Differential expression on a daily basis of plastid sigma factor genes from the moss Physcomitrella patens. Regulatory interactions among PpSig5, the circadian clock, and blue light signaling mediated by cryptochromes

The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisubunit bacteria-type plastid RNA polymerase. We studied here whether or not three genes, PpSig1, PpSig2, and PpSig5 encoding plastid sigma factors, are controlled by the circadian clock and/or by blue light signaling in the moss Physcomitrella patens. Among the three PpSig genes, only PpSig5 was clearly controlled by the circadian clock. In contrast to the differential regulation on a daily timescale, a pulse of blue light induced the expression of all the three PpSig genes. This induction was significantly reduced in a knockout mutant that lacked the blue light photoreceptor cryptochromes PpCRY1a and PpCRY1b, indicating that PpCRY1a and/or PpCRY1b mediate the blue light signal that induces the expression of the PpSig genes. In a daily cycle of 12-h blue light/12-h dark, the timing of peak expression of PpSig5 and a chloroplast gene psbD, encoding the D2 subunit of photosystem II, advanced in the cryptochrome mutant relative to those in the wild type, suggesting the presence of regulatory interactions among the expression of PpSig5 and psbD, the circadian clock, and the blue light signaling mediated by the cryptochrome(s).     

Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii

Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a yellow-in-the-dark phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.     

Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.     

Mutations in CHLOROPLAST RNA BINDING provide evidence for the involvement of the chloroplast in the regulation of the circadian clock in Arabidopsis

The Arabidopsis circadian system regulates the expression of up to 36% of the nuclear genome, including many genes that encode photosynthetic proteins. The expression of nuclear-encoded photosynthesis genes is also regulated by signals from the chloroplasts, a process known as retrograde signaling. We have identified CHLOROPLAST RNA BINDING (CRB), a putative RNA-binding protein, and have shown that it is important for the proper functioning of the chloroplast. crb plants are smaller and paler than wild-type plants, and have altered chloroplast morphology and photosynthetic performance. Surprisingly, mutations in CRB also affect the circadian system, altering the expression of both oscillator and output genes. In order to determine whether the changes in circadian gene expression are specific to mutations in the CRB gene, or are more generally caused by the malfunctioning of the chloroplast, we also examined the circadian system in mutations affecting STN7, GUN1, and GUN5, unrelated nuclear-encoded chloroplast proteins known to be involved in retrograde signaling. Our results provide evidence that the functional state of the chloroplast may be an important factor that affects the circadian system.     

同源重组置换衣藻叶绿体chlL基因及其同质化鉴定

设计以 aad A基因置换衣藻 chl L基因并作为转基因衣藻筛选标记 ,将 chl L基因两侧的基因片段 clp P- trn L- pet B- chl L5′- 3′作为同源片段 ,构建衣藻叶绿体同源重组质粒 p64D.通过改进的基因枪法转化程序 ,将此质粒轰击入衣藻叶绿体 .经抗性筛选、暗培养及 PCR、Southern- blotting等鉴定 ,证实衣藻叶绿体基因组中大部分 chl L结构基因已被外源基因 aad A所置换     

Localization and function of SulP,a nuclear-encoded chloroplast sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Recent work [H.-C. Chen et al. (2003) Planta 218:98-106] reported on the genomic, proteomic, phylogenetic and evolutionary aspects of a putative nuclear gene ( SulP) encoding a chloroplast sulfate permease in the model green alga Chlamydomonas reinhardtii. In this article, evidence is provided for the envelope localization of the SulP protein and its function in the uptake and assimilation of sulfate by the chloroplast. Localization of the SulP protein in the chloroplast envelope was concluded upon isolation of C. reinhardtii chloroplasts, followed by fractionation into envelope and thylakoid membranes and Western blotting of these fractions with specific polyclonal antibodies raised against the recombinant SulP protein. The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the photosystem-II D1 reaction-center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in C. reinhardtii is discussed along with its impact on the repair of photosystem-II from a frequently occurring photo-oxidative damage and potential use for the elucidation of the H(2)-evolution-related metabolism in this green alga.     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.