| In the chloroplast,the normal operation of photosynthesis needs the synergy of multiple photosynthetic protein complexes such as Photosystem I（PSI）,Photosystem II（PSII）and Ru Bis CO.After entering the chloroplast,the nuclear-encoded chloroplast proteins can perform functions by being properly folded and located.Among the photosynthesis-related nuclear encoding proteins,photosynthetic antenna complex LHC II is the most abundant protein in thylakoids of chloroplast.There are lots of unknown areas to be studied in the regulation of the accumulation and homeostasis balance of photosynthesis-related nuclear encoding proteins during chloroplast development in higher plants.To illustrate the homeostasis regulation of nuclear-encoded chloroplast proteins,we used an Arabidopsis thaliana mutant pga1-1 with pale green leaf color screened by EMS mutagenesis in our laboratory.PGA1 encodes chloroplast envelope protein AtFtsH12,a member of the Fts H family that locates in the inner membrane of chloroplasts.Our findings are as follows:1.PGA1 encodes AtFtsH12.Two binary plant expression vectors,p Cambia1300-p AtFtsH12:g AtFtsH12-GFP and p Cambia1300-p UBQ10-AtFtsH12,were constructed,and then were applied for the complement of pga1-1.It was confirmed that the mutation in AT1G79560/AtFtsH12 gene indeed caused the pga1-1 phenotype.The missense mutation site G703R is located in the GAD motif of ATPase domain of AtFtsH12.2.AtFtsH12 is involved in the regulation of chloroplast development in Arabidopsis thaliana.The 1-D BN-PAGE and the 2-D SDS-Urea-PAGE analyses found that the accumulation of photosynthetic protein complexes in pga1-1 was significantly reduced compared with those in the WT.The immunoblotting with total protein of 2-weeks-old plants further revealed that the general reduction of photosynthetic proteins and the most affected accumulation of light-harvesting proteins.The immunoblotting analyses with deetiolated seedlings suggested that there are a strongly decline of the accumulation rate of chloroplast protein homeostasis during de-etiolation in pga1-1.3.The purification and confirmation of AtFtsH12 polyclonal antibody.The prokaryotic expression system of p ET28a-p T7:AtFtsH12-Loop-His was established and the fusion protein AtFtsH12-Loop-His was expressed by IPTG induction.AtFtsH12-loop-His protein was purified and antiserum AtFtsH12 was prepared by immunizing rabbits.High efficiency of AtFtsH12 polyclonal antibody was obtained by antigen-affinity purified antiserum,and endogenous AtFtsH12 was detected in total protein of WT,pga1-1 and its different complementation lines.4.AtFtsH12 formed large protein complexes on the chloroplast envelope.By observing the fluorescence signal of mesophyll protoplast of pga1-1 p AtFtsH12:g AtFtsH12-GFP and chloroplast fractionation,AtFtsH12 was confirmed to be localized in chloroplast envelope of Arabidopsis thaliana.The results of the 1-D BN-PAGE and the 2-D SDS-Urea-PAGE analyses found that AtFtsH12G703R don’t affect the oligomerzation of its large complexes.The increased AtFtsH12G703R represents a compensating response to the defective chloroplast development in pga1-1.5.AtFtsH12 is involved in the accumulation of PEP complex and expression of PEP-dependent chloroplast genes.The q RT-PCR analyses showed that the accumulation of psb A and rbc L transcripts transcribed by PEP decreased in pga1-1 compared with those in WT during photomorphogenesis.Interestingly,the rpo Tp gene,encoding the NEP in pga1-1was nearly twice as up-regulated as that of WT at 24 h,while the expression of rpo B,the core subunit of PEP catalyzed by NEP,was only slightly down-regulated at the RNA level,and the accumulation of p TAC2 encoding an auxiliary subunit of PEP was almost not affected.The results of BN-PAGE and Western Blot analysis showed a significant decrease of the accumulation of PEP complexes in pga1-1 compared with those in WT6.AtFtsH12 regulates the accumulation of cytosol-translated chloroplast proteins.The results of Yeast two-hybrid suggest that the Loop domain of AtFtsH12 directly interacted with the N-terminus of Lhc B2 precursor protein.The fluorescence signal in 4-weeks-old pga1-1 showed that the GFP protein labeled after the c TP could not fully enter the chloroplast.The fluorescence signal of hybrid plant pga1-1 35S:FNRctp-GFP showed that the accumulation of FNRctp-GFP decreased significantly.GFP protein accumulation occurred in cytosol of 4-weeks-old pga1-1 35S:FNRctp-GFP protoplasts.7.The pga1-1 mutation triggers cytosolic protein stress response and the chloroplast unfolded protein response.The q RT-PCR analyses showed that ct Hsp70 and cp Hsp70,the marker genes of cytosolic protein stress response and chloroplast unfolded protein response were up-regulated at both transcript level and protein level in pga1-1 than that of WT.In addition,the expression of Hsf A2,encoding the Heat Shock Transcription Factor A2（Hsf A2）involved in chloroplast unfold protein response（cp UPR）,was also up-regulated in pga1-1.Meanwhile,the accumulation of protease Clp P1 in pga1-1 was significantly increased.These results indicate that the balance of cytosolic and chloroplast protein homeostasis was affected in pga1-1. |
