant Growth Regulation

The effect of increasing concentrations of C ­ dCl2 for two days (48 h), until the maximum limit of 1.5 mM C ­ dCl2 on rice cultivars, has been shown in our earlier publication

(Roychoudhury and others 2012) with respect to oxidative damage indices and activation of defense mechanisms in the form of osmolytes and antioxidants. A comprehensive biochemical and physiological analysis for deciphering the genotypic variation in Cd tolerance was undertaken and Nonabokra was found to exhibit higher Cd tolerance as compared to IR-29 (Roychoudhury and others 2012). The present communication is actually an extension of the previous work, because we have moved a step forward to focus upon the time-kinetic expression pattern of key stress-responsive genes from diverse metabolic pathways under maximum Cd concentration (1.5 mM) using semi-quantitative RT-PCR in two different rice varieties, that is, IR-64 and Nonabokra. Both the microarray data and semi-quantitative RT-PCR analysis involved early developmental stages of rice plants. The heat map and hierarchical clustering showed the relative abundance of rice stress-responsive genes in the leaves and roots of IR-64 and Nonabokra under Cd stress (Figs. 2f, 3e), which is consistent with the results obtained from semiquantitative RT-PCR. The genes examined were clustered together on the basis of their transcript abundance at different time points upon exposure to Cd stress, which showed

Fig. 2  Relative expression of genes encoding antioxidant enzymes (CAT, SOD, APX, GR) (a), osmolytes (BADH1, P5CS, PDH) (b), PA biosynthetic (SAMDC, SPDS, SPMS) and catabolic (DAO) enzymes (c), TFs (TRAB-1, WRKY-71) and LEA (Osem) (d), and RbcS (e) under cadmium stress (1.5 mM ­CdCl2) for 6/12/24 h in the leaves of 10-day-old seedlings of sensitive (IR-64) and tolerant (Nonabokra) rice; the actin gene was used as an internal control. The vertical bars

indicate the standard deviation of three biological replicates. The data represented are means of three observations (n = 3) ± SE. The heat map and hierarchical cluster display the differential expression profile of various stress-responsive genes under cadmium stress. The fold change of expression is represented by the color bar with green, black, and red colors, representing lowest, medium, and highest expression levels, respectively (f). (Color figure online)

others 2010) was found to be more up regulated by ABA and Cd. The PDH transcript was up regulated during Cd exposure (Yooyongwech and others 2012). Among PA biosynthetic genes, SAMDC and SPDS were up regulated under all the stress conditions (Basu and others 2014; Saha and Giri 2017). The TRAB-1 transcript level was high under all the stresses except cold. The WRKY-71 transcript was induced by all the stresses. The Osem seemed to be highly induced under ABA in rice seedlings; however, the expression was reduced under Cd stress. These data indicated that there is a high level of cross-talk between diverse abiotic stresses and ABA is supposed to play a crucial role in Cd tolerance by modulating the expression of stress-responsive genes, possibly through cross-talk between multiple metabolic pathways.

Expression Profiling of Stress‑Responsive Genes from Rice Using Semi‑quantitative RT‑PCR Under Cadmium (1.5 mM ­CdCl2) Stress

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Journal of Plant Growth Regulation

Fig. 3  Relative expression of genes encoding antioxidant enzymes (CAT, SOD, APX, GR) (a), osmolytes (BADH1, P5CS, PDH) (b), PA biosynthetic (SAMDC, SPDS, SPMS) and catabolic (DAO) enzymes (c), TFs (TRAB-1), WRKY-71, and LEA (Osem) under cadmium stress (1.5  mM C ­ dCl2) (d), for 6/12/24  h in the roots of 10-day-old seedlings of sensitive (IR-64) and tolerant (Nonabokra) rice; the actin gene was used as an internal control. The vertical bars indicate the

standard deviation of three biological replicates. The data represented are means of three observations (n = 3) ± SE. The heat map and hierarchical cluster display the differential expression profiles of various stress-responsive genes under cadmium stress. The fold change of expression is represented by the color bar with green, black, and red colors, representing lowest, medium, and highest expression levels, respectively (e). (Color figure online)

the relatedness of the various data. The mean expression values per gene derived from the results of semi-quantitative RT-PCR are provided in Table S3 (electronic supplementary material). In silico analyses of the upstream region of all these genes showed the presence of abscisic acid responsive elements (ABREs) (data not shown), pointing towards their activation by ABA.

