Evolution Analysis And Functional Identifications Of Plant Fibrillin Family Proteins In Rice Chloroplasts

Chloroplast is a multifunctional organelle,which is not only a prime place for photosynthesis,but also regulates the growth and development through the biosynthesis and metabolism pathways in plants.Plastoglobles(PGs)are the monolayer membrane with several lipoproteins,which are attached to the thylakoid membrane,and formed in the intense budding process of the thylakoid outer membranes in chloroplasts.Previous studies have shown that PGs were ubiquitous in plants and their size and number were related to the growth,development and the stress resistance of plants.In addition,there were about 30 core proteins in PGs,which were mainly involved in the lipid metabolism pathways.Of all the core proteins,the fibrillin(FBN)protein family is constituent of important and abundant proteins in PGs of chloroplasts.In plants.FBNs protein family possess the conserved plastid lipid-associated protein(PAP)domain,and participate in metabolites transport and lipid metabolisms of dicotyledous plastids.However,function of most FBN proteins in monocotyledon plants had not been studied yet.In this study,we mainly studied the function of FBN family proteins in rice chloroplasts from the perspectives of bioinformatics analysis,physiology,biochemistry and molecular biology,phylogeny and structure of the FBN family proteins were analyzed.Then,the biological functions of OsFBN1 and OsFBN7 in PGs development and heat stress tolerance were fully identified in rice.The main results are as follows:1.Bioinformatics and structural analysis of FBNs family proteins in plants.A total of121 FBN family proteins were identified from 10 representative plant species and phylogenetic trees were constructed.Phylogenetic analysis showed that 121 FBN proteins were divided into 24 clades,which were further subdivided into three groups:terrestrial-specific group,algae-specific group and intermididate group;structures of rice FBN genes were different,although all the FBN genes encoded one N-terminal chloroplast transport peptide(CTP)domain and a plastid lipid-related protein(PAP)domain.In rice,there are 11 FBN members,each of which had a CTP domain and a PAP domian.CTPs from FBNs in rice had a capacity to transport preproteins into chloroplasts except for OsFBN8 by the transient subcellular localization experiment in tobacco system;PAP domains of FBNs contained a highly conserved motif(Yx D motif,Y and D are completely conserved tyrosine and aspartic acid residues).Through site-directed mutation and in vitro lipid binding activity analysis of these two amino acids in this motif,the in vitro lipids binding activity of the PAP domain in OsFBN2 and OsFBN4 to C12-C22 fatty acids was found to be affected by the conserved Yx D motif;The q RT-PCR analysis showed that transcript levels of OsFBN family genes were induced by high and low temperature stress to varying degrees.2.Functional idenfitication of OsFBN1 in PGs formation and heat stress tolerance in rice.Subcellular localization experiments in the tobacco system showed that CTP domains from both CTP domain only and OsFBN1 precursor protein can expedite the protein transport into chloroplasts;The in vitro lipid binding activity assays showed that mature protein of OsFBN1 was specifically bound to C18-and C20-fatty acids in vitro;Tillers number,panicle length,seed-setting rate and JA content in OsFBN1 overexpressing transgenic lines changed under heat stress compared with that in WT(ZH11);Both OsFBN1 overexpression and heat stress treatment induced the increase in the number and size of PGs in chloroplasts;OsFBN1 overexpression also affected the transcription levels of those genes involved in JA synthesis,isoprene lipid metabolism and photosynthesis in rice.These results showed that,OsFBN1 was involved in the response to heat stress and played important role in the development of PGs and lipid metabolism in chloroplasts in rice.3.Functional identification of OsFBN7 gene in the PGs formation and heat stress response in rice.OsFBN7 overexpression inhibited the growth of rice seedlings by decreasing the light energy utilization of photosystem II(PSII)in chloroplasts;OsFBN7 overexpression promoted the accumulation of PGs assembly into clusters in chloroplasts at 5-leaf stage and flowering stage;Yeast two-hybrid(Y2H)screening,Bi FC and Co-IP assays indicated that,OsFBN7 specifically interacted with ?-ketoacyl ACP synthetase I in rice(Os KAS Ia and Os KAS Ib);OsFBN7 overexpression promoted the enzymatic activity of KAS I,decreased the enzymatic activity of KAS II,and had no effect on the enzymatic activity of KAS III in flag leaves at flowering stage;Lipid metabolomics detections of chloroplasts suggested that OsFBN7 overexpression increased the contents and accumulations of diglycerol(DAG)and glycolipids(MGDG and DGDG)in chloroplasts.significantly.Therefore,functional model of OsFBN7 to regulate the formation of PGs in chloroplasts were proposed: OsFBN7 promoted the biosynthesis of fatty acids by interacting with Os KAS Ia/ Ib,a key enzyme in the de novo fatty acid synthesis pathway in chloroplasts,to significantly increase the contents and accmulations of DAG,MGDG and DGDG,as well as the formation of PGs in rice.In summary,in this study,evolutionary and structure and characteristics of FBN familys from ten representative plant species including green algae to higher plants were systematically analyzed,and the transcriptional levels of FBN family genes in rice in response to extreme temperature stress were examined;OsFBN1 and OsFBN7 were further selected for their function identifiecations in the ormations of PGs and heat stress resistance;OsFBN1 was identified as being involved in the heat stress response in rice and playing important roles in the PGs development and lipid metabolism in chloroplasts;In addition,OsFBN7 overexpression promoted the accumulation of PGs and the fatty acid synthesis by interacting with proteins Os KAS Ia/Ib in chloroplasts.All these new findings will provide a novel clue to further study of molecular mechanism of FBN family genes in lipids synthesis in chloroplasts and heat stress tolerance in rice.