ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat-and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE −273 to −280 ) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast onehybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpBcytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic).Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry.
Genome wide transcriptional changes by cold stress, heat stress and oxidative stress in rice seedlings were analyzed. Heat stress resulted in predominant changes in transcripts of heat shock protein and heat shock transcription factor genes, as well as genes associated with synthesis of scavengers of reactive oxygen species and genes that control the level of sugars, metabolites and auxins. Cold stress treatment caused differential expression of transcripts of various transcription factors including desiccation response element binding proteins and different kinases. Transcripts of genes that are part of calcium signaling, reactive oxygen scavenging and diverse metabolic reactions were differentially expressed during cold stress. Oxidative stress induced by hydrogen peroxide treatment, resulted in significant up-regulation in transcript levels of genes related to redox homeostasis and down-regulation of transporter proteins. ROS homeostasis appeared to play central role in response to temperature extremes. The key transcription factors that may underlie the concerted transcriptional changes of specific components in various signal transduction networks involved are highlighted. Co-ordinated expression pattern and promoter architectures based analysis (promoter models and overrepresented transcription factor binding sites) suggested potential regulons involved in stress responses. A considerable overlap was noted at the level of transcription as well as in regulatory modules of differentially expressed genes.
BackgroundClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues.ResultsNine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300), 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770) and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210). Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Δhsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene imparted appreciably high level tolerance to the mutant yeast cells.ConclusionsRice class I Clp ATPase gene family is constituted of 9 members. Of these 9, only 3 belonging to ClpB group are heat stress regulated. Distribution of ClpB proteins to different cell organelles indicates that their functioning might be critical in different cell locations. From the complementation assays, OsClpD1 appears to be more effective than OsClpB-cyt protein in rescuing the thermosensitive defect of the yeast ScΔhsp104 mutant cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.