Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reduc-tase, type C （NTRC） in the light. Here we show overexpression of NTRC in Arabidopsis （NTRC°E） resulting in...Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reduc-tase, type C （NTRC） in the light. Here we show overexpression of NTRC in Arabidopsis （NTRC°E） resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants （ntrcl） exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the protein＇s biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure.. Complexes of higher molecular weight （HMW） showed stronger activity as a holdase chaperone, while low molecular weight （LMW） species exhibited weaker holdase chaperone activity but stronger disulfide reductase and fol-dase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser （C217/454S-NTRC） led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°E plants. However, after prolonged incubation under heat shock, NTRC°E plants tolerated the stress to a higher degree than C217/454S-NTRC°E plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.展开更多
Plastid-encoded RNA polymerase （PEP） is closely associated with numerous factors to form PEP complex for plastid gene expression and chloroplast development. However, it is not clear how PEP complex are regulated in...Plastid-encoded RNA polymerase （PEP） is closely associated with numerous factors to form PEP complex for plastid gene expression and chloroplast development. However, it is not clear how PEP complex are regulated in chloroplast. Here, one thioredoxin-like fold protein, Arabidopsis early chloroplast biogenesis 1 （AtECB1）, an allele of MRL7, was identified to regulate PEP function and chloroplast biogenesis. The knockout lines for AtECB1 displayed albino phenotype and impaired chloroplast development. The transcripts of PEP-dependent plastid genes were barely detected, suggesting that the PEP activity is almost lost in atecbl-1. Although AtECB1 was not identified in PEP complex, a yeast two-hybrid assay and pull-down experiments demonstrated that it can interact with Trx Z and FSD3, two intrinsic subunits of PEP complex, respectively. This indicates that AtECB1 may play a regulatory role for PEP-dependent plastid gene expression through these two subunits. AtECB1 contains a βαβαββα structure in the thioredoxin-like fold domain and lacks the typical C-X-X-C active site motif. Insulin assay demonstrated that AtECB1 harbors disulfide reductase activity in vitro using the purified recombinant AtECB1 protein. This showed that this thioredoxin-like fold protein, AtECB1 also has the thioredoxin activity. AtECB1 may play a role in thioredoxin signaling to regulate plastid gene expression and chloroplast development.展开更多
文摘Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reduc-tase, type C （NTRC） in the light. Here we show overexpression of NTRC in Arabidopsis （NTRC°E） resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants （ntrcl） exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the protein＇s biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure.. Complexes of higher molecular weight （HMW） showed stronger activity as a holdase chaperone, while low molecular weight （LMW） species exhibited weaker holdase chaperone activity but stronger disulfide reductase and fol-dase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser （C217/454S-NTRC） led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°E plants. However, after prolonged incubation under heat shock, NTRC°E plants tolerated the stress to a higher degree than C217/454S-NTRC°E plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.
文摘Plastid-encoded RNA polymerase （PEP） is closely associated with numerous factors to form PEP complex for plastid gene expression and chloroplast development. However, it is not clear how PEP complex are regulated in chloroplast. Here, one thioredoxin-like fold protein, Arabidopsis early chloroplast biogenesis 1 （AtECB1）, an allele of MRL7, was identified to regulate PEP function and chloroplast biogenesis. The knockout lines for AtECB1 displayed albino phenotype and impaired chloroplast development. The transcripts of PEP-dependent plastid genes were barely detected, suggesting that the PEP activity is almost lost in atecbl-1. Although AtECB1 was not identified in PEP complex, a yeast two-hybrid assay and pull-down experiments demonstrated that it can interact with Trx Z and FSD3, two intrinsic subunits of PEP complex, respectively. This indicates that AtECB1 may play a regulatory role for PEP-dependent plastid gene expression through these two subunits. AtECB1 contains a βαβαββα structure in the thioredoxin-like fold domain and lacks the typical C-X-X-C active site motif. Insulin assay demonstrated that AtECB1 harbors disulfide reductase activity in vitro using the purified recombinant AtECB1 protein. This showed that this thioredoxin-like fold protein, AtECB1 also has the thioredoxin activity. AtECB1 may play a role in thioredoxin signaling to regulate plastid gene expression and chloroplast development.
