Arabidopsis DnaJ-Like Zinc Finger Protein AtOR Regulates Plastid Development Through Its Interaction With The BHLH Transcription Factor TCP14

In natural condition,seedlings have to struggle out of the dark soil for the sunshine.Cotyledon will open,expand and turn green after exposing to the light.This crucial event that plants perceive light and cotyledons turn green is so-called de-etiolation.In the cotyledon of dark grown seedlings,proplastid will develop into etioplast where prolamellar body(PLB)forms with membranous structure harboring carotenoids,chlorophyll precursor protochlorophyllide,protochlorophyllide oxidoreductases(POR)and some other proteins.Etioplasts will convert into chloroplasts once perceive the light.Both structural change and biochemical variation will happen during this switch,including the formation of thylakoid membrane structure,the accumulation of carotenoids and chlorophylls,and the synthesis and importing of proteins for photosynthesis,etc.Previously we reported an Orange(Or)gene in cauliflower.Or encodes a DnaJ-like cysteine-rich zinc finger protein which is highly conserved in land plants.Functional characterization and transgenic studies both showed that Or is capable of turning non-pigmented plastids into chromoplasts with high accumulation of carotenoids,although the molecular mechanism is still unclear.Here we identified a bHLH transcription factor TCP 14 as a novel partner of the Arabidopsis OR ortholog(AtOR)by yeast two-hybrid screening.We also confirmed the in vitro and in planta interaction between these two proteins by different approaches.The overexpression of AtOr induced the expression of genes for carotenoid metabolism,sequestration and plastid division,and also showed enhanced ratios of major carotenoid components(?-carotene,lutein,violaxanthin and neoxanthin)against chlorophyll a comparing with that in the wild type seedlings.However,AtOr overexpression repressed the expression of genes for key enzymes for chlorophyll biosynthesis and for chlorophyll binding proteins.Ultrastructure of plastids in AtOr overexpression lines also revealed a delay in the development of thylakoid membranes.When TCP 14 is knocked out,no distinct phenotype was observed.Most of the genes which AtOr regulated with a Col-0 WT background did not show significant response to AtOr overexpression,nor silencing,with a tcp14-1 background.It is TCP 14 that directly binds to the promoter region of related genes,and AtOR conducts its function by attaching to TCP 14 through protein-protein interaction.Our genetic analysis revealed that the functioning of OR relies on the presence of TCP 14.Moreover,our studies showed that AtOR is a co-repressor of TCP 14,and the expression of AtOr is stimulated by TCP 14.In this way,these two proteins might form a regulatory loop controling the development of etioplast.