# Expression Profiling of Abiotic Stress-Inducible Genes in response to Multiple Stresses in Rice (Oryza sativa L.) Varieties with Contrasting Level of Stress Tolerance

Authors: Supratim Basu; Aryadeep Roychoudhury
Journal: BioMed Research International
 (2014)

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## Abstract

The present study considered transcriptional profiles and protein expression analyses from shoot and/or root tissues under three abiotic stress conditions, namely, salinity, dehydration, and cold, as well as following exogenous abscisic acid treatment, at different time points of stress exposure in threeindica rice varieties, IR-29 (salt sensitive), Pokkali, and Nonabokra (both salt tolerant). The candidate genes chosen for expression studies were HKT-1, SOS-3, NHX-1, SAPK5, SAPK7, NAC-1, Rab16A, OSBZ8, DREBP2, CRT/DREBP, WRKY24, and WRKY71, along with the candidate proteins OSBZ8, SAMDC, and GST. Gene expression profile revealed considerable differences between the salt-sensitive and salt-tolerant rice varieties, as the expression in the latter was higher even at the constitutive level, whereas it was inducible only by corresponding stress signals in IR-29. Whether in roots or shoots, the transcriptional responses to different stressors peaked following 24 h of stress/ABA exposure, and the transcript levels enhanced gradually with the period of exposure. The generality of stress responses at the transcriptional level was therefore time dependent. Heat map data also showed differential transcript abundance in the three varieties, correlating the observation with transcript profiling. In silico analysis of the upstream regions of all the genes represented the existence of conserved sequence motifs in single or multiple copies that are indispensable to abiotic stress response. Overall, the transcriptome and proteome analysis undertaken in the present study indicated that genes/proteins conferring tolerance, belonging to different functional classes, were overrepresented, thus providing novel insight into the functional basis of multiple stress tolerance in indica rice varieties. The present work will pave the way in future to select gene(s) for overexpression, so as to generate broad spectrum resistance to multiple stresses simultaneously.


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## Body

## 1. Introduction

Abiotic stresses like salinity, water deficit, chilling, and heavy metals adversely affect the growth and several physiological processes of plants. In general, low temperature mainly results in mechanical constraint, whereas salinity and drought exert their malicious effect by disrupting the ionic and osmotic equilibrium of the cell. The detrimental effects of excess salts are the consequences of water deficit that results from decreased osmotic/water potential of soil solution due to high solute concentration in the soil, as well as ion-specific stresses due to altered Na+/K+ ratios and Na+/Cl− ratios that are inimical to the plants [1]. The phytohormone abscisic acid (ABA) regulates desiccation tolerance in seeds as well as in vegetative tissue [2]. The endogenous ABA concentration increases in different plant tissues during drought, salinity, or cold induced oxidative stress. To cope with the unfavorable growth conditions, plants respond with a series of morphological, biochemical, and molecular adaptations, aiming at safeguarding the basic metabolic activities. It is now well known that the stress signal is first perceived at the membrane level by the receptors and then transduced into the cell to switch on the stress responsive genes for mediating stress tolerance. A number of genes have been reported to be induced by drought, high salinity, and low temperature stresses, and their products are thought to function in stress tolerance and response [3]. Many stress-inducible genes are responsive to both water stress and low temperature; some are induced only by water stress, while several genes respond only to low temperature. The stress induced changes in gene expression in turn may participate in the generation of hormones like ABA, salicylic acid, and ethylene. These gene products may amplify the initial signal and initiate a second round of signaling that may follow the same pathway or use altogether different components of signaling pathway. Under dehydrating conditions, when ABA levels are high, the endogenous hormone exerts a strong positive effect on root growth and a slight negative effect on shoot growth. The overall effect is a dramatic increase in the root: shoot ratio at low water potentials which along with the effect of ABA on stomatal closure helps the plants to cope with water stress. The long-term effects are mediated by ABA-induced gene expression. The ABA or stress-induced genes that are presumed to contribute to adaptive aspects of induced tolerance include genes encoding proteases, chaperonins, enzymes of sugar, proline or other compatible solute metabolism, enzyme encoding S-adenosylmethionine decarboxylase (SAMDC) catalyzing spermidine biosynthesis, ion and water channel proteins, enzymes that detoxify active oxygen species (like glutathione-S transferase, GST), and regulatory proteins such as transcription factors (TFs) and protein kinases [4]. The HKT-1 is the K+ transporter that maintains high affinity K+ loading and hence proper K+ level, crucial for cell osmoregulation and turgor maintenance during dehydration [5]. The salt overly sensitive 3 (SOS-3) is a calcium sensor protein that activates further downstream SOS-2 and SOS-1, along with a tonoplast Na+/H+ antiporter called NHX-1 [6]. Protein phosphorylation play pivotal roles in ABA signaling, with ABA-activated SnRK2 protein kinases like SAPK (SAPK1-10) showing varying expression patterns with various stresses [7]. The basic leucine zipper (bZIP) group of TFs like OSBZ8 was earlier reported to target the late embryogenesis abundant (LEA) protein, Rab16A, during salinity stress inindica rice varieties [8], through their binding to 8 bp conserved sequence called abscisic acid responsive element (ABRE). Another TF called NAC-1 characterized in rice [9] was reported to improve drought resistance and salt tolerance upon overexpression. The CRT/DREBP group of proteins are mostly regulated by cold, whereas some other group of DREBPs (DRE-binding proteins) are induced by drought and high salt stress, with the cross-talk between drought-inducible DREBP and cold-inducible DREBP occurring on a cis-acting element called dehydration responsive element (DRE) [10]. In recent times, another group of TFs, called WRKY, that binds to the 6 bp cognate binding site, designated as W boxes (TTGACC/T) of abiotic stress-inducible genes, act as major hubs at multiple levels in ABA-responsive signaling web [11]. AnAGAMOUS-LIKE 6 (AGL6) gene calledMADS6 interacts with other floral homeotic genes, playing essential role in floral development of rice [12]. All these genes having similar expression patterns contain common motifs in their promoter regions. These common promoter motifs are the key signatures for a family of coregulated genes and are usually present in the regions where complex protein interactions occur. However, in some cases, single motifs can bind to various TFs, thereby bringing the genes under multiple regulatory controls. Extensive studies on upstream regions of yeast promoters suggest that regulatory elements are commonly present in those regions. To achieve a more comprehensive understanding of the gene expression patterns of rice in response to ABA or salinity, drought, and cold stress, we performed gene profiling analysis of some of the key representative genes as mentioned above, using IR-29 (salt-sensitive) and Pokkali and Nonabokra (both salt tolerant) rice varieties. Since it is known that Nonabokra, Pokkali, and IR-29 have very high, moderate, and low levels of endogenous ABA, respectively, we wanted to compare the level of expression of stress-inducible genes in response to salinity, drought, cold and exogenous ABA treatment. A careful observation will reveal that the representative genes that have been selected for expression profiling in our study are from different categories: (i) protein kinases (SAPK5 andSAPK7) that activate the downstream TFs through phosphorylation; (ii) TFs belonging to distinct groups, namely,OSBZ8 (bZIP ABRE-binding factor),NAC-1 (containing DNA binding domain similar to the three proteins NAM (no apical meristem), ATAF1-2 and CUC2 (cup-shaped cotyledon),WRKYs (W-box binding factor), andDREBP andCRT/DREBP (both DRE-binding factors); (iii) target gene forOSBZ8, namely,Rab16A; (iv) ion transporters (HKT-1 (cell membrane-bound),SOS-3 (calcium-dependent), andNHX-1 (vacuolar membrane-bound)) that regulate ion homeostasis and K+/Na+ balance. Although the above genes chosen for our study have already been suggested earlier to play a role in tolerance to a particular type of abiotic stress, the precise role of each of these proteins and their cross-talks with other stress pathways is not clearly deciphered. The main purpose of our study was not to highlight the potential role of each protein in tolerance to a particular stress. Rather, we focused more on the comparative transcriptome profiling of all these diverse upstream and downstream components of stress signaling cascade in response to multiple stresses (like salinity, drought, and cold, as well as exogenous ABA application, which mimics oxidative stress), between salt-sensitive and salt-tolerant rice varieties. Rice indeed exhibits a wide genotypic variation with respect to stress tolerance, but the molecular-genetic mechanism of their tolerance is yet to be deciphered. There are very limited studies in rice on the comparative expression profiling of the same gene to multiple stresses simultaneously for different durations of stress exposure. A study of this kind would provide us a holistic knowledge regarding the integrated signaling circuit operative at the cellular level for rendering plant tolerance and survival. The inducibility pattern of diverse groups of genes with increase in the duration of stress exposure is also undoubtedly a valuable knowledge in stress genomics, especially for gene manipulation programs.


