Characterization Of Physiological Significance Of D1 Protein Maturation In Arabidopsis

Photosynthesis,which convert inorganic matter into organic matter by assimilating solar energy,provides the material basis for the formation,development,prosperity and maintenance of the biosphere.It is known as “the most important chemical reaction on earth”.Photosystem ?(PS?),a multimeric protein complex in the thylakoid membrane,catalyzes the light-driven oxidation of water and the reduction of plastoquinone,which play a critical role of photosynthetic electron transport chain.D1 protein,as the core of PS? complex,plays a critical role in PS? activity.In most oxygenic photosynthetic organisms,D1 is synthesized as precursor form(pD1),which is cleaved by C-terminal peptidase(CTP)to generating mature D1 protein.The maturation of D1 protein is one of the key steps for the assembly of PS? complex and photo-damage repair.At present,it remains unclear that the biological significance of D1 protein maturation and the physiological function of pD1 C-terminal peptides.We explored these two issues through experimental analysis of both CTP protease and D1 protein.The CTP proteins from oxygenic photosynthetic organisms were firstly performed phylogenetic tree analysis.It showed thatCtpAs of eukaryotes and prokaryotes evolved from the most recent a common ancestor;ProkaryoticCtpBs andCtpCs are clustered in a monophyletic clade and share a close relationship withCtpA while eukaryoticCtpBs andCtpCs evolved in independent lineages,indicating thatCtpA has diverged early fromCtpB andCtpC during evolution implying distinct roles for the Ctps.The transcription level analysis showed that three Arabidopsis CTP genes displayed different expression levels,which implied the functional differences again.We further analyzed these three Ctp-deficient mutants in Arabidopsis.The deficiency of AtCtpA(At4G17740)could lead to the accumulation of pD1 protein and lethal phenotype while its homologous,AtCtpB(At3G57480)and AtCtpC(At5G46390),single or double mutants did not cause any significant changes in plant growth phenotype and pD1 protein accumulation.It seemed that only AtCtpA protein was responsible for the maturation of pD1 protein.The Ctpprotein family was fall into four clades based on the phylogenetic tree analysis.We analyzed the function of Ctps from Synechococcus elongatus PCC7942,Chlamydomonas reinhardtii,Physcomitrella patens,and Arabidopsis thaliana,four model organisms representing different evolutionary levels.TheCtpAs with relative low sequence similarity were found to be functional conserved and could convert pD1 into mature form to rescue the lethal phenotype of AtCtpA-deficient in Arabidopsis.However,allCtpB orCtpC proteins did not have the function of processing pD1 protein.We deduced thatCtpA should be developed the function of processing pD1 protein during evolution after diverging withCtpB andCtpC,whileCtpB and CtpC did not in whole evolution process.The activity of above-mentioned fourCtpA proteases was tested with two substrates from Arabidopsis thaliana and cyanobacteria pD1,respectively,showing thatCtpA from higher plants exhibited the strongest processing activity against both substrates,which may be an adaptation to the rapid turnover of photo-damaged D1 protein under excessive light environment.Taken together,Ctp A is the only protease that achieves the function of processing pD1 during evolution.In order to explore the physiological function of pD1 C-terminal short peptides,we fused mature D1 proteins with transit peptides localized to chloroplast stroma and transferred it into AtCtpA-deficient mutants.The results showed that the transit peptide localized to chloroplast stroma could transfer the nucleus-encoded fusion proteins into the chloroplast stroma,and the exogenous mature D1 proteins could successfully assemble the active PS? complex.Compared with the wild type,there was no significant difference in the growth phenotype in this transgenic plant.Photosynthetic parameter analysis showed that PS? activity of transgenic plants could make the same light response as wild type,but it was slightly lower than that of the wild type due to the endogenous pD1 protein encoded by chloroplast genome.We further fused the mature AtCtpA protease with the transit peptide localized to the chloroplast stroma and transferred it into the mutant with AtCtpA deletion to make a mature D1 protein in chloroplast stroma,which could also be successfully assembled into the functional PS? complex,and the PS? activity showed the same light response as the wild type.These results indicate that the AtCtpA localized to chloroplast stroma could process pD1;and the premature D1 in chloroplast stroma could perform normally function.Therefore,we conclude that C-terminal extension of pD1 protein does not play an important physiological function in photosynthesis.Taken together,all data indicate that the mature D1 protein is necessary for oxygenic photosynthetic organisms and a prerequisite for the formation of active PS? complex,Ctp A may be only protease responsible for processing pD1,and the pD1 C-terminal extension seems not physiological significance.We speculated that the original PS? contained a pD1/D2 type anoxygenic reaction center,whileCtpA evolved a specific function of processing pD1 C-terminal extension,which lead to the transformation of pD1/D2 to D1/D2 type PS?.Therefore,we believe that the specialization ofCtpA function may be a key factor in the emergence of oxygenic photosynthetic organisms,and the C-terminal extension of pD1 may be a historical legacy in the evolutionary process.