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Comparative analysis of gene expression in response to cold stress in diverse rice genotypes
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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Comparative analysis of gene expression in response to cold stress in diverse rice genotypes Gabriela Peres Moraes de Freitas a, b, Supratim Basu a, Venkategowda Ramegowda a, Eugenia Bolacel Braga b, Andy Pereira a, * a b
Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA Department of Botany, Federal University of Pelotas, Pelotas, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2016 Accepted 1 February 2016 Available online xxx
Cold stress is a major factor affecting rice (Oryza sativa) growth and productivity, limiting its distribution worldwide. Rice production is affected primarily due to its vulnerability to cold stress at seedling stage, as well as reproductive stage leading to spikelet sterility. We report here the analysis of 21 diverse rice genotypes from the USDA mini-core collection for cold tolerance and categorized their tolerance levels on the basis of reduction in growth measured by root and shoot length. The screening identified 12 cold tolerant genotypes from which six tolerant genotypes were characterized at the vegetative stage for cold tolerance and gas-exchange parameters. Two tolerant and two sensitive genotypes were used further for gene expression analysis. Lipid Transfer Protein (LTP) genes showed a clear difference in expression between cold tolerant and sensitive genotypes suggesting that they are good candidates for engineering cold tolerance in rice. Nipponbare was identified as a cold tolerant genotype with stress tolerance mechanism potentially operating via both ABA dependent and independent pathways. © 2016 Elsevier Inc. All rights reserved.
Keywords: ABA Cold Photosynthesis ROS Transcription factor
1. Introduction Cold stress, classified as freezing (<0 C) or chilling (0e15 C) plays a key role in determining the growing season and geographical distribution of plants [1]. The sensitivity and symptoms of plant responses to cold stress varies with the growth stage. Plants subjected to cold stress at the germination stage show delayed and lower percentage of germination, while at the vegetative stage symptoms are expressed through yellowing of leaves, lower stature, and decreased tillering of the rice plants [2]. Exposure of plants to cold stress severely affects the photosynthetic machinery, more specifically the ultrastructure of chloroplasts, altering the light harvesting chlorophyll antenna complexes [3] and/or modifying thylakoid structures [4]. The restriction of photosynthetic processes by cold temperatures thereby leads to a lack of plant energy resources. Cold stress induced ROS accumulation [5] is capable of causing severe damage to various cellular components such as altering membrane lipid composition due to
* Corresponding author. 115 Plant Sciences Building Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA. E-mail address: [email protected] (A. Pereira).
excess accumulation of malondialdehyde (MDA), structural proteins and enzymes. The survival of plants under low temperatures involves two distinct mechanisms: chilling tolerance and cold acclimation. Chilling tolerance can be defined as the intrinsic ability of the plant to survive under low temperature without injury or damage [6] while cold acclimation is the enhanced potential of the plant to tolerate the physical and physiochemical abnormalities of cold stress [7]. Plants upon perceiving cold stress stimulus become proactive to restore normal metabolite levels, and most importantly, metabolic fluxes [8]. The modification of metabolic responses via increased accumulation of antioxidants and osmolytes is coupled to enhanced tolerance, achieved through an intricate stress signaling network. Previous studies on cold tolerance have identified several regulons comprising of transcription factor (TF) CBF/DREB (Crepeat binding factors, also known as DEHYDRATION-RESPONSIVE ELEMENT-BINDING protein or DREBs) and its cold-inducible target genes, KIN (cold-induced), or LTI (low-temperature-induced) [9,10]. In addition to the TFs, several other genes like FRO1 (FROSTBITE 1) encoding ferric reduction oxidase 1, and OsFAD2 encoding fatty acid desaturase 2 have been identified in Arabidopsis and rice, respectively, that contribute to cold tolerance through increased expression of cold responsive (COR) genes or by maintaining
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Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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Fig. 1. Phenotypic changes of rice seedlings in response to cold stress. Dehusked and sterilized seeds were germinated in ½ MS medium for 7 days. Cold stress was applied by keeping the plants at 10 C for 96 h. Plants kept at 28 C served as control. Cold stress tolerance of different genotypes was estimated by measuring the shoot and root length. Response of different rice genotypes to cold stress was indicated by reduction in (A) Shoot length, and (B) Root length relative to the control.
