Summary: The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca²⁺ ([Ca²⁺]_ext)‐induced [Ca²⁺]_cyt transients and stomatal closure in Arabidopsis. CAS (Ca²⁺ sensing receptor) is a plant‐specific putative Ca²⁺‐binding protein that was originally proposed to be a plasma membrane‐localized external Ca²⁺ sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N‐terminus acidic Ca²⁺‐binding region is likely exposed to the stromal side of the membrane. The phenotype of T‐DNA insertion CAS knockout mutants and cDNA mutant‐complemented plants revealed that CAS is essential for stomatal closure induced by external Ca²⁺. In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca²⁺. Furthermore, using the transgenic aequorin system, we showed that [Ca²⁺]_ext‐induced [Ca²⁺]_cyt transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane‐localized CAS is essential for [Ca²⁺]_ext‐induced [Ca²⁺]_cyt transients and stomatal closure.

Summary: Eubacterial‐type multi‐subunit plastid RNA polymerase (PEP) is responsible for the principal transcription activity in chloroplasts. PEP is composed of plastid‐encoded core subunits and one of multiple nuclear‐encoded sigma factors that confer promoter specificity on PEP. Thus, the replacement of sigma factors associated with PEP has been assumed to be a major mechanism for the switching of transcription patterns during chloroplast development. The null mutant (sig6‐1) of plastid sigma factor geneAtSIG6exhibited a cotyledon‐specific pale green phenotype. Light‐dependent chloroplast development was significantly delayed in thesig6‐1mutant. Genetic complementation of the mutant phenotype by theAtSIG6cDNA demonstrated that AtSIG6 plays a key role in light‐dependent chloroplast development. Northern and array‐based global analyses for plastid transcripts revealed that the transcript levels of most PEP‐dependent genes were greatly reduced in thesig6‐1mutant, but that the accumulation of nuclear‐encoded RNA polymerase (NEP)‐dependent transcripts generally increased. As the PEPαsubunit and PEP‐dependenttrnVaccumulated at normal levels in thesig6‐1mutant, theAtSIG6knockout mutant probably retained functional PEP, and the transcriptional defects are likely to have been directly caused byAtSIG6deficiency. Most of the AtSIG6‐dependent genes are preceded byσ⁷⁰‐type promoters comprised of conserved −35/−10 elements. Thus, AtSIG6 may act as a major general sigma factor in chloroplasts during early plant development. On the other hand, the mutant phenotype was restored in older seedlings. Arabidopsis probably contains another late general sigma factor, the promoter specificity of which widely overlaps with that of AtSIG6.

Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.

Chloroplasts are semiautonomous organelles which possess their own genome and gene expression system. However, extant chloroplasts contain only limited coding information, and are dependent on a large number of nucleus-encoded proteins. During plant evolution, chloroplasts have lost most of the prokaryotic DNA binding proteins and transcription regulators that were present in the original endosymbiont. Thus, chloroplasts have a unique hybrid transcription system composed of the remaining prokaryotic components, such as a prokaryotic RNA polymerase as well as nucleus-encoded eukaryotic components. Recent proteomic and transcriptomic analyses have provided insights into chloroplast transcription systems and their evolution. Here, we review chloroplast-specific transcription systems, focusing on the multiple RNA polymerases, eukaryotic transcription regulators in chloroplasts, chloroplast promoters, and the dynamics of chloroplast nucleoids.

Light is one of the most important environmental factors regulating expression of photosynthesis genes. The plastid psbD gene encoding the photosystem II reaction center protein D2 is under the control of a unique blue light responsive promoter (BLRP) that is transcribed by a bacterial-type plastid RNA polymerase (PEP). Promoter recognition of PEP is mediated by one of the six nuclear-encoded σ factors in Arabidopsis . The replacement of the plastid σ factor associated with PEP may be the major mechanism for switching of plastid transcription pattern in response to environmental and developmental signals. This study demonstrates that AtSig5 is a unique σ factor that is essential for psbD BLRP activity. A T-DNA insertional mutant with reduced AtSIG5 expression resulted in loss of primary transcripts from the psbD BLRP. Furthermore, transient overexpression of AtSig5 in dark-adapted protoplasts specifically elevated psbD and psbA transcription activities. On the other hand, overproduction of AtSig2 enhanced the transcription of psbA gene and trnE operon, but not psbD transcription. The AtSIG5 gene is phylogenetically distinct from other plastid σ factors, and its expression is induced exclusively by blue light. We propose that AtSig5 acts as a mediator of blue light signaling that specifically activates the psbD BLRP in response to blue light in Arabidopsis .

Plastid transcription is mediated by two distinct types of RNA polymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type RNAP (NEP). Recent genomic and proteomic studies revealed that higher plants have lost most prokaryotic transcription regulators and have acquired eukaryotic-type proteins during plant evolution. However, in vivo dynamics of chloroplast RNA polymerases and eukaryotic-type plastid nucleoid proteins have not been directly characterized experimentally. Here, we examine the association of the α-subunit of PEP and eukaryotic-type protein, plastid transcriptionally active chromosome 3 (pTAC3) with transcribed regions in vivo by using chloroplast chromatin immunoprecipitation (cpChIP) assays. PEP α-subunit preferentially associates with PEP promoters of photosynthesis and rRNA genes, but not with NEP promoter regions, suggesting selective and accurate recognition of PEP promoters by PEP. The cpChIP assays further demonstrate that the peak of PEP association occurs at the promoter-proximal region and declines gradually along the transcribed region. pTAC3 is a putative DNA-binding protein that is localized to chloroplast nucleoids and is essential for PEP-dependent transcription. Density gradient and immunoprecipitation analyses of PEP revealed that pTAC3 is associated with the PEP complex. Interestingly, pTAC3 associates with the PEP complex not only during transcription initiation, but also during elongation and termination. These results suggest that pTAC3 is an essential component of the chloroplast PEP complex. In addition, we demonstrate that light-dependent chloroplast transcription is mediated by light-induced association of the PEP–pTAC3 complex with promoters. This study illustrates unique dynamics of PEP and its associated protein pTAC3 during light-dependent transcription in chloroplasts.