ABSTRACT
Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.
Subject(s)
Arabidopsis , Heat-Shock Proteins , Plant Proteins , Thermotolerance , Arabidopsis/metabolism , DNA, Complementary/metabolism , Gene Expression Regulation, Plant , Heat-Shock Proteins/metabolism , Hot Temperature , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Thermotolerance/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Heat shock protein 70 (Hsp70) chaperones are highly conserved and essential proteins with diverse cellular functions, including plant abiotic stress tolerance. Hsp70 proteins have been linked with basal heat tolerance in plants. Hsp101 likewise is an important chaperone protein that plays a critical role in heat tolerance in plants. We observed that Arabidopsis hsc70-1 mutant seedlings show elevated basal heat tolerance compared with wild-type. Over-expression of Hsc70-1 resulted in increased heat sensitivity. Hsp101 transcript and protein levels were increased during non-heat stress (HS) and post-HS conditions in hsc70-1 mutant seedlings. In contrast, Hsp101 was repressed in Hsc70-1 over-expressing plants after post-HS conditions. Hsc70-1 showed physical interaction with HsfA1d and HsfA1e protein in the cytosol under non-HS conditions. In transient reporter gene analysis, HsfA1d, HsfA1e and HsfA2 showed transcriptional response on the Hsp101 promoter. HsfA1d and HsfA2 transcripts were at higher levels in hsc70-1 mutant compared with wild-type. We provide genetic evidence that Hsc70-1 is a negative regulator affecting HsfA1d/A1e/A2 activators, which in turn regulate Hsp101 expression and basal thermotolerance.