membrane fluidity [11,12]. Rice is more vulnerable to cold stress at the reproductive stage not only showing spikelet sterility but also incomplete panicle exertion and spikelet abortion [13]. During the course of evolution, the continuous selection and breeding efforts have led to the development of rice cultivars that are abiotic stress tolerant. Some rice genotypes have evolved naturally which are tolerant to cold, salinity and other stresses, but are low yielders, however serve as excellent genetic resources for stress tolerance. Previous research has identified 40 quantitative trait loci (QTLs) in different combinations contributing to cold tolerance [14]. Hence, it becomes essential to identify naturally occurring cold tolerant rice genotypes with diverse mechanisms from the rice germplasm for developing cold tolerant rice. In this study we screened a diverse panel of rice genotypes for cold tolerance at the seedling stage. Selected genotypes were tested for cold tolerance response parameters at the vegetative stage followed by the gene expression
analysis to dissect the potential cold tolerance mechanisms operating in these genotypes. 2. Materials and methods 2.1. Screening for cold stress tolerance at vegetative stage For cold stress treatment, the plants were kept at 10 C for 48 h. Leaf rolling, a symptom of cold stress, was monitored. Measurement of photosynthesis and photochemical efficiency of PSII during steady state illumination were taken on the second fully expanded leaf using a portable photosynthesis meter (LI-6400XT; LI-COR) at a CO2 concentration of 370 mmol mol1, light intensity of 1000 mmol m2s1, and 55%e60% RH. Instantaneous water use efficiency (iWUE) was calculated using data obtained from the photosynthesis meter as ¼ (photosynthesis/transpiration rate). RWC was measured with modifications as described [15] in the
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
G.P. Moraes de Freitas et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
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Fig. 2. Effect of cold stress on physiological processes in different rice genotypes. Control and cold-treated plants showing response in Photosynthesis (A), Stomatal Conductance (B), Transpiration (C) and Biomass (D). The data are expressed as mean of five observations. Asterisks indicate significance at P 0.01 compared with the control as analyzed by the Student's t-test.
leaves used from where photosynthesis was measured. Leaf fragments of same size were cut, their fresh weight was measured and hydrated immediately to full turgidity in deionized water for 6 h. After 6 h the leaf fragments were blotted on paper towels and the fully turgid weight was taken. Turgid leaf samples were then ovendried at 80 C for 72 h and weighed to determine dry weight. RWC percentage was measured as: RWC (%) ¼ (fresh weight dry weight)/(turgid weight dry weight) 100. For growth conditions see supplemental methods.
2.2. Gene expression analysis The qRT-PCR experiments were conducted using GoTaq® qPCR Master Mix (Promega) with Ubiquitin as internal reference and other gene-specific primers (Supplementary Table 1). Increasing temperature (0.5 C 10 s1) from 55 C to 95 C was used for melt curve analysis. Un-transcribed 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 of Ubiquitin and the relative fold change was calculated using the equation (2)DDCt [16]. The expression analysis was performed with three biological replicates. The average data from qPCR analysis was imported into TM4 microarray software suite [17].
3. Results and discussion 3.1. Screening of rice genotypes for cold tolerance at seedling stage Root and shoot length of 22 genotypes from the USDA mini-core collection was measured at the end of cold stress (Supplementary Table 2). The genotypes were categorized as highly tolerant, semi-tolerant and sensitive based on the percentage reduction in root and shoot length under cold (Fig. 1A and B), where 0e29% reduction was categorized as highly tolerant, 30e49% semitolerant; and >50% as sensitive. This screening identified eight sensitive, four semi-tolerant and nine tolerant rice genotypes, out of which six representative genotypes were selected for further analysis (Fig. 2). 3.2. Effect of cold stress on physiological parameters at the vegetative stage Exposure of plants to environmental stresses such as drought stress reduces stomatal conductance to CO2 as well as photosynthesis [2,18,19]. In addition to drought stress, it has been shown previously that rice plants on being exposed to low temperature show increased membrane permeability, inhibition of chlorophyll biosynthesis and broken chloroplasts that eventually affect photosynthesis. Thus, the ability of rice plants to sustain CO2 levels and maintain a steady photosynthesis rate under stress conditions is a tolerance response mechanism with great potential for
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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3.3. Expression of LTPs in response to cold stress
Fig. 3. Effect of cold stress on gene expression in rice genotypes. Relative expression of Lipid Transfer Protein genes and genes conferring cold stress tolerance in rice. The data are mean of three biological replicates and are expressed as ± SE.