## 2. Materials and Methods

### 2.1. Cis-Acting Regulatory Elements in the Promoter Regions of the Concerned Genes

The promoter regions of the selected genes comprising 910 bp upstream and 90 bp downstream sequences obtained from Plant Promoter Db were used as an input sequence for identifying cis-acting regulatory elements (CAREs). The software programs used were PLACE (http://www.dna.affrc.go.jp/PLACE/) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The common regulatory motifs in the upstream sequences of these genes were identified by NSITE-PL.


### 2.2. Plant Materials, Growth Conditions, and Stress Treatments

Seeds ofOryza sativa L. cv. IR-29, Pokkali, and Nonabokra were surface-sterilized with 0.1% (w/v) HgCl2 for 20 min, washed extensively, imbibed in deionised water for 6 to 8 h, and allowed to germinate over water-soaked sterile gauge in Petri dishes at 37°C in dark for 3 days. The germinated seedlings were grown in presence of 0.25X Murashige and Skoog (MS) complete media at 32°C under 16 h light and 8 h dark photoperiodic cycle with 50% relative humidity and 700 μmol photons m−2 s−1 for the desired period in a plant growth chamber (NIPPON, LHP-100-RDS, Tokyo, Japan). For stress imposition, the plants were treated with 0.25X MS medium supplemented with 200 mM NaCl or 100 μM ABA or 20% (w/v) polyethylene glycol (PEG) or the seedlings were exposed to 4°C for the desired periods. Following stress treatment, the plants were washed thoroughly; roots and leaves were harvested; samples of equal fresh weight were frozen in liquid nitrogen and immediately homogenized for the preparation of total RNA or total protein.


### 2.3. Semi-Quantitative RT- (Reverse Transcriptase-) Polymerase Chain Reaction (PCR) Analyses

Total RNA was isolated from the young leaf tissues or roots of control and salt-treated (200 mM NaCl)/ABA-treated (100μM)/PEG-treated (20%)/cold-treated (4°C) seedlings of IR-29, Pokkali, and Nonabokra [13]. RNA samples were treated with RNase-free DNase I (Boehringer Mannheim). The cDNA was synthesized using 5 μg of total RNA. The semiquantitative RT-PCR experiments were conducted in triplicates using GoTaq qPCR Master Mix (Promega) withactin as standard and other gene-specific primers in a CFX-96 Bio-Rad thermocycler (Bio-Rad). Increasing temperature from 55°C to 95°C was used for melt curve analysis. The untranscribed RNA was also run as negative control. The relative difference in expression for each sample in individual experiments was determined by normalizing the Ct value for each gene against the Ct value ofactin and was calculated relative to a calibrator using the equation 2
-
Δ
Δ
Ct [14]. The data was imported in TM4 microarray software suite [15], normalized using GC-RMA algorithm to generate the heat map. Average of three biological replicates was used to get each expression value. The RT-PCR withactin gene was performed as an internal control to normalize the data.


