Abstract
Chloroplasts have their own DNA and gene expression systems. Transcription in chloroplasts is regulated by two types of RNA polymerase, nuclear-encoded plastid RNA polymerase (NEP) and plastid-encoded plastid RNA polymerase (PEP), and multiple sigma factors for PEP. To study transcriptional regulation in chloroplasts, a molecular genetic approach has extensively been used. However, this method may include indirect effects, and it cannot be applied to the analysis of factors essential to survival. These limitations make understanding specific regulation by transcription factors difficult. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool for obtaining information on transcription-factor binding sites; it can directly detect dynamic changes in their interaction patterns in vivo. To further understand transcriptional regulation in chloroplasts, we here established a ChIP-based method in Arabidopsis thaliana and analyzed the binding pattern of a chloroplast sigma factor, SIG1. We found that SIG1 specifically binds to newly identified target promoters as well as to a set of promoters of genes whose mRNA expression is dependent on OsSIG1 in rice and that this binding changed in response to high-light stress. These results suggested that the ChIP-based approach is very useful in understanding transcriptional regulation of chloroplast genes and can overcome several problems posed by conventional methods.
Highlights
Chloroplasts are plant organelles that originated from an endosymbiotic event involving an ancestral oxygen-evolving photosynthetic cyanobacterium
Antiserum specificity was checked by immunoblot analysis. It has been reported in a previous study that SIG1 detected around 50 kDa [18], major single band around 40 kDa, suggesting a mature protein of SIG1, could be detected in our experimental condition (Figure 1b), and we used this antibody for further immunoblot and Chromatin immunoprecipitation (ChIP) analyses
The basal SIG1 binding level to its target promoters under normal light was significant; SIG1 binding was dramatically decreased by high-light exposure (Figure 3). This binding pattern observed in all five promoters, while other promoters showed no major changes in binding activity. These results indicated that SIG1 binds strongly to target promoters under normal light conditions, and it can be released in response to high-light stress
Summary
Chloroplasts are plant organelles that originated from an endosymbiotic event involving an ancestral oxygen-evolving photosynthetic cyanobacterium. Chloroplasts have their own genomes and transcription-translation machineries of cyanobacterial origin. The chloroplast genomes of most higher plants are circular, double-stranded DNA molecules containing about 120 genes required for photosynthesis, gene expression, and some metabolic functions [1,2]. These genes encode only some of the chloroplast proteins; most other proteins required for chloroplast functions are encoded by the nuclear genome [3].
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