improvement of leaf-level photosynthesis contributing to greater yield. The difference in ability of plants to cope with photosynthetic and leaf gas-exchange capacities has been shown to be attributed to the genetic variation in rice germplasm [20]. Fv/Fm is used to study the effect of stress on PSII in dark adapted state [21]. In our experiments, the rice genotypes showed a significant reduction in photosynthesis with Nipponbare showing the least (35.7%), M202 a known tolerant with 42% reduction and Secano do Brazil (75.3%) with maximum reduction (Fig. 1A and Supplementary Fig. 1A and B). A similar pattern was observed for stomatal conductance, transpiration and biomass (Fig. 1B and C). Fv'/Fm' was also significantly affected by cold stress (Supplementary Fig. 1C). RWC is the measure of plant water status in terms of physiological consequence of stress exposure. Cold stress also significantly affected the RWC of different rice genotypes, with Secano do Brazil showing least RWC (33.4%) and M202 the maximum (65.8%) after exposure to cold stress (Supplementary Fig. 1D). In the light of these responses of the different rice genotypes to cold stress we selected 2 cold tolerant (M202 and Nipponbare) and 2 sensitive genotypes (Cypress and Secano do Brazil) for a time-course analysis of gene expression in response to cold stress at 3 h, 6 h, 24 h and 48 h.
LTPs abundantly present in higher plants are basic, 9-kDa proteins that enhance the transfer of phospholipids between membranes and also can bind acyl chains. Biochemically, LTPs are characterized by an elevated isoelectric point (~9) and a conserved motif of 8 cysteine residues. LTPs comprise of a signal peptide suggesting that they can play a role in secretory pathways, and also be involved in membrane biogenesis and regulation of the intracellular fatty acid pools. LTPs have been suggested to be involved in cutin formation, embryogenesis, defense against phytopathogens, and adaptation of plants to various environmental conditions [22]. Genes encoding LTP proteins are differentially regulated under various environmental conditions including cold stress. Previously, genotypic differences have been shown in the expression of LTP genes in response to cold stress. A clear example is blt4.1 from barley that is induced in response to cold stress in winter cultivars but not in spring cultivars [23]. Moreover, it has been shown that overexpression of LTP3 increased freezing tolerance in Arabidopsis [24]. The LTPs were therefore studied for their role in response to cold stress in diverse rice germplasm at different time points (3 h, 6 h, 24 h and 48 h). The expression of LTP7, LTP8, LTP12 and LTP25 show early and maximal induction after 3 h and decrease with time in tolerant M202 and Nipponbare, while in Cypress and Secano do Brazil there is an increased expression with time (Fig. 3). LTP10, and LTP26 were induced maximally and early after 3 h in M202, while for Nipponbare and Cypress there was an increase with time (Fig. 3 and Supplementary Fig. 2B and C). LTP23 was induced early in all the genotypes. M202 and Nipponbare showed the highest accumulation after 48 h, for Cypress maximum transcript accumulation was seen after 6 h and Secano do Brazil showed maximum accumulation after 6 h. LTP14 was induced maximally in Cypress and Secano do Brazil while for Nipponbare and M202 maximum accumulation was obtained after 48 h and 3 h respectively. LTP13 was induced maximally in Cypress while showed increased transcript accumulation with time (Fig. 3 and Supplementary Fig. 2A). Furthermore, we also studied the expression of Oryza sativa Drought-Induced LTP (OsDIL) encoding a lipid transfer protein that have been shown to be induced by cold and which when overexpressed in rice gives drought tolerance. Our results are consistent with previous observation and have seen that it is induced maximally after 3 h in M202 while for Cypress maximum induction is after 48 h [25] (Supplementary Fig.2D). 3.4. Role of transcription factor and metabolic pathway genes in cold stress acclimation On exposure to environmental stress, plants trigger a battery of genes controlling the stress induced signaling cascade. The accumulation of ROS in response to cold stress activates TFs that operate via the ABA-dependent or ABA-independent pathway. Induction of CBF/DREBs help in ROS detoxification, membrane transport, osmolyte biosynthesis, phosphoinositide metabolism [26]. Overexpression of OsICE1 and OsICE2 encoding the MYC-like bHLH transcriptional activator was shown to impart freezing tolerance by inducing expression of the CBF regulon [27,28]. Nipponbare and M202 showed the highest and maximal induction of OsICE1 after 6 h, in Cypress the induction after 6 h, and in Secano do Brazil a slight induction after 3 h that decreased with time. OsICE2 was induced early in Nipponbare after 3 h and decreased with time while M202 showed slight induction after 6 h while Cypress showed induction after 6 h and reaching maximum after 24 h while Secano do Brazil showed maximum induction after 6 h (Fig. 3 and Supplementary Fig. 3A). Plant phytohormone ABA plays a key role in various aspects of
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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Fig. 4. Model for cold tolerance mechanism in rice. The regulatory cascade of signal transduction following cold-induced damage and perception by transcription factors (rectangles) and downstream changes in gene regulation and biochemical responses leading to expression of cold tolerance.
