Structural And Functional Characterization Of The Geranylgeranyl Diphosphate Synthase (GGPPS) In Rice

The world population is expected to reach 8.5 billion by 2030.The increased demand for food supply has to be resolved by higher plant productivity,which in turn,mostly depends upon photosynthesis.Chlorophyll is the most essential light absorbing pigment for photosynthesis.The biosynthetic pathway of chlorophyll has been wellstudied and found to exclusively occur in plastids in higher plants.Chlorophyll molecule is composed by a hydrophilic tetrapyrrole chromophore for light harvesting and a hydrophobic phytyl side chain for the assembly of chlorophyll with the apoproteins of antenna and core complexes of the photosystems and for the formation of thylakoid lamellae.The tetrapyrrole biosynthesis pathway has been extensively studied,however,the supply of GGPP from the isoprenoid pathway for phytyl biosynthesis is largely neglected.Rice is one of the most important crops in the world,providing more than 20%of total calories consumed worldwide.In plants,the isoprenoid pathway produces geranylgeranyl diphosphate(GGPP)by GGPP synthase(GGPPS)in chloroplast as a common precursor which is shared by vital downstream metabolic branches,including the biosynthesis of chlorophylls,carotenoids,gibberellins,etc.However,the molecular mechanisms regulating the allocation of GGPP among these branches are largely unknown.In this study,we identified 12 candidate genes for GGPPS homologs from the rice genome,and found that OsGGPPS(Os07g39270)is the only GGPPS that is functionally active in rice chloroplast.Yeast two hybrid(Y2H)assay and bimolecular flourescent complementation(BiFC)assay showed that OsGGPPS forms homodimer in stroma and interacts with a small subunit protein(OsSSU,Os02g44780)in thylakoid membrane to form a heterodimer.Prenyltransferase assay indicated that heterodimeric OsSSU/OsGGPPS produces GGPP more specifically.By resolving crystal structures of both homodimeric and heterodimeric OsGGPPS,we found that binding of OsSSU does not change the structure of OsGGPPS.Through site-directed mutagenesis of residues on the dimer interfaces,we demonstrated that OsSSU has stronger affinity with OsGGPPS to form heterodimer.The stronger interaction between OsSSU and OsGGPPS probably contributes to the stabilization of the heterodimer thus exhibiting a higher enzymatic efficiency.Our further analysis revealed that this OsSSU/OsGGPPS heterodimer exists in a large protein complex,together with GGPP reductase(OsGGR),an enzyme that reduces GGPP for the phytyl side chain of chlorophyll,and an anchor protein OsLIL3,in the thylakoid membrane.The phenotypes of our transgenic rice seedlings suggest that OsSSU functions in recruiting OsGGPPS from stroma to the thylakoid membrane for supplying GGPP for chlorophyll biosynthesis.Based on the results of this study,we proposed the mechanism that regulates GGPP supply in rice chloroplast.In stroma,OsGGPPS forms homodimer and provides GGPP for gibberellins,etc.In the thylakoid membrane,OsGGPPS interacts with OsSSU to form heterodimer and associate with OsGGR/OsLIL3...