feature to ward off the more pronounced oxidative damage and ensure survival. The maximum transcript levels for APX, CAT, SOD, and GR in the leaves of Nonabokra were observed after 24 h. It is quite evident from Table S3 that in IR-64 leaves, the expressions of CAT, SOD, APX, and GR were increased by 2.7-, 1.9-, 2.1-, and 1.6-folds at 6 h of Cd stress as compared with control. Considering that the antioxidative enzymes are directly regulated by Cd-induced ROS levels, the much greater enhancement in APX, CAT, SOD, and GR transcripts in IR-64 at the initial stages of Cd exposure (6 h) may be indicative of a rapid signaling response, suggesting that these enzymes play a synergistic active role in ROS scavenging, immediately when the sensitive variety gets exposed to the metal stress (Fig. 2a). Therefore, early induction of antioxidant genes in the sensitive cultivar could act as an adaptive strategy to withstand deleterious effects of Cd stress. The activity of antioxidant enzymes, that is, peroxidase, CAT and SOD, in the leaves of Brassica juncea increased significantly in response to Cd (Irfan and others 2014). In B. juncea, the higher activity of antioxidative enzymes offers a greater detoxification efficiency which provides better resistance to a plant variety against heavy metal-induced oxidative stress (Mohamed and others 2012).

Genes Encoding Antioxidants Regarding the antioxidants, we have selected the most common scavenging enzymes like CAT (Higo and Higo 1996), SOD (Sakamoto and others 1992), APX (Chou and others 2012) and GR (Chou and others 2012), which have been well characterized with respect to tolerance mechanisms. The transcript levels of APX, CAT, SOD, and GR showed more pronounced induction in the leaves of IR-64 with increase in duration of stress, the maximum transcript level being recorded after 24 h of stress exposure for APX, CAT, and SOD, whereas for GR, the maximum transcript accumulation in IR-64 was recorded after 12 h (Fig. 2a). The significantly greater induction in transcripts for antioxidative enzymes in the sensitive variety appears to be a noteworthy adaptive

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In roots, the maximal induction in CAT transcript with stress was evident in both the varieties mostly after 24 h. However, a slight down regulation of CAT expression after 12 h of stress was noted in the roots of both the varieties. The SOD expression in roots followed a differential pattern in the two varieties, with the maximum level after 12 h in IR-64, and after 24 h in Nonabokra. The GR transcript in the roots of both the varieties showed a gradual decline with progressive increase in duration of stress exposure, attaining the minimum level at the highest time point (24 h) of stress exposure. The transcript level was comparatively higher in IR-64 roots during prolonged stress, suggesting that GR renders powerful intrinsic protection against oxidative damage in the roots of the sensitive variety (Fig. 3a). Because roots are the primary tissues to face major oxidative damages during stress, the maintenance of higher transcript levels of GR is an aspect that may be part of the more efficient tolerance mechanism of this variety to Cd through GSH regeneration and its involvement in the biosynthesis of phytochelatin (Roychoudhury and others 2012).