### 2.4. Protein Immunoblot Analyses

Buffer-soluble protein extracts were prepared from the young leaf tissues of control and NaCl (200 mM)/ABA (100μM)/PEG (20%)/cold (4°C) treated seedlings of IR-29, Pokkali, and Nonabokra [16]. After transferring 50 μg of total protein from each sample electrophoretically to a PVDF membrane, it was blocked with 1% (w/v) nonfat dried milk in 1X Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 for 2 h at room temperature (25°C), washed repeatedly with the same buffer, and incubated with antibody against either OSBZ8 or SAMDC or GST, each at 1 : 1000 dilution, overnight at 4°C. The membrane was next incubated with goat anti-rabbit IgG alkaline phosphatase conjugate at 1 : 1000 dilutions. The cross-reacted bands of OSBZ8 or SAMDC or GST were detected using 4-nitroblue-tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrates.


## 2.1. Cis-Acting Regulatory Elements in the Promoter Regions of the Concerned Genes

The promoter regions of the selected genes comprising 910 bp upstream and 90 bp downstream sequences obtained from Plant Promoter Db were used as an input sequence for identifying cis-acting regulatory elements (CAREs). The software programs used were PLACE (http://www.dna.affrc.go.jp/PLACE/) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The common regulatory motifs in the upstream sequences of these genes were identified by NSITE-PL.


## 2.2. Plant Materials, Growth Conditions, and Stress Treatments

Seeds ofOryza sativa L. cv. IR-29, Pokkali, and Nonabokra were surface-sterilized with 0.1% (w/v) HgCl2 for 20 min, washed extensively, imbibed in deionised water for 6 to 8 h, and allowed to germinate over water-soaked sterile gauge in Petri dishes at 37°C in dark for 3 days. The germinated seedlings were grown in presence of 0.25X Murashige and Skoog (MS) complete media at 32°C under 16 h light and 8 h dark photoperiodic cycle with 50% relative humidity and 700 μmol photons m−2 s−1 for the desired period in a plant growth chamber (NIPPON, LHP-100-RDS, Tokyo, Japan). For stress imposition, the plants were treated with 0.25X MS medium supplemented with 200 mM NaCl or 100 μM ABA or 20% (w/v) polyethylene glycol (PEG) or the seedlings were exposed to 4°C for the desired periods. Following stress treatment, the plants were washed thoroughly; roots and leaves were harvested; samples of equal fresh weight were frozen in liquid nitrogen and immediately homogenized for the preparation of total RNA or total protein.


## 2.3. Semi-Quantitative RT- (Reverse Transcriptase-) Polymerase Chain Reaction (PCR) Analyses

Total RNA was isolated from the young leaf tissues or roots of control and salt-treated (200 mM NaCl)/ABA-treated (100μM)/PEG-treated (20%)/cold-treated (4°C) seedlings of IR-29, Pokkali, and Nonabokra [13]. RNA samples were treated with RNase-free DNase I (Boehringer Mannheim). The cDNA was synthesized using 5 μg of total RNA. The semiquantitative RT-PCR experiments were conducted in triplicates using GoTaq qPCR Master Mix (Promega) withactin as standard and other gene-specific primers in a CFX-96 Bio-Rad thermocycler (Bio-Rad). Increasing temperature from 55°C to 95°C was used for melt curve analysis. The untranscribed RNA was also run as negative control. The relative difference in expression for each sample in individual experiments was determined by normalizing the Ct value for each gene against the Ct value ofactin and was calculated relative to a calibrator using the equation 2
-
Δ
Δ
Ct [14]. The data was imported in TM4 microarray software suite [15], normalized using GC-RMA algorithm to generate the heat map. Average of three biological replicates was used to get each expression value. The RT-PCR withactin gene was performed as an internal control to normalize the data.


## 2.4. Protein Immunoblot Analyses

Buffer-soluble protein extracts were prepared from the young leaf tissues of control and NaCl (200 mM)/ABA (100μM)/PEG (20%)/cold (4°C) treated seedlings of IR-29, Pokkali, and Nonabokra [16]. After transferring 50 μg of total protein from each sample electrophoretically to a PVDF membrane, it was blocked with 1% (w/v) nonfat dried milk in 1X Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 for 2 h at room temperature (25°C), washed repeatedly with the same buffer, and incubated with antibody against either OSBZ8 or SAMDC or GST, each at 1 : 1000 dilution, overnight at 4°C. The membrane was next incubated with goat anti-rabbit IgG alkaline phosphatase conjugate at 1 : 1000 dilutions. The cross-reacted bands of OSBZ8 or SAMDC or GST were detected using 4-nitroblue-tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrates.