plant growth and development [29]. ABA is known to be the best trigger for abiotic stress signaling in crop plants inducing other signaling components that are positive and negative regulators of ABA signaling, such as phosphatases, kinases and TFs and genes encoding enzymes involved in the synthesis of osmoprotectants [30,31]. NAC and basic leucine zipper (bZIP) TFs have been implicated to play diverse role in modulating stress response [32,33]. Previous reports have shown that SNAC1, and OsbZIP72 confer abiotic stress tolerance when overexpressed in rice [34,35]. OsNAP was induced slightly after 3 h in Nipponbare and M202 induced maximally after 6 h and maintains its induced level after 24 and 48 h while Cypress the induction was observed after 6 h increased to maximum after 24 h and Secano do Brazil on the contrary shows slight induction after 6 h and goes down completely after 48 h (Fig. 3). These results are consistent with previous observations where it has been reported that overexpression in rice gives stress tolerance through ABA dependent pathway [36]. OsABF2 another bZIP TF that imparts abiotic stress tolerance in rice [37] was induced maximally after 3 h in Nipponbare and M202 while it subsided after 48 h with little expression in Cypress and Secano do Brazil (Supplementary Fig. 3E). WRKY TFs, another broad class of TF superfamily comprising of 98e102 members are induced by abiotic stresses and has been proposed to play important roles in rice adaptation to abiotic stresses [38]. OsWRKY45 and OsWRKY76 when overexpressed in
rice show enhanced tolerance to low temperature by ABA signaling pathway and scavenging the free radicals [39,40]. OsWRKY45 and OsWRKY76 were induced early and maximally after 3 h in M202 while in Nipponbare, OsWRKY45 was induced in time dependent manner and OsWRKY76 was induced maximally after 3 h decreasing with time (Supplementary Fig. 3C and D). Besides these, there are other TF families like OsMYB4 belonging to MYB superfamily or OsCOIN belonging to Zinc finger family with a RING domain have been shown to play important roles in cold stress tolerance through increased accumulation of osmolytes [41,42]. M202 showed the highest expression of OsCOIN after 3 h decreasing with time while OsMYB4 was induced with time in all the genotypes with Secano do Brazil showing the highest expression e (Fig. 3 and Supplementary Fig. 3F). To study the effect of cold stress on the cell cycle in response to cold stress in different rice genotypes we studied the expression of OsRAN2 that has been shown to confer cold tolerance by maintaining cell division through promoting the normal export of intranuclear tubulin [43]. OsRAN2 was induced maximally in Nipponbare and M202 after 3 h and maintaining its level after 6 h decreasing with time while for Cypress and Secano do Brazil maximum induction was observed after 48 h (Fig. 3). OsSPX1 that has been shown to confer stress tolerance in rice by modulating the ROS scavenging pathway was induced maximally after 3 h in M202 while for other genotypes it was induced maximally after 48 h
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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(Supplementary Fig. 3B). In conclusion, this research identified cold tolerant rice genotypes from a diverse panel of germplasm. We have made a comprehensive analysis of LTP expression in response to cold stress response, showing a clear difference in expression between cold tolerant and sensitive genotypes and suggesting a role in engineering cold tolerance in rice. Differential expression analysis of cold responsive TFs and genes established Nipponbare as a cold tolerant genotype showing similar stress responsive gene expression as cold tolerant M202 with stress tolerance signaling operating via both ABA dependent and independent pathways. Finally, we developed a representative model of cold stress signaling in rice from our data on gene expression analysis (Fig. 4). Using the molecular markers derived from this study we can screen a wide variety of rice germplasm for identifying cold tolerant rice that can be used for breeding programs. Acknowledgments This work was supported by the National Science Foundation awards DBIe0922747 and ABI1062472. This work was conducted during a scholarship supported by the International Cooperation Program CAPES/COFECUB at the Federal University of Pelotas Financed by CAPES e Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil.
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Transparency document
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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.02.004.
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Appendix A. Supplementary data [24]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.02.004. Author contributions Gabriela Peres Moraes de Freitas and Supratim Basu conducted the stress experiments and gene expression studies and wrote the manuscript, Venkategowda Ramegowda and Eugenia Bolacel Braga guided stress physiology experiments, and Andy Pereira designed experiments and edited the manuscript.