Genes Encoding Osmolytes Cd stress up regulated the expression of the P5CS gene in the leaves of both the varieties with respect to the untreated (control) condition; the enhanced transcript level showed a positive correlation with the increased duration of stress exposure, reaching the maximum level after 24 h. However, the transcript level was conspicuously higher in the tolerant cultivar at any time point of stress exposure, and even at the constitutive level. Although the expression of the PDH gene (encoding proline dehydrogenase, catalyzing the oxidation of Pro to P5C) was initially up regulated with Cd stress (after 6 h) in IR-64, several-folds down regulation could be noted in both the varieties, especially Nonabokra, with prolonged stress exposure, indicating that Pro accumulation or maintenance of a higher endogenous Pro level is a pre-requisite in Cd tolerance. However, the PDH transcript was less in Nonabokra, as compared to IR-64, at any time point of Cd stress. Similar induction in P5CS was observed in the roots of Cd-stressed seedlings of both the varieties, and the transcript level increased with duration of stress exposure, attaining a maximum after 12 h in IR-64 and 24 h in Nonabokra. The maximum transcript level was recorded in IR-64 after 12 h of Cd stress. The down regulation of PDH transcripts with increased duration of Cd stress was likewise noted in the roots of both the varieties. IR-64 showed higher PDH expression in the roots during 6 h of Cd stress exposure, thus showing a similar pattern as in leaves (Fig. 3b). The higher levels of Pro accumulation and P5CS mRNA expression were earlier reported in the salt-tolerant compared to the salt-sensitive cultivar of rice (Zhu and others 1998). The inhibition in PDH activity under dehydration

stress has also been observed earlier, so that the level of Pro is regulated at the transcriptional level by P5CS and PDH (Hayashi and others 2000). Antisense suppression of PDH is characterized by higher Pro content and cytoplasmic osmotic pressure (Kochetov and others 2004). The induction of BADH1 transcripts in the leaves of both IR-64 and Nonabokra was noteworthy, reaching the maximum level after 24 h of stress exposure (Fig. 2b). Similar induction in BADH1 expression was also noted in roots; however, the expression was the maximum during the initial phase (6 h) of Cd stress, and thereafter decreased with the duration of stress, attaining the minimum level after 12 h in IR-64 and 24 h in Nonabokra (Fig. 3b). The observation made by Fan and others (2012) emphasized the fact that plant tolerance to various abiotic stresses is improved through overexpression of the BADH1 gene in stress-sensitive cultivars. The transgenics also showed increased accumulation of Pro with a synchronized up regulation of ROS-scavenging genes, possibly through some unknown interconnecting cross-talk pathway. A similar mechanism involving up regulation of BADH1 in both rice varieties probably operates in our case, showing a direct correlation between Cd-tolerant capacity of each variety and BADH1 gene expression level.

Genes Encoding PA‑Metabolic Enzymes In our experiment, SAMDC expression in the leaves was induced to a higher extent in Nonabokra, especially with prolonged stress, that is, 12 and 24 h, as compared to IR-64 (Fig. 2c). In roots, the expression during the initial phase (6 h) of Cd stress was higher in Nonabokra, whereas for IR-64, maximum induction was noted after 12 h of stress imposition (Fig.  3c). The SPDS expression profile was different for the sensitive and tolerant variety. Although the transcript level in IR-64 peaked at 12 h of stress and then showed a sharp decrease at 24 h, maximum transcript accumulation in Nonabokra was noted after 24 h of stress application (Fig. 2c). In roots, early (6 h) induction of SPDS transcripts was noted in Nonabokra, whereas more prominent expression was noted in the sensitive variety IR-64 up to 12 h of stress exposure, following which a retarded expression was evident. SPMS showed a uniform pattern of expression in leaves of both the varieties, registering a gradual increase in the transcript level with increased time of Cd stress exposure. In Nonabokra roots, the SPMS transcript was more up regulated during 12 h, whereas early (6 h) induction was noted in IR-64 and maintained its level after 12 h of stress exposure (Fig. 3c). Higher endogenous Spd and Spm levels, reported in salt-tolerant rice, in comparison to the sensitive cultivars, were positively correlated with greater increase in antioxidant enzymes and hence more closely associated with stress tolerance in plants (Quinet and others 2010; Do and others 2013; Paul and others 2017).