## 3. Results and Discussion

Salinity, drought, and low temperature are the key environmental factors affecting plant species distribution and crop productivity. The responses of plants to various abiotic stresses have been an important subject of physiological studies. The major events of plant response to dehydration stresses are perception and transduction of the stress signals through signaling components, resulting in activation of a large number of stress-related genes and synthesis of diverse functional proteins that finally lead to various physiological and metabolic responses. The determination of the biological functions of these candidate genes is among the greatest challenges for postgenomic research, which is targeted mostly towards determination of their expression profiles. A multidisciplinary approach involving identification of novel genes and studies on structural similarity, expression profile, and mutant phenotypes is required for assignment of gene functions. In the present communication, we have initiated transcriptional monitoring of stress-inducible genes from rice in response to dehydration, high salinity, low temperature, and ABA application. Such an understanding will provide us with the basis of effective engineering strategies to improve stress tolerance.The gene products are normally classified into two groups. The first group consists of functional proteins or proteins that probably function in stress tolerance. They can be late embryogenesis abundant (LEA) proteins (like Rab16A that belongs to group 2), dehydration-inducible (RD and ERD) proteins, cold-acclimation (COR) proteins, ion transporters (HKT-1) and channel proteins (NHX-1), osmoprotectant biosynthesis-related genes and reactive oxygen species- (ROS-) detoxification enzymes (peroxidase, catalase, superoxide dismutase, or glutathione-S-transferase), and molecular chaperones. The LEA proteins are thought to play a role as desiccation protectants and have been shown to be involved in protecting macromolecules, such as enzymes and lipids. The second group consists of regulatory proteins, namely, MYB, NAC, bZIP (like OSBZ8, TRAB-1) and DREBP or CRT/DREBP family of TFs, protein kinases (SAPKs), and protein phosphatases [17].Plant species show varietal differences so far as their tolerance mechanism is concerned, with the tolerant varieties showing better resistance than the susceptible ones. In general, rice plants are more sensitive to salinity stress at young seedling stages than at reproductive stages. Some traditional cultivars and landraces of rice are more tolerant than many elite cultivars to various abiotic stresses. These resistant genotypes are considered to be good sources of tolerance traits. The examples of traditional genotypes that are tolerant to high salinity are the Indian landraces, Nonabokra, and Pokkali [4]. One aspect of response to stress is at the transcriptional level, which involves alteration in expression of genes. Since the molecular regulation and function of most of these genes to diverse stresses are yet to be fully understood, we carried out analyses of expression profiles of these genes during 0, 6, 12, or 24 h durations of salinity, dehydration, cold and ABA treatment, their tissue-specific expression under stress, and their differential expression pattern in salt-sensitive (IR-29) and salt-tolerant (Pokkali and Nonabokra) rice varieties. In the nutshell, taking rice (the major staple food in Asian countries) as our model crop, this work focuses on (i) comparative transcriptome profile of stress-inducible genes between salt-sensitive and salt tolerantindica rice varieties and (ii) gene expression profile in response to multiple stresses at different time points.


### 3.1.In Silico Identification of Cis-Acting Elements in the Promoters and Heat Maps of the Genes Examined

The ABA-inducibility of the stress-responsive genes is conferred by the presence of single or multiple copies of ABREs, 8–10 bp conserved sequence with ACGT core, to which the bZIP group of TFs bind. However, gene expression during drought or cold stress may occur in ABA-independent pathway as well, and such genes are known to possess DREs (TACCGACAT) and similar cis-acting sequences called C-repeat (CRT) and low temperature responsive element (LTRE), with all these sequences containing a TGG/ACCGAC motif, to which different groups of DREBPs bind [3, 18]. A putative MYB (TAACTG) motif was earlier reported to act as cis-acting DRE [19]. The MYB proteins, a superfamily of TFs, that play regulatory roles in developmental processes and defense responses in plants are reported to bind to the consensus sequences of MYB-cis-acting elements. The WRKY TFs, reported to mediate abiotic plant responses to freezing, wounding, oxidative stress, drought, salinity, cold, and heat, bind to 6 bp cis-elements called W-box (TTGACC/T). In order to study the differential regulation of a few selected genes in the salt-sensitive and salt-tolerant rice varieties, we have primarily performed anin silico analysis of the promoter regions of those genes using PLACE database and PlantCARE, which revealed the presence of a number of putative cis-elements, namely, ABRE, DRE, MYB, W-box, and LTRE, associated with various environmental signals like salinity, drought, cold, and ABA (Table 1). The presence of such elements in the upstream region of these stress-inducible genes suggests that the genes may be regulated by ABRE-binding factors (ABFs) or DREBPs, MYBs, LTREs, and so forth. In the light of this observation, we have actually exposed the rice plants to different abiotic stress treatments and studied the expression of several target genes. Although bioinformatic identification of particular DNA sequence motifs may not demonstrate a functionally active site, thein silico analyses detecting the presence of particular cis-acting sequence(s) give an indication beforehand regarding the nature of the stress signal that might induce the expression of the particular gene; for example, presence of one or multiple ABREs in the upstream region shows that the gene will be triggered by ABA treatment, salinity, drought, and so on. To gain insight into the effect of the varying conditions/treatments on transcript expression profiles, we performed a hierarchical clustering analysis of all the transcripts in control as well as the treated samples across different experiments. This clustering revealed the relatedness of the various data. The differential expression of genes obtained from semiquantitative RT-PCR analysis was represented as heat map and was clustered. The heat map generated forin silico expression profiling showed the differential transcript abundance of the candidate genes in the root and leaf of IR-29, Pokkali, and Nonabokra (Figures 1(a), 1(b), 1(c), 1(d), and 1(e)) which is consistent with the results obtained from semiquantitative RT-PCR. Furthermore, here we have also shown that the genes under investigations cluster together based on their induction at different time points upon exposure to various stresses. The expression analysis of genes under study was represented in the scale of 0 to 2 in the blue-black-yellow colour scheme.Table 1
Cis-acting regulatory elements in the promoter regions of the concerned genes.
cDNAs
TATA
CAAT
ABRE
DRE
MYB
W-Box
LTRE
HKT-1
3
9
0
0
4
4
0
SOS-3
2
5
0
0
2
5
0
NHX-1
3
4
5
2
1
6
4
SAPK5
2
1
0
0
6
0
0
SAPK7
1
8
1
0
1
3
4
NAC-1
1
4
1
2
5
8
16
Rab16A
2
13
2
2
5
3
12
DREBP2
N/A
N/A
0
4
4
0
5
CRT/DREBP
1
5
8
3
8
5
2
OSBZ8
2
5
1
0
0
12
1
WRKY24
N/A
9
0
0
10
16
0
WRKY71
5
2
0
1
3
15
1Heat map representation of the differential expression of the candidate genes in response to NaCl (a), PEG-mediated dehydration (b), cold (c), and ABA (d) in the leaves ((a)–(d)) and roots (e). The expression of the genes is represented in the heat map in the colour scale of 0–2 in blue-yellow-black colour scheme. The genes are represented in columns while conditions are shown in rows.
(a)
(b)
(c)
(d)
(e)