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Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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Comparative analysis of gene expression in response to cold stress in diverse rice genotypes Gabriela Peres Moraes de Freitas a, b, Supratim Basu a, Venkategowda Ramegowda a, Eugenia Bolacel Braga b, Andy Pereira a, * a b
Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA Department of Botany, Federal University of Pelotas, Pelotas, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2016 Accepted 1 February 2016 Available online xxx
Cold stress is a major factor affecting rice (Oryza sativa) growth and productivity, limiting its distribution worldwide. Rice production is affected primarily due to its vulnerability to cold stress at seedling stage, as well as reproductive stage leading to spikelet sterility. We report here the analysis of 21 diverse rice genotypes from the USDA mini-core collection for cold tolerance and categorized their tolerance levels on the basis of reduction in growth measured by root and shoot length. The screening identified 12 cold tolerant genotypes from which six tolerant genotypes were characterized at the vegetative stage for cold tolerance and gas-exchange parameters. Two tolerant and two sensitive genotypes were used further for gene expression analysis. Lipid Transfer Protein (LTP) genes showed a clear difference in expression between cold tolerant and sensitive genotypes suggesting that they are good candidates for engineering cold tolerance in rice. Nipponbare was identified as a cold tolerant genotype with stress tolerance mechanism potentially operating via both ABA dependent and independent pathways. © 2016 Elsevier Inc. All rights reserved.
Keywords: ABA Cold Photosynthesis ROS Transcription factor
1. Introduction Cold stress, classified as freezing (<0 C) or chilling (0e15 C) plays a key role in determining the growing season and geographical distribution of plants [1]. The sensitivity and symptoms of plant responses to cold stress varies with the growth stage. Plants subjected to cold stress at the germination stage show delayed and lower percentage of germination, while at the vegetative stage symptoms are expressed through yellowing of leaves, lower stature, and decreased tillering of the rice plants [2]. Exposure of plants to cold stress severely affects the photosynthetic machinery, more specifically the ultrastructure of chloroplasts, altering the light harvesting chlorophyll antenna complexes [3] and/or modifying thylakoid structures [4]. The restriction of photosynthetic processes by cold temperatures thereby leads to a lack of plant energy resources. Cold stress induced ROS accumulation [5] is capable of causing severe damage to various cellular components such as altering membrane lipid composition due to
* Corresponding author. 115 Plant Sciences Building Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA. E-mail address: [email protected] (A. Pereira).
excess accumulation of malondialdehyde (MDA), structural proteins and enzymes. The survival of plants under low temperatures involves two distinct mechanisms: chilling tolerance and cold acclimation. Chilling tolerance can be defined as the intrinsic ability of the plant to survive under low temperature without injury or damage [6] while cold acclimation is the enhanced potential of the plant to tolerate the physical and physiochemical abnormalities of cold stress [7]. Plants upon perceiving cold stress stimulus become proactive to restore normal metabolite levels, and most importantly, metabolic fluxes [8]. The modification of metabolic responses via increased accumulation of antioxidants and osmolytes is coupled to enhanced tolerance, achieved through an intricate stress signaling network. Previous studies on cold tolerance have identified several regulons comprising of transcription factor (TF) CBF/DREB (Crepeat binding factors, also known as DEHYDRATION-RESPONSIVE ELEMENT-BINDING protein or DREBs) and its cold-inducible target genes, KIN (cold-induced), or LTI (low-temperature-induced) [9,10]. In addition to the TFs, several other genes like FRO1 (FROSTBITE 1) encoding ferric reduction oxidase 1, and OsFAD2 encoding fatty acid desaturase 2 have been identified in Arabidopsis and rice, respectively, that contribute to cold tolerance through increased expression of cold responsive (COR) genes or by maintaining
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Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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Fig. 1. Phenotypic changes of rice seedlings in response to cold stress. Dehusked and sterilized seeds were germinated in ½ MS medium for 7 days. Cold stress was applied by keeping the plants at 10 C for 96 h. Plants kept at 28 C served as control. Cold stress tolerance of different genotypes was estimated by measuring the shoot and root length. Response of different rice genotypes to cold stress was indicated by reduction in (A) Shoot length, and (B) Root length relative to the control.