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The PAs were documented to execute roles in heavy metal tolerance by acting as ROS scavengers, enhancing the antioxidant enzyme systems, accumulating Pro, and modulating the endogenous PA level by modifying the PA biosynthetic and catabolic enzymes (Zhao and Yang 2008). In our study, DAO expression was higher at the initial phase (6 h) of Cd stress in both the varieties, with more expression in the sensitive variety. With increased duration of stress, significant down regulation of DAO expression was observed after 24 h in Nonabokra, and after 12 h in IR-64 (Fig. 2c). The induction of DAO expression in roots was several folds higher in IR-64 after 6 and 12 h of Cd stress treatment, as compared to Nonabokra. However, there was a remarkable induction of DAO expression in Nonabokra roots after 24 h of stress, reaching almost the same level as in IR-64 (Fig. 3c). The activities of DAO increased significantly with the increase in Cd concentration in Potamogeton crispus (Yang and others 2010). In vitro shoots of a transgenic European pear, overexpressing MdSPDS1 showed attenuated susceptibility to heavy metal (Cu, Cd, Pb, Al, and Zn) (Wen and others 2010) stress, signifying the role of Spd in heavy metal tolerance.

Genes Encoding TFs and LEA Transcription factor Responsible for ABA Regulation 1 (TRAB-1), the rice bZIP factor, binds to the ABREs of target stress-inducible LEA genes like Osem and activates transcription through ABA-mediated signaling (Hattori and others 1995). The expression of TRAB-1 was up regulated by exogenously applied ABA in leaves, roots, and suspensioncultured cells of rice (Hobo and others 1999). Osem, a rice gene homologous to wheat Em, is expressed in vegetative tissues of rice in response to ABA (Hobo and others 1999). The expression of the genes encoding the WRKY-71 protein is induced by ABA. In ABA-treated, salt-tolerant rice variety Pokkali, the expression of WRKY-71 was enhanced (Basu and Roychoudhury 2014). The induction in TRAB-1, Osem, and WRKY-71 with Cd stress in the present investigation was significantly prominent in the leaves of both IR-64 and Nonabokra, and the transcript level peaked at the highest time point of stress exposure (24 h) (Fig. 2d). The difference in the level of TRAB-1 transcript between the two varieties was clear at the highest time point (24 h) of stress, with a higher level in IR-64, which is in accordance with the previous result (Oono and others 2014). The pattern of WRKY-71 expression was almost uniform in both the varieties at all the time points of Cd stress. For Osem, such a difference was noted during the early phase (6 h) of Cd stress, with Nonabokra registering a higher level of gene expression (Fig. 2d). In case of roots of Cd-treated seedlings, the TRAB1 expression, though induced after 6 h of stress, remained constant until 24 h for both varieties. Though WRKY-71 expression was largely induced by Cd stress in both the

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varieties, the induction was visibly sharper in the sensitive variety IR-64, particularly after 12 h of stress. For Osem, a sharp enhancement in expression was evident after 12 h in IR-64, followed by a drastic reduction in expression with enhanced time of stress exposure, whereas the induced Osem expression with Cd stress in Nonabokra was still maintained at 24 h (Fig. 3d).

Gene Encoding Ribulose‑1, 5‑Bisphosphate Carboxylase Small Subunit (RbcS) With respect to the expression of the RbcS (encoding the small subunit of RuBisCo, the major photosynthesizing enzyme) gene (Suzuki and others 2007), the transcript level in leaves was lowered with gradually increased duration of Cd stress; the extent of lowering was considerably higher in the sensitive variety IR-64, signifying greater sensitivity of the enzyme to Cd stress, as compared to Nonabokra, where the extent of reduction in the transcript level was lower at any time point of Cd stress (Fig. 2e).