### 3.2. Monitoring the Expression Profile in response to NaCl Stress (200 mM)

With respect to salinity stress, the genesHKT-1,SOS-3,SAPK5, andOSBZ8 showed better induction in expression in the leaves of all the three varieties, with increase in duration of stress, the maximum induction being recorded at 24 h of NaCl treatment. With the exception ofCRT/DREBP, the expression of all the genes in leaves was recorded the maximum in Nonabokra, with all the genes showing constitutive expression. Though the tolerant cultivar Pokkali also showed gene expression at the constitutive level, the induction was considerably high in the susceptible variety IR-29, especially for the genes likeHKT-1,SAPK5,Rab16A, andOSBZ8 (Figure 2(a)). In roots, the genesHKT-1,SAPK5, andDREBP2 showed high level of expression with salinity stress in all the three varieties. Of the three varieties, Nonabokra again showed the highest level of expression of all the genes tested. The induction inHKT-1,SAPK5,Rab16A, andDREBP2 was recorded especially in the salt-sensitive cultivar (Figure 2(b)). TheMADS6 gene was unaffected by stress treatment, though the level of the transcript was detected higher in the leaves of the tolerant varieties Pokkali and Nonabokra (Figure 2(a)).Semiquantitative RT-PCR analysis showing differential gene expression in leaf tissues (a) and roots (b) of 12-day-old seedlings of IR-29 (salt sensitive), Pokkali, and Nonabokra (both salt tolerant) rice cultivars in response to salinity stress (200 mM NaCl); salt stress was imposed for 6/16/24 hours (in IR-29 and Pokkali) and for 6/24 hours (in Nonabokra) to detect transcript level in leaves, whereas for roots, the durations of stress treatment were 24 hours (in IR-29 and Pokkali) and 6/24 hours (in Nonabokra); the expression of each gene was compared relative to its expression in control C (untreated) sample (0 h).
(a)
(b)


### 3.3. Monitoring the Expression Profile in response to PEG (20%)-Mediated Dehydration Stress

With dehydration stress, especially the transcripts ofSOS-3,SAPK5, andOSBZ8 were greatly accumulated in the leaves of all the varieties; the maximum accumulation being recorded after 24 h of stress in both IR-29 and Pokkali, although the induction over control was higher for the salt-sensitive variety for all the genes exceptHKT-1. Nonabokra exhibited constitutive expression of all the genes, with lesser inducibility. TheMADS6 gene showed no variation in expression with dehydration stress, as compared to control (Figure 3(a)). In case of roots,HKT-1,SOS-3,SAPK5,OSBZ8, andCRT/DREBP showed better inducibility with dehydration stress. Moreover, all the genes, exceptDREBP2, showed higher expression in Pokkali and Nonabokra, following 24 h stress treatment, although the induction over control was sharper in IR-29. The genesSAPK7,Rab16A,NAC-1, andDREBP2 showed very weak response to dehydration stress in roots. TheMADS6 gene expression was almost unaffected by stress (Figure 3(b)).Semiquantitative RT-PCR analysis showing differential gene expression in leaf tissues (a) and roots (b) of 12-day-old seedlings of IR-29 (salt sensitive), Pokkali and Nonabokra (both salt tolerant) rice cultivars in response to PEG (20%)-mediated dehydration; stress was imposed for 6/24 hours to detect the transcript level in leaves, whereas for roots, the durations of stress treatment were 24 hours (in IR-29 and Pokkali) and 6/24 hours (in Nonabokra); the expression of each gene was compared relative to its expression in control C (untreated) sample (0 h).
(a)
(b)


### 3.4. Monitoring the Expression Profile in response to Cold (4°C) Stress

With cold stress, the genes likeSOS-3,SAPK5,SAPK7, andDREBP2 showed better expression in the leaves of all the three varieties, though the expression level was more pronounced in the two tolerant cultivars, where the expression was constitutive. The genes likeHKT-1,NHX-1, andRab16A showed better response in Nonabokra than Pokkali. TheMADS6 gene showed uniform expression in all the varieties, being unaffected by stress imposition (Figure 4(a)). In case of roots, the three varieties showed similar expression level for all the genes, following 24 h stress treatment, though the transcript level was higher in Pokkali at the background condition (Figure 4(b)).Semiquantitative RT-PCR analysis showing differential gene expression in leaf tissues (a) and roots (b) of 12-day-old seedlings of IR-29 (salt sensitive), Pokkali, and Nonabokra (both salt tolerant) rice cultivars in response to cold (4°C); stress was imposed for 6/24 hours to detect the transcript level in leaves, whereas for roots, the durations of stress treatment were 24 hours (in IR-29 and Pokkali) and 6/24 hours (in Nonabokra); the expression of each gene was compared relative to its expression in control C (untreated) sample (0 h).
(a)
(b)


### 3.5. Monitoring the Expression Level with ABA (100μM) Treatment

The inducibility by ABA was clear from the gene expression pattern in both IR-29 and Pokkali, the genes likeHKT-1,NHX-1,NAC-1,SAPK5,Rab16A,DREBP2,CRT/DREBP, andWRKY71 showing better response andSAPK7 orWRKY24 with feeble induction (Figure 5(a)). The gene expression was constitutive in Pokkali, thus showing a uniform trend. In case of both roots and leaves of ABA-treated Nonabokra seedlings, the induction in expression of all the genes (exceptNAC-1,WRKY24, andMADS6) over control was noteworthy (Figure 5(b)).Semiquantitative RT-PCR analysis showing differential gene expression in response to ABA (100μM) in leaf tissues of 12-day-old seedlings of IR-29 (salt sensitive) and Pokkali (salt tolerant) rice cultivars (a); comparative gene expression in the roots and leaves of 12-day-old seedlings of Nonabokra (b); the expression of each gene was compared relative to its expression in control (untreated) sample (0 h).
(a)
(b)


