The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.     

Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein

Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI-atpH intergenic region (i) blocks both 5'→3' and 3'→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5'-terminus in conjunction with a generic 5'→3' exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10's ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins.     

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20–60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6–12 bp perfect repeats, or 10–16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomology-mediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle''s ability to repair broken chromosomes.     

基于叶绿体psbA-trnH基因间区序列鉴定肉苁蓉属植物

肉苁蓉的干燥肉质茎是常用中药材, 药典收录基源植物有管花肉苁蓉和荒漠肉苁蓉两种, 但是市场上经常会与黄花列当、草苁蓉、盐生肉苁蓉及沙苁蓉等混用。本文对不同类群肉苁蓉及混淆品的叶绿体psbA-trnH基因间区进行PCR扩增并测序, 获得了该区间的完整序列, 用K-2P法建立了系统进化树, 聚类结果与形态分类相符。所得结果显示, psbA-trnH片段序列在肉苁蓉及混淆品种间存在丰富的变异, 肉苁蓉属各种的种间遗传距离从0.077% 到0.743%, 种内遗传距离从0% 到0.007%, 种内和种间存在明显的“barcoding gap”。肉苁蓉属各种与黄花列当的遗传距离为0.979% 至1.149%, 与草苁蓉的遗传距离为1.066% 至1.224%。研究结果表明psbA-trnH基因间区可以作为条形码来鉴定肉苁蓉及其混淆品。     

Horizontal transfer of chloroplast genomes between plant species

The genomes of DNA-containing cell organelles (mitochondria, chloroplasts) can be laterally transmitted between organisms, a process known as organelle capture. Organelle capture often occurs in the absence of detectable nuclear introgression, and the capture mechanism is unknown. Here, we have considered horizontal genome transfer across natural grafts as a mechanism underlying chloroplast capture in plants. By grafting sexually incompatible species, we show that complete chloroplast genomes can travel across the graft junction from one species into another. We demonstrate that, consistent with reported phylogenetic evidence, replacement of the resident plastid genome by the alien genome occurs in the absence of intergenomic recombination. Our results provide a plausible mechanism for organelle capture in plants and suggest natural grafting as a path for horizontal gene and genome transfer between sexually incompatible species.     

Cell-to-cell movement of plastids in plants

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.     

TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import

TIC236 is an essential component of the translocon for protein import into chloroplasts, as evidenced by the embryonic lethality of the knockout mutant. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen exploring CRL function found multiple dominant TIC236 gain-of-function (tic236-gf) mutations that abolished crl-induced phenotypes. Moreover, CRL associates with TIC236, and a tic236-knockdown mutant exhibited multiple lesions similar to the crl mutant, supporting their shared functionality. Consistent with the defective plastid division phenotype of crl, CRL interacts with the transit peptides of proteins essential in plastid division, with tic236-gf mutations reinforcing their import via increased TIC236 stability. Ensuing reverse genetic analyses further revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Collectively, our data shed light on the links between plastid protein import, plastid division, and plant stress responses.

Chloroplasts evolved from a gram-negative cyanobacterial endosymbiont, with most cyanobacterial genes having been transferred to the host plant genome. Therefore, thousands of nuclear-encoded chloroplast proteins are posttranslationally imported into chloroplasts, orchestrated by outer and inner envelope membrane (OEM and IEM) translocons, respectively termed TOC and TIC. Although an array of translocon proteins has been identified (1, 2), it has been unclear how TOC and TIC are linked across the two envelope membranes separated by an intermembrane space. The recent discovery of the TIC236 protein solved this long-standing question (3). TIC236 is an integral IEM protein associated with TIC components. Its C-terminal domain, located in the intermembrane space, directly interacts with the N-terminal polypeptide transport-associated (POTRA) domains of TOC75-III (hereafter TOC75), the channel protein in the TOC complex. TOC75 and TIC236 are homologs of bacterial TamA (TRANSLOCON ASSEMBLY MODULE A) and TamB, respectively, which together function in bacterial outer membrane protein biogenesis and protein export (3–5).Plastid division occurs in developing cells to ensure an optimal number of plastids is in place before cell division, requiring the import of a suite of plastid-division machinery (PDM) proteins. The loss of any vital PDM components results in gigantic plastids and a drastically reduced plastid number per cell (6). Surprisingly, several Arabidopsis mutants deficient in plastid division—including crumpled leaf (crl), accumulation and replication of chloroplasts6 (arc6), plastid division2 (pdv2), and filamenting temperature-sensitive z1 (ftsz1)—develop foliar cell death (7), resembling lesion-mimicking mutants (LMM) that exhibit a light-dependent hypersensitive response–like cell death (8, 9). Like LMM, crl and other plastid division mutants constantly up-regulate immune-related genes (7, 10). The gigantic chloroplasts of crl mutants also induce an abnormal cell cycle, with increased endoreduplication activity leading to stunted growth (11). Previous studies have indicated that autoimmune responses, abnormal cell cycle, and growth inhibition are likely mediated by a process called retrograde signaling [i.e., signaling from the gigantic chloroplasts back to the nucleus (7, 10, 11)].CRL is a nuclear-encoded chloroplast OEM protein. Its short N-terminal region resides in the intermembrane space, followed by a transmembrane domain and a cytosolic chromophore lyase CpcT/CpeT domain characterized from a cyanobacterial CpcT bilin lyase (12). Although the lyase domain retains phycocyanobilin-binding aptitude, there is no apparent correlation between phycocyanobilin-binding ability and crl-induced lesions in Arabidopsis (13), indicating that CRL has gained a divergent function.