Functional Analysis Of A Chlorophyll Synthesis Gene CBD1 (Chlorophyll Biosynthetic Deficiency1) In Arabidopsis Thaliana

Photosynthesis is a fundamental process that supports organic life in the biosphere by conversion of solar energy into chemical energy and carbon food.Chloroplasts are unique site for the reactions of photosynthesis in land plants,algae and some protists,which making it responsible for much of the world's primary productivity.As the most abundant pigment in photosynthetic organisms,chlorophyll(Chl)plays vital roles in harvesting light energy in the antenna systems and driving electron transfer in the reaction centers.Thus,it is not surprising that chl biosynthesis is str:ictly regulated.The deficiency in Chl biosynthesis would lead to leaf coloration.In this study,we isolated an Arabidopsis mutant with pale-green leaves from the T-DNA inserted mutant library in our lab,and named it as cbdl(chlorophyll biosynthetic deficiency 1).We utilized various methods including bioinformatics,plant molecular genetics,chloroplast genomics and spectrometry analysis to explore the function of CBD1.The important results were as follows.1.CBD1 is a conserved multi-transmembrane protein localized in chloroplastCBD1 encodes a protein with 6 transmembrane domains,and has a predicted cTP cleavage site at its N-terminal.CBD1 contains 3 functional conserved motifs,which assigned it to a Type ? CAAX superfamily.CBD1 is well conserved in fern,moss and angiosperm,but is absent in archaebacteria and gymnosperm based on bioinformatics and phylogenic analysis.In functional analysis of genes co-expressed with CBD1,we inferred that CBD1 might be involved in photosynthesis and tetrapyrrole biosynthetic pathway.Through transient expression in Arabidopsis protoplast and histochemical GUS staining,we identified that CBD1 was localized in thylakoid membrane and expressed in green tissues only.2.CBD1 is involved in Chl biosynthetic pathwayCbd1 exhibited a pale-green phenotype in normal growth condition,and the Chl content was decreased by 30%-40%compared with WT,especially the Ch1 b,which resulted in an increased ratio of Chl a/b.When dark-grown WT and cbd1 etiolated seedlings were exposed to light,cbd1 showed a slower rate in Ch1 accumulation.In analysis of gene expression and intermediates levels in Ch1 biosynthetic pathway using qRT-PCR and spectrometry,we detected that the light-responsive expression levels of several key genes in this pathway were altered in cbd1.When DP(Fe chelator)and ALA(precursor in Ch1 biosynthetic pathway)were added to the growth medium,cbd1 showed a remarkably accumulation of Mg-ProtoIX.3.CBD1 affects the photosynthetic assembly and efficiencySince chloroplast development relies on both coordinated synthesis of Chls and integration of thylakoid proteins,the alterations of thylakoid proteins between WT and cbd1 were analyzed using BN-PAGE,SDS-PAGE and immunoblot.The accumulation of PsaA+B and PsaE as well as decrease of LHCII-T indicated that CBD1 might be responsible for the assembly of PSI supercomplex and LHCII-T.In photosynthetic efficiency assessment using Ch1 fluorometer FMS2 and Dual-PAM100,cbd1 showed a relatively higher photochemical efficiency of PSI and PS?,and also a higher heat loss.4.CBD1 alters the ion homeostasis in chloroplast.Expression of CBD1 could complement bacterial strain MM281 lacking Mg2+uptake capability.In ICP-MS analysis of the ion profiles in every 109 intact chloroplasts and thylakoids,cbd1 showed a 40%lower Mg2+content than WT.5.CBD1 is genetically interacted with GUN5The cbd1gun5 double mutant was obtained through hybridization and exhibited a yellowish and dwarf phenotype.Double mutant cbd1gun5 demonstrated a severe inhibition of Ch1 biosynthesis and assembly of photosystem apparatus.Through transmission electronic microscopy analysis,we found cbd1gun5 contains only poorly developed chloroplast and thylakoid.Based on results above,we came to the conclusion that CBD1 participated in chlorophyll biosynthetic pathway,and was responsible for the Mg acquisition and transfer in this process.Collectively,efforts on CBD1 not only revealed the biological functions of a previously unknown protein,but also provided new insights into researches concerning the regulatory mechanisms of the Ch1 biosynthetic pathway.