membrane fluidity [11,12]. Rice is more vulnerable to cold stress at the reproductive stage not only showing spikelet sterility but also incomplete panicle exertion and spikelet abortion [13]. During the course of evolution, the continuous selection and breeding efforts have led to the development of rice cultivars that are abiotic stress tolerant. Some rice genotypes have evolved naturally which are tolerant to cold, salinity and other stresses, but are low yielders, however serve as excellent genetic resources for stress tolerance. Previous research has identified 40 quantitative trait loci (QTLs) in different combinations contributing to cold tolerance [14]. Hence, it becomes essential to identify naturally occurring cold tolerant rice genotypes with diverse mechanisms from the rice germplasm for developing cold tolerant rice. In this study we screened a diverse panel of rice genotypes for cold tolerance at the seedling stage. Selected genotypes were tested for cold tolerance response parameters at the vegetative stage followed by the gene expression
analysis to dissect the potential cold tolerance mechanisms operating in these genotypes. 2. Materials and methods 2.1. Screening for cold stress tolerance at vegetative stage For cold stress treatment, the plants were kept at 10 C for 48 h. Leaf rolling, a symptom of cold stress, was monitored. Measurement of photosynthesis and photochemical efficiency of PSII during steady state illumination were taken on the second fully expanded leaf using a portable photosynthesis meter (LI-6400XT; LI-COR) at a CO2 concentration of 370 mmol mol1, light intensity of 1000 mmol m2s1, and 55%e60% RH. Instantaneous water use efficiency (iWUE) was calculated using data obtained from the photosynthesis meter as ¼ (photosynthesis/transpiration rate). RWC was measured with modifications as described [15] in the
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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Fig. 2. Effect of cold stress on physiological processes in different rice genotypes. Control and cold-treated plants showing response in Photosynthesis (A), Stomatal Conductance (B), Transpiration (C) and Biomass (D). The data are expressed as mean of five observations. Asterisks indicate significance at P 0.01 compared with the control as analyzed by the Student's t-test.
leaves used from where photosynthesis was measured. Leaf fragments of same size were cut, their fresh weight was measured and hydrated immediately to full turgidity in deionized water for 6 h. After 6 h the leaf fragments were blotted on paper towels and the fully turgid weight was taken. Turgid leaf samples were then ovendried at 80 C for 72 h and weighed to determine dry weight. RWC percentage was measured as: RWC (%) ¼ (fresh weight dry weight)/(turgid weight dry weight) 100. For growth conditions see supplemental methods.
2.2. Gene expression analysis The qRT-PCR experiments were conducted using GoTaq® qPCR Master Mix (Promega) with Ubiquitin as internal reference and other gene-specific primers (Supplementary Table 1). Increasing temperature (0.5 C 10 s1) from 55 C to 95 C was used for melt curve analysis. Un-transcribed 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 of Ubiquitin and the relative fold change was calculated using the equation (2)DDCt [16]. The expression analysis was performed with three biological replicates. The average data from qPCR analysis was imported into TM4 microarray software suite [17].
3. Results and discussion 3.1. Screening of rice genotypes for cold tolerance at seedling stage Root and shoot length of 22 genotypes from the USDA mini-core collection was measured at the end of cold stress (Supplementary Table 2). The genotypes were categorized as highly tolerant, semi-tolerant and sensitive based on the percentage reduction in root and shoot length under cold (Fig. 1A and B), where 0e29% reduction was categorized as highly tolerant, 30e49% semitolerant; and >50% as sensitive. This screening identified eight sensitive, four semi-tolerant and nine tolerant rice genotypes, out of which six representative genotypes were selected for further analysis (Fig. 2). 3.2. Effect of cold stress on physiological parameters at the vegetative stage Exposure of plants to environmental stresses such as drought stress reduces stomatal conductance to CO2 as well as photosynthesis [2,18,19]. In addition to drought stress, it has been shown previously that rice plants on being exposed to low temperature show increased membrane permeability, inhibition of chlorophyll biosynthesis and broken chloroplasts that eventually affect photosynthesis. Thus, the ability of rice plants to sustain CO2 levels and maintain a steady photosynthesis rate under stress conditions is a tolerance response mechanism with great potential for
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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3.3. Expression of LTPs in response to cold stress
Fig. 3. Effect of cold stress on gene expression in rice genotypes. Relative expression of Lipid Transfer Protein genes and genes conferring cold stress tolerance in rice. The data are mean of three biological replicates and are expressed as ± SE.
