Immunophilin CYN28 is required for accumulation of photosystem II and thylakoid FtsH protease in Chlamydomonas.

Excess light causes severe photodamage to photosystem II (PSII) where the primary charge separation for electron transfer takes place. Dissection of mechanisms underlying the PSII maintenance and repair cycle in green algae promotes the usage of genetic engineering and synthetic biology to improve photosynthesis and biomass production. In this study, we systematically analyzed the high light (HL) responsive immunophilin genes in Chlamydomonas (Chlamydomonas reinhardtii) and identified one chloroplast lumen-localized immunophilin, CYN28, as an essential player in HL tolerance. Lack of CYN28 caused HL hypersensitivity, severely reduced accumulation of PSII supercomplexes and compromised PSII repair in cyn28. The thylakoid FtsH (filamentation temperature-sensitive H) is an essential AAA family metalloprotease involved in the degradation of photodamaged D1 during the PSII repair cycle and was identified as one potential target of CYN28. In the cyn28 mutant, the thylakoid FtsH undergoes inefficient turnover under HL conditions. The CYN28-FtsH1/2 interaction relies on the FtsH N-terminal proline residues and is strengthened particularly under HL. Further analyses demonstrated CYN28 displays peptidyl-prolyl isomerase (PPIase) activity, which is necessary for its physiological function. Taken together, we propose that immunophilin CYN28 participates in PSII maintenance and regulates the homeostasis of FtsH under HL stress via its PPIase activity.

Functional Relationship of Arabidopsis AOXs and PTOX Revealed via Transgenic Analysis.

Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.

Diverged Early From CtpB and CtpC, CtpA Has Evolved to Process D1 Precursor in Oxygenic Photosynthetic Organisms.

C-terminal peptidase (Ctp) cleaves the C-terminal extension of the D1 precursor (pD1) to form mature D1. Among the three homologs CtpA, CtpB, and CtpC in photosynthetic organisms only the first is capable of processing pD1 while the roles of CtpB and CtpC remain elusive. Phylogenetic analysis of Ctps from photosynthetic organisms revealed that CtpA has diverged early from CtpB and CtpC during evolution implying distinct roles for the Ctps. Analysis of Arabidopsis Ctp-deficient mutants revealed that pD1 processing was not affected in atctpb, atctpc, or atctpbatctpc mutants, demonstrating that AtCtpA, not AtCtpB or AtCtpC, is responsible for cleaving the pD1 C-terminal extension. Ectopic expression of CtpAs from Synechococcus elongatus, Chlamydomonas reinhardtii, and Physcomitrella patens in atctpa rescued the lethal phenotype of the mutant indicating that SeCtpA, CrCtpA, and PpCtpA could process pD1 in Arabidopsis. Enzyme activity assays showed that PpCtpA and CrCtpA could convert pD1 into mature D1 in vitro. In contrast, expressing CtpB or CtpC from Arabidopsis, C. reinhardtii, or P. patens in atctpa did not rescue its D1 maturation deficiency, and enzyme activity assays also showed that neither CtpB nor CtpC could process pD1 in vitro. Taken together, we conclude that the function of pD1 processing by CtpA is conserved in photosynthetic organisms. It is possible that among other factors CtpA developed this function to initiate the formation of the oxygenic D1/D2 type PSII complex during evolution whereas CtpB or CtpC have other roles that are still unclear.