### 3.6. Monitoring the Expression Level of OSBZ8, SAMDC, and GST Proteins in response to Multiple Stresses

All the three proteins examined were found to be expressed in response to salinity, ABA, drought, and cold stress. The SAMDC protein level was found to be comparatively higher with ABA treatment and particularly induced after 24 h of cold treatment. The antioxidative enzyme GST was better triggered with dehydration, cold, and ABA treatment, especially for longer duration of stress exposure. The OSBZ8 protein expression on the other hand was nearly uniform with all the stresses (Figure6).Immunoblot analysis to study the protein expression in response to salinity (200 mM NaCl), PEG (20%)-mediated dehydration, ABA (100μM), and cold (4°C) using total protein isolated from 12-day-old leaves of Nonabokra and the antibodies raised against (a) OSBZ8 (38 kDa), (b) SAMDC (38 kDa), and (c) GST (28 kDa).
(a)
(b)
(c)


## 3.1.In Silico Identification of Cis-Acting Elements in the Promoters and Heat Maps of the Genes Examined

The ABA-inducibility of the stress-responsive genes is conferred by the presence of single or multiple copies of ABREs, 8–10 bp conserved sequence with ACGT core, to which the bZIP group of TFs bind. However, gene expression during drought or cold stress may occur in ABA-independent pathway as well, and such genes are known to possess DREs (TACCGACAT) and similar cis-acting sequences called C-repeat (CRT) and low temperature responsive element (LTRE), with all these sequences containing a TGG/ACCGAC motif, to which different groups of DREBPs bind [3, 18]. A putative MYB (TAACTG) motif was earlier reported to act as cis-acting DRE [19]. The MYB proteins, a superfamily of TFs, that play regulatory roles in developmental processes and defense responses in plants are reported to bind to the consensus sequences of MYB-cis-acting elements. The WRKY TFs, reported to mediate abiotic plant responses to freezing, wounding, oxidative stress, drought, salinity, cold, and heat, bind to 6 bp cis-elements called W-box (TTGACC/T). In order to study the differential regulation of a few selected genes in the salt-sensitive and salt-tolerant rice varieties, we have primarily performed anin silico analysis of the promoter regions of those genes using PLACE database and PlantCARE, which revealed the presence of a number of putative cis-elements, namely, ABRE, DRE, MYB, W-box, and LTRE, associated with various environmental signals like salinity, drought, cold, and ABA (Table 1). The presence of such elements in the upstream region of these stress-inducible genes suggests that the genes may be regulated by ABRE-binding factors (ABFs) or DREBPs, MYBs, LTREs, and so forth. In the light of this observation, we have actually exposed the rice plants to different abiotic stress treatments and studied the expression of several target genes. Although bioinformatic identification of particular DNA sequence motifs may not demonstrate a functionally active site, thein silico analyses detecting the presence of particular cis-acting sequence(s) give an indication beforehand regarding the nature of the stress signal that might induce the expression of the particular gene; for example, presence of one or multiple ABREs in the upstream region shows that the gene will be triggered by ABA treatment, salinity, drought, and so on. To gain insight into the effect of the varying conditions/treatments on transcript expression profiles, we performed a hierarchical clustering analysis of all the transcripts in control as well as the treated samples across different experiments. This clustering revealed the relatedness of the various data. The differential expression of genes obtained from semiquantitative RT-PCR analysis was represented as heat map and was clustered. The heat map generated forin silico expression profiling showed the differential transcript abundance of the candidate genes in the root and leaf of IR-29, Pokkali, and Nonabokra (Figures 1(a), 1(b), 1(c), 1(d), and 1(e)) which is consistent with the results obtained from semiquantitative RT-PCR. Furthermore, here we have also shown that the genes under investigations cluster together based on their induction at different time points upon exposure to various stresses. The expression analysis of genes under study was represented in the scale of 0 to 2 in the blue-black-yellow colour scheme.Table 1
Cis-acting regulatory elements in the promoter regions of the concerned genes.
cDNAs
TATA
CAAT
ABRE
DRE
MYB
W-Box
LTRE
HKT-1
3
9
0
0
4
4
0
SOS-3
2
5
0
0
2
5
0
NHX-1
3
4
5
2
1
6
4
SAPK5
2
1
0
0
6
0
0
SAPK7
1
8
1
0
1
3
4
NAC-1
1
4
1
2
5
8
16
Rab16A
2
13
2
2
5
3
12
DREBP2
N/A
N/A
0
4
4
0
5
CRT/DREBP
1
5
8
3
8
5
2
OSBZ8
2
5
1
0
0
12
1
WRKY24
N/A
9
0
0
10
16
0
WRKY71
5
2
0
1
3
15
1Heat map representation of the differential expression of the candidate genes in response to NaCl (a), PEG-mediated dehydration (b), cold (c), and ABA (d) in the leaves ((a)–(d)) and roots (e). The expression of the genes is represented in the heat map in the colour scale of 0–2 in blue-yellow-black colour scheme. The genes are represented in columns while conditions are shown in rows.
(a)
(b)
(c)
(d)
(e)