improvement of leaf-level photosynthesis contributing to greater yield. The difference in ability of plants to cope with photosynthetic and leaf gas-exchange capacities has been shown to be attributed to the genetic variation in rice germplasm [20]. Fv/Fm is used to study the effect of stress on PSII in dark adapted state [21]. In our experiments, the rice genotypes showed a significant reduction in photosynthesis with Nipponbare showing the least (35.7%), M202 a known tolerant with 42% reduction and Secano do Brazil (75.3%) with maximum reduction (Fig. 1A and Supplementary Fig. 1A and B). A similar pattern was observed for stomatal conductance, transpiration and biomass (Fig. 1B and C). Fv'/Fm' was also significantly affected by cold stress (Supplementary Fig. 1C). RWC is the measure of plant water status in terms of physiological consequence of stress exposure. Cold stress also significantly affected the RWC of different rice genotypes, with Secano do Brazil showing least RWC (33.4%) and M202 the maximum (65.8%) after exposure to cold stress (Supplementary Fig. 1D). In the light of these responses of the different rice genotypes to cold stress we selected 2 cold tolerant (M202 and Nipponbare) and 2 sensitive genotypes (Cypress and Secano do Brazil) for a time-course analysis of gene expression in response to cold stress at 3 h, 6 h, 24 h and 48 h.
LTPs abundantly present in higher plants are basic, 9-kDa proteins that enhance the transfer of phospholipids between membranes and also can bind acyl chains. Biochemically, LTPs are characterized by an elevated isoelectric point (~9) and a conserved motif of 8 cysteine residues. LTPs comprise of a signal peptide suggesting that they can play a role in secretory pathways, and also be involved in membrane biogenesis and regulation of the intracellular fatty acid pools. LTPs have been suggested to be involved in cutin formation, embryogenesis, defense against phytopathogens, and adaptation of plants to various environmental conditions [22]. Genes encoding LTP proteins are differentially regulated under various environmental conditions including cold stress. Previously, genotypic differences have been shown in the expression of LTP genes in response to cold stress. A clear example is blt4.1 from barley that is induced in response to cold stress in winter cultivars but not in spring cultivars [23]. Moreover, it has been shown that overexpression of LTP3 increased freezing tolerance in Arabidopsis [24]. The LTPs were therefore studied for their role in response to cold stress in diverse rice germplasm at different time points (3 h, 6 h, 24 h and 48 h). The expression of LTP7, LTP8, LTP12 and LTP25 show early and maximal induction after 3 h and decrease with time in tolerant M202 and Nipponbare, while in Cypress and Secano do Brazil there is an increased expression with time (Fig. 3). LTP10, and LTP26 were induced maximally and early after 3 h in M202, while for Nipponbare and Cypress there was an increase with time (Fig. 3 and Supplementary Fig. 2B and C). LTP23 was induced early in all the genotypes. M202 and Nipponbare showed the highest accumulation after 48 h, for Cypress maximum transcript accumulation was seen after 6 h and Secano do Brazil showed maximum accumulation after 6 h. LTP14 was induced maximally in Cypress and Secano do Brazil while for Nipponbare and M202 maximum accumulation was obtained after 48 h and 3 h respectively. LTP13 was induced maximally in Cypress while showed increased transcript accumulation with time (Fig. 3 and Supplementary Fig. 2A). Furthermore, we also studied the expression of Oryza sativa Drought-Induced LTP (OsDIL) encoding a lipid transfer protein that have been shown to be induced by cold and which when overexpressed in rice gives drought tolerance. Our results are consistent with previous observation and have seen that it is induced maximally after 3 h in M202 while for Cypress maximum induction is after 48 h [25] (Supplementary Fig.2D). 3.4. Role of transcription factor and metabolic pathway genes in cold stress acclimation On exposure to environmental stress, plants trigger a battery of genes controlling the stress induced signaling cascade. The accumulation of ROS in response to cold stress activates TFs that operate via the ABA-dependent or ABA-independent pathway. Induction of CBF/DREBs help in ROS detoxification, membrane transport, osmolyte biosynthesis, phosphoinositide metabolism [26]. Overexpression of OsICE1 and OsICE2 encoding the MYC-like bHLH transcriptional activator was shown to impart freezing tolerance by inducing expression of the CBF regulon [27,28]. Nipponbare and M202 showed the highest and maximal induction of OsICE1 after 6 h, in Cypress the induction after 6 h, and in Secano do Brazil a slight induction after 3 h that decreased with time. OsICE2 was induced early in Nipponbare after 3 h and decreased with time while M202 showed slight induction after 6 h while Cypress showed induction after 6 h and reaching maximum after 24 h while Secano do Brazil showed maximum induction after 6 h (Fig. 3 and Supplementary Fig. 3A). Plant phytohormone ABA plays a key role in various aspects of
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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Fig. 4. Model for cold tolerance mechanism in rice. The regulatory cascade of signal transduction following cold-induced damage and perception by transcription factors (rectangles) and downstream changes in gene regulation and biochemical responses leading to expression of cold tolerance.