Monitoring the Level of Accumulation of TRAB‑1 Protein in Response to Cadmium (1.5 mM ­CdCl2) Stress The ABRE-binding bZIP TF, TRAB-1, has been characterized as the master regulator in ABA-mediated transcription during seed development; the ABA-inducible expression of TRAB-1 in leaves and roots has also been reported earlier (Hobo and others 1999), which corresponds with our results. The TRAB-1 protein was found to be accumulated in both the cultivars; however, Nonabokra showed higher accumulation of TRAB-1 at the basal or constitutive level and during early (6 h) exposure of Cd stress; the level remained higher at 12 h, but declined at 24 h of stress treatment (Fig. 4). In IR-64, the TRAB-1 was more accumulated during 12 h and the maximum induction was noted at 24 h of stress imposition (Fig. 4). The gradual increase of TRAB-1 with time could be the adaptive strategy by the sensitive cultivar during stress encounter. The decrease in accumulation of TRAB-1 protein at 24 h in Nonabokra could be due to the inherent tolerant nature of the cultivar. Because of its preadaptive nature, Nonabokra may not require further synthesis of TRAB-1 protein with prolonged duration of stress, thereby restoring the protein accumulation to the basal level. Though an increased TRAB-1 transcript level was detected in Nonabokra after 24 h of stress, the decreased TRAB-1 protein accumulation and hence translational repression of the protein could be due to the involvement of some regulatory miRNA which can silence the mRNA transcript, thereby playing a pivotal role in gene regulation for better adaptation under stress conditions. Such regulation has been reported earlier by Brodersen and others (2008) in Arabidopsis,

Journal of Plant Growth Regulation

Fig. 4  Immunoblot analysis of TRAB-1 protein accumulation in response to cadmium stress (1.5 mM C ­ dCl2) for 6/12/24 h using total protein isolated from the leaves of 10-day-old seedlings of sensitive (IR-64) and tolerant (Nonabokra) rice

where miRNA-guided silencing constituted a widespread translational inhibitory component. Identification of such miRNA and deciphering their mechanistic involvement in regulating gene expression during Cd stress will probably enable us to delve deeper into the mechanism of regulation of TRAB-1 as well as other stress-inducible proteins for Cd tolerance. Overall, the inducibility of TRAB-1 together with the subsequent accumulation of the corresponding protein during Cd stress suggests that it could be the key signaling component in Cd stress responses, especially in the sensitive variety.

Conclusion We have performed a comprehensive transcript profiling of diverse groups of stress-inducible genes in rice varieties under a high level of Cd stress. The MPSS analysis along with microarray expression data under different developmental stages and stress conditions suggested the associated physiological function of these genes and a possible cross-talk between Cd and other abiotic stresses. Semiquantitative RT-PCR-based expression data suggested a difference in stress responses between sensitive and tolerant varieties. The pattern of expression for a particular gene, as observed in leaves, varied in most cases from that in roots, pointing towards a tissue-specific response. Moreover, the higher accumulation of TRAB-1 transcript and protein in the tolerant cultivar Nonabokra in response to Cd stress suggested that TRAB-1 is a nodal component in the Cd signaling pathway and could be mediating crosstalk between diverse metabolic pathways to withstand Cd stress. To the best of our knowledge, we first report here the role of TRAB-1 protein in response to Cd stress in rice cultivars. However, to acquire in-depth knowledge about the functional role of TRAB-1 in Cd-stress tolerance, overexpression of TRAB-1 through the transgenic approach requires attention in future. The expression of diverse groups of genes encoding antioxidative enzymes, osmolytes, and TFs can therefore be correlated with Cd tolerance, being controlled primarily through their

transcriptional activation and corresponding translation of the enzymes or proteins of diverse metabolic pathways in rice. Acknowledgements  The financial support from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India through the research grant (SR/FT/LS-65/2010), and from Council of Scientific and Industrial Research (CSIR), Government of India, through the project [38(1387)/14/EMR-II] to Dr. Aryadeep Roychoudhury is gratefully acknowledged. The authors are thankful to University Grants Commission (UGC), Government of India, for providing Junior Research Fellowship to Saikat Paul.

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