Limits...
Expression profiling of abiotic stress-inducible genes in response to multiple stresses in rice (Oryza sativa L.) varieties with contrasting level of stress tolerance.

Basu S, Roychoudhury A - Biomed Res Int (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop, Soil, and Environmental Sciences, University of Arkansas, 115 Plant Sciences Building, Fayetteville, AR 72701, USA.

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 three indica 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.

Show MeSH

Related in: MedlinePlus

Heat 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109260&req=5

fig1: Heat 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.

Mentions: 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 an in 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, the in 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 for in 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.


Expression profiling of abiotic stress-inducible genes in response to multiple stresses in rice (Oryza sativa L.) varieties with contrasting level of stress tolerance.

Basu S, Roychoudhury A - Biomed Res Int (2014)

Heat 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109260&req=5

fig1: Heat 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.
Mentions: 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 an in 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, the in 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 for in 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop, Soil, and Environmental Sciences, University of Arkansas, 115 Plant Sciences Building, Fayetteville, AR 72701, USA.

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 three indica 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.

Show MeSH
Related in: MedlinePlus