plant growth and development [29]. ABA is known to be the best trigger for abiotic stress signaling in crop plants inducing other signaling components that are positive and negative regulators of ABA signaling, such as phosphatases, kinases and TFs and genes encoding enzymes involved in the synthesis of osmoprotectants [30,31]. NAC and basic leucine zipper (bZIP) TFs have been implicated to play diverse role in modulating stress response [32,33]. Previous reports have shown that SNAC1, and OsbZIP72 confer abiotic stress tolerance when overexpressed in rice [34,35]. OsNAP was induced slightly after 3 h in Nipponbare and M202 induced maximally after 6 h and maintains its induced level after 24 and 48 h while Cypress the induction was observed after 6 h increased to maximum after 24 h and Secano do Brazil on the contrary shows slight induction after 6 h and goes down completely after 48 h (Fig. 3). These results are consistent with previous observations where it has been reported that overexpression in rice gives stress tolerance through ABA dependent pathway [36]. OsABF2 another bZIP TF that imparts abiotic stress tolerance in rice [37] was induced maximally after 3 h in Nipponbare and M202 while it subsided after 48 h with little expression in Cypress and Secano do Brazil (Supplementary Fig. 3E). WRKY TFs, another broad class of TF superfamily comprising of 98e102 members are induced by abiotic stresses and has been proposed to play important roles in rice adaptation to abiotic stresses [38]. OsWRKY45 and OsWRKY76 when overexpressed in
rice show enhanced tolerance to low temperature by ABA signaling pathway and scavenging the free radicals [39,40]. OsWRKY45 and OsWRKY76 were induced early and maximally after 3 h in M202 while in Nipponbare, OsWRKY45 was induced in time dependent manner and OsWRKY76 was induced maximally after 3 h decreasing with time (Supplementary Fig. 3C and D). Besides these, there are other TF families like OsMYB4 belonging to MYB superfamily or OsCOIN belonging to Zinc finger family with a RING domain have been shown to play important roles in cold stress tolerance through increased accumulation of osmolytes [41,42]. M202 showed the highest expression of OsCOIN after 3 h decreasing with time while OsMYB4 was induced with time in all the genotypes with Secano do Brazil showing the highest expression e (Fig. 3 and Supplementary Fig. 3F). To study the effect of cold stress on the cell cycle in response to cold stress in different rice genotypes we studied the expression of OsRAN2 that has been shown to confer cold tolerance by maintaining cell division through promoting the normal export of intranuclear tubulin [43]. OsRAN2 was induced maximally in Nipponbare and M202 after 3 h and maintaining its level after 6 h decreasing with time while for Cypress and Secano do Brazil maximum induction was observed after 48 h (Fig. 3). OsSPX1 that has been shown to confer stress tolerance in rice by modulating the ROS scavenging pathway was induced maximally after 3 h in M202 while for other genotypes it was induced maximally after 48 h
Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004
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(Supplementary Fig. 3B). In conclusion, this research identified cold tolerant rice genotypes from a diverse panel of germplasm. We have made a comprehensive analysis of LTP expression in response to cold stress response, showing a clear difference in expression between cold tolerant and sensitive genotypes and suggesting a role in engineering cold tolerance in rice. Differential expression analysis of cold responsive TFs and genes established Nipponbare as a cold tolerant genotype showing similar stress responsive gene expression as cold tolerant M202 with stress tolerance signaling operating via both ABA dependent and independent pathways. Finally, we developed a representative model of cold stress signaling in rice from our data on gene expression analysis (Fig. 4). Using the molecular markers derived from this study we can screen a wide variety of rice germplasm for identifying cold tolerant rice that can be used for breeding programs. Acknowledgments This work was supported by the National Science Foundation awards DBIe0922747 and ABI1062472. This work was conducted during a scholarship supported by the International Cooperation Program CAPES/COFECUB at the Federal University of Pelotas Financed by CAPES e Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil.
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Transparency document
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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.02.004.
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Appendix A. Supplementary data [24]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.02.004. Author contributions Gabriela Peres Moraes de Freitas and Supratim Basu conducted the stress experiments and gene expression studies and wrote the manuscript, Venkategowda Ramegowda and Eugenia Bolacel Braga guided stress physiology experiments, and Andy Pereira designed experiments and edited the manuscript.
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Please cite this article in press as: G.P. Moraes de Freitas, et al., Comparative analysis of gene expression in response to cold stress in diverse rice genotypes, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.004