Binding affinities and interactions among different heat shock element types and heat shock factors in rice (Oryza sativa L.)

Abstract

Binding of heat shock factors (Hsfs) to heat shock elements (HSEs) leads to transcriptional regulation of heat shock genes. Genome-wide, 953 rice genes contain perfect-type, 695 genes gap-type and 1584 genes step-type HSE sequences in their 1-kb promoter region. The rice genome contains 13 class A, eight class B and four class C Hsfs (OsHsfs) and has OsHsf26 (which is of variant type) genes. Chemical cross-linking analysis of in vitro synthesized OsHsf polypeptides showed formation of homotrimers of OsHsfA2c, OsHsfA9 and OsHsfB4b proteins. Binding analysis of polypeptides with oligonucleotide probes containing perfect-, gap-, and step-type HSE sequences showed that OsHsfA2c, OsHsfA9 and OsHsfB4b differentially recognize various model HSEs as a function of varying reaction temperatures. The homomeric form of OsHsfA2c and OsHsfB4b proteins was further noted by the bimolecular fluorescence complementation approach in onion epidermal cells. In yeast two-hybrid assays, OsHsfB4b showed homomeric interaction as well as distinct heteromeric interactions with OsHsfA2a, OsHsfA7, OsHsfB4c and OsHsf26. Transactivation activity was noted in OsHsfA2c, OsHsfA2d, OsHsfA9, OsHsfC1a and OsHsfC1b in yeast cells. These differential patterns pertaining to binding with HSEs and protein-protein interactions may have a bearing on the cellular functioning of OsHsfs under a range of different physiological and environmental conditions. Structured digital abstractl HSFA2C binds to HSFA2C by cross-linking study (View interaction) l HSFA2C physically interacts with HSFA2C by bimolecular fluorescence complementation (View interaction) l HSFB4B physically interacts with HSFB4B by bimolecular fluorescence complementation (View interaction) l HSFA2A physically interacts with HSFB4B by two hybrid (View interaction) l HSFB4B binds to HSFB4B by cross-linking study (View interaction) l HSFB4B physically interacts with HSF26 by two hybrid (View interaction) l HSFA9 binds to HSFA9 by cross-linking study (View interaction) l HSFA7 physically interacts with HSFB4B by two hybrid (View interaction) l HSFB4B physically interacts with HSFB4C by two hybrid (View interaction) l HSFB4B physically interacts with HSFB4B by two hybrid (View interaction) Abbreviations 3-AT, 3-amino-1,2,4-triazole; BiFC, bimolecular fluorescence complementation; EGS, ethylglycol bis(succinimidylsuccinate); EMSA, electrophoretic mobility shift assay; HS, heat shock; HSE, heat shock element; Hsf, heat shock transcription factor; Hsp, heat shock protein.

OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.)

“…OsHsfA2c thus appears to be an important player in the rice heat shock response. Earlier work has documented that OsHsfA2c is one of the most rapidly induced Hsf genes in response to heat stress, that OsHsfA2c possesses transactivation activity, that OsHsfA2c shows trimer formation activity from its monomeric forms, and that OsHsfA2c binds to perfect-type HSE (Mittal et al 2009;Mittal et al 2011). This study further noted that OsHsfB4b binds with OsClpB-cyt based on yeast two-hybrid and BiFC assays.…”

“…As OsHsfB4b also showed binding to OsHsfA2c, there is a possibility that a supra complex of OsHsfB4b, OsHsfA2c, and OsClpB-cyt is formed. Additionally, OsHsfB4b also interacts with OsHsfA7, OsHsfA2a, OsHsfB4c, and OsHsf26 proteins (Mittal et al 2011). Thereby, OsHsfB4b appears to be a critical protein in governing OsHsf interactions.…”

“…6a, c). Our earlier work has shown that OsHsfB4b is induced in response to heat stress, OsHsfB4b lacks transactivation activity and shows trimer formation activity and maximum binding activity to perfect-type HSE in electrophoretic mobility shift assays (Mittal et al 2009;Mittal et al 2011). It may be inferred that HS-induced OsHsfA2c binds to OsClpB-cyt promoter, possibly controlling synthesis of the OsClpB-cyt transcript.…”

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments

“…OsHSFA2c is the TF with the highest correlation between transcript abundance and TFA in our entire data set (correlation = 0.92). Moreover, the binding of OsHSFA2c is temperature regulated in vitro (Mittal et al, 2011), supporting its role as a heat responsive transcriptional regulator acting through the HSE.…”

Intergenic Sequence between Arabidopsis Caseinolytic Protease B-Cytoplasmic/Heat Shock Protein100 and Choline Kinase Genes Functions as a Heat-Inducible Bidirectional Promoter

“…The consensus sequence that demarcates HSEs in eukaryotic heat-inducible promoters is defined by altering units of 59-nGAAn-39, upstream of the TATA box (Mittal et al, 2011;Scharf et al, 2012). The organization of this consensus sequence separates HSEs into three groups: perfect, gapped, and stepped (Yamamoto et al, 2005).…”