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
The long-term response （LTR） to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB （enco...The long-term response （LTR） to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB （encoding the P700 apoproteins of photosystem I） and psbA （encoding the D1 protein of photosystem II） and requires the action of plastidlocalized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase （PEP） is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.展开更多
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.
文摘The long-term response （LTR） to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB （encoding the P700 apoproteins of photosystem I） and psbA （encoding the D1 protein of photosystem II） and requires the action of plastidlocalized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase （PEP） is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.