## 3.2. Monitoring the Expression Profile in response to NaCl Stress (200 mM)

With respect to salinity stress, the genesHKT-1,SOS-3,SAPK5, andOSBZ8 showed better induction in expression in the leaves of all the three varieties, with increase in duration of stress, the maximum induction being recorded at 24 h of NaCl treatment. With the exception ofCRT/DREBP, the expression of all the genes in leaves was recorded the maximum in Nonabokra, with all the genes showing constitutive expression. Though the tolerant cultivar Pokkali also showed gene expression at the constitutive level, the induction was considerably high in the susceptible variety IR-29, especially for the genes likeHKT-1,SAPK5,Rab16A, andOSBZ8 (Figure 2(a)). In roots, the genesHKT-1,SAPK5, andDREBP2 showed high level of expression with salinity stress in all the three varieties. Of the three varieties, Nonabokra again showed the highest level of expression of all the genes tested. The induction inHKT-1,SAPK5,Rab16A, andDREBP2 was recorded especially in the salt-sensitive cultivar (Figure 2(b)). TheMADS6 gene was unaffected by stress treatment, though the level of the transcript was detected higher in the leaves of the tolerant varieties Pokkali and Nonabokra (Figure 2(a)).Semiquantitative RT-PCR analysis showing differential gene expression in leaf tissues (a) and roots (b) of 12-day-old seedlings of IR-29 (salt sensitive), Pokkali, and Nonabokra (both salt tolerant) rice cultivars in response to salinity stress (200 mM NaCl); salt stress was imposed for 6/16/24 hours (in IR-29 and Pokkali) and for 6/24 hours (in Nonabokra) to detect transcript level in leaves, whereas for roots, the durations of stress treatment were 24 hours (in IR-29 and Pokkali) and 6/24 hours (in Nonabokra); the expression of each gene was compared relative to its expression in control C (untreated) sample (0 h).
(a)
(b)


## 3.3. Monitoring the Expression Profile in response to PEG (20%)-Mediated Dehydration Stress

With dehydration stress, especially the transcripts ofSOS-3,SAPK5, andOSBZ8 were greatly accumulated in the leaves of all the varieties; the maximum accumulation being recorded after 24 h of stress in both IR-29 and Pokkali, although the induction over control was higher for the salt-sensitive variety for all the genes exceptHKT-1. Nonabokra exhibited constitutive expression of all the genes, with lesser inducibility. TheMADS6 gene showed no variation in expression with dehydration stress, as compared to control (Figure 3(a)). In case of roots,HKT-1,SOS-3,SAPK5,OSBZ8, andCRT/DREBP showed better inducibility with dehydration stress. Moreover, all the genes, exceptDREBP2, showed higher expression in Pokkali and Nonabokra, following 24 h stress treatment, although the induction over control was sharper in IR-29. The genesSAPK7,Rab16A,NAC-1, andDREBP2 showed very weak response to dehydration stress in roots. TheMADS6 gene expression was almost unaffected by stress (Figure 3(b)).Semiquantitative RT-PCR analysis showing differential gene expression in leaf tissues (a) and roots (b) of 12-day-old seedlings of IR-29 (salt sensitive), Pokkali and Nonabokra (both salt tolerant) rice cultivars in response to PEG (20%)-mediated dehydration; stress was imposed for 6/24 hours to detect the transcript level in leaves, whereas for roots, the durations of stress treatment were 24 hours (in IR-29 and Pokkali) and 6/24 hours (in Nonabokra); the expression of each gene was compared relative to its expression in control C (untreated) sample (0 h).
(a)
(b)


## 3.4. Monitoring the Expression Profile in response to Cold (4°C) Stress

With cold stress, the genes likeSOS-3,SAPK5,SAPK7, andDREBP2 showed better expression in the leaves of all the three varieties, though the expression level was more pronounced in the two tolerant cultivars, where the expression was constitutive. The genes likeHKT-1,NHX-1, andRab16A showed better response in Nonabokra than Pokkali. TheMADS6 gene showed uniform expression in all the varieties, being unaffected by stress imposition (Figure 4(a)). In case of roots, the three varieties showed similar expression level for all the genes, following 24 h stress treatment, though the transcript level was higher in Pokkali at the background condition (Figure 4(b)).Semiquantitative RT-PCR analysis showing differential gene expression in leaf tissues (a) and roots (b) of 12-day-old seedlings of IR-29 (salt sensitive), Pokkali, and Nonabokra (both salt tolerant) rice cultivars in response to cold (4°C); stress was imposed for 6/24 hours to detect the transcript level in leaves, whereas for roots, the durations of stress treatment were 24 hours (in IR-29 and Pokkali) and 6/24 hours (in Nonabokra); the expression of each gene was compared relative to its expression in control C (untreated) sample (0 h).
(a)
(b)


## 3.5. Monitoring the Expression Level with ABA (100μM) Treatment

The inducibility by ABA was clear from the gene expression pattern in both IR-29 and Pokkali, the genes likeHKT-1,NHX-1,NAC-1,SAPK5,Rab16A,DREBP2,CRT/DREBP, andWRKY71 showing better response andSAPK7 orWRKY24 with feeble induction (Figure 5(a)). The gene expression was constitutive in Pokkali, thus showing a uniform trend. In case of both roots and leaves of ABA-treated Nonabokra seedlings, the induction in expression of all the genes (exceptNAC-1,WRKY24, andMADS6) over control was noteworthy (Figure 5(b)).Semiquantitative RT-PCR analysis showing differential gene expression in response to ABA (100μM) in leaf tissues of 12-day-old seedlings of IR-29 (salt sensitive) and Pokkali (salt tolerant) rice cultivars (a); comparative gene expression in the roots and leaves of 12-day-old seedlings of Nonabokra (b); the expression of each gene was compared relative to its expression in control (untreated) sample (0 h).
(a)
(b)


## 3.6. Monitoring the Expression Level of OSBZ8, SAMDC, and GST Proteins in response to Multiple Stresses

All the three proteins examined were found to be expressed in response to salinity, ABA, drought, and cold stress. The SAMDC protein level was found to be comparatively higher with ABA treatment and particularly induced after 24 h of cold treatment. The antioxidative enzyme GST was better triggered with dehydration, cold, and ABA treatment, especially for longer duration of stress exposure. The OSBZ8 protein expression on the other hand was nearly uniform with all the stresses (Figure6).Immunoblot analysis to study the protein expression in response to salinity (200 mM NaCl), PEG (20%)-mediated dehydration, ABA (100μM), and cold (4°C) using total protein isolated from 12-day-old leaves of Nonabokra and the antibodies raised against (a) OSBZ8 (38 kDa), (b) SAMDC (38 kDa), and (c) GST (28 kDa).
(a)
(b)
(c)


## 4. Conclusion

The intercellular or endogenous ABA concentration in leaf increased 10–50 folds within a few hours of the onset of water deficit, caused by high osmoticum, high NaCl, or drying. It has been reported that in barley and bean, drought stress could result in high levels of ABA by 57–160 times, compared with those of controls. During osmotic shock (150 mM NaCl), the peak ABA concentration was 30-fold higher for Nonabokra and sixfold higher for Pokkali, the two salt-tolerant rice varieties, as compared to the salt-sensitive Taichung Native-1 (TN-1) [20]. The maximum level of accumulation oflea transcripts and LEA proteins coincided with the maximum accumulation of endogenous ABA. A cDNA cloneoslea3 encoding a group 3 LEA protein from the roots of rice seedlings [21] was found to accumulate to higher levels in the roots of two salt-tolerant varieties, compared to a salt-sensitive variety in response to ABA. Exogenous application of ABA and exposure to salt shock (150 mM NaCl) rapidly inducedoslea3 transcript accumulation in seedling roots. Theoslea3 expression was compared for the salt-tolerant variety Pokkali and the salt-sensitive cultivar TN-1. Higher maximal mRNA levels were found in the roots of the tolerant variety, also declining less rapidly upon sustained salt shock, concomitant with a delayed drop in shoot water content. The results suggested that a differential regulation ofoslea3 expression is an aspect of the varietal differences in salt stress tolerance. In accordance with these earlier observations, the analysis of our transcriptomal data revealed considerable varietal differences, so far as responses of genes to multiple environmental stresses are concerned. Our observation showed that all the candidate genes incurred variations in their expression patterns in response to one or more stresses so that the varietal difference in expression of all the genes and proteins was noteworthy with the different stressors applied in our experiments. In case of the tolerant cultivars Pokkali and Nonabokra (with higher endogenous ABA level), the transcript and protein levels were higher even at the constitutive level, whereas it was inducible only by corresponding stress signals in IR-29; the transcript levels enhanced with the period of stress exposure, becoming progressively maximum at 24 h of stress in both roots and leaves. TheHKT,SAPK5, andRab16A were the three upregulated genes in both the roots and leaves of all the varieties examined. The higher expression ofSOS-3,NHX-1, andSAPK5 genes was evidenced in all the varieties during dehydration stress, while all the genes were upregulated to a considerable extent during cold stress and exogenous ABA treatment. The enhancement in SAMDC, GST, and OSBZ8 protein expression with all the stresses explains the importance of the polyamine spermidine, the quenching of reactive oxygen species by GST, and the triggering of downstream target genes by the trans-acting factor OSBZ8, towards stress tolerance in the different rice varieties.Most of the previous reports involving laboratory-based studies have focused on analyzing the mechanism of stress tolerance in isolation, namely, determining the effect of an individual stress and studying the upregulated and downregulated genes in response to that particular stress for a certain period of exposure. However, the situation is entirely different in the actual field conditions where a plant may be challenged with multiple stresses simultaneously. A gene proved to be salt responsive may or may not provide sufficient response to some other forms of stress. Thus, it is necessary to have a more holistic approach to study the behavior and/or effect of the same gene(s) (whose role in tolerance to a particular stress is known) to multiple stresses (which is the actual situation in the agricultural field), and analyzing the concentration-dependent or time-dependent regulation of gene expression, mediated by the stressors. If a particular gene is found to be elevated by multiple stresses with prolonged duration of stress exposure, it can be well presumed that the gene can be employed in genetic engineering program to generate crop plants showing broad spectrum resistance, rather than resistance to a particular stress only. Thus, the upregulation of signal transduction elements and changes in transcriptome level following different stress treatments accentuate the importance of these responses that are induced as early as 6 h and further enhanced with the duration of stress, helping the plants to ward off the stress-induced damage. This variability in gene expression patterns implies that a complex network of pathways regulates different physiological functions for acclimatizing towards multiple challenges. The description of this succession of changes in gene expression over time also establishes connections among various interrelated pathways and commonality of signals in diverse stresses. The results presented in this study clearly provide a new insight into the role of different rice genes during multiple abiotic stresses. It brings out a clear-cut difference between sensitive and tolerant varieties regarding the level or regulation of gene expression during multiple stresses. Although differential gene expression may suggest a potential role of gene products in the stress response, more thorough genetic studies like knockout/knockdown of gene expression or complementation of relevant mutants are required to confirm the role. We intend to carry out in future a further detailed and thorough analysis using more contemporary high throughput techniques like RNA sequencing. Further analysis of these stress-inducible genes using transgenic approach will provide more information about not only the function of the stress-inducible genes involved in stress tolerance, but also novel cis-acting promoter elements constituting these genes involved in high salinity, drought, cold, or ABA-responsive gene expression. The present work will pave the way in future to select gene(s) for overexpression, so as to generate broad spectrum resistance to multiple stresses simultaneously.



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*Source: 706890-2014-07-07.xml*