Fructokinase-like Protein 1 And 2 Interact With Thioredoxin Z To Regulate Chloroplast Development In Rice

Chloroplast genes are transcribed by two RNA polymerases that are similar to the prokaryotic cells,the plastid-encoded RNA polymerase（PEP）and the nucleus-encoded RNA polymerase（NEP）.PEP transcribes genes that are involved in photosynthesis,whereas NEP transcribes housekeeping genes.Recently,we isolated and characterized the rice heat-stress sensitive albino mutant hsa1,Map-based cloning revealed that HSA1 encodes a fructokinase-like protein FLN2,which is essential for PEP activity and the regulation of gene expression of PEP.However,how do fructosekinase-like proteins regulate the molecular mechanism of Chloroplast biosynthesis in rice remains unknown.After homologous alignment,we found that HSA1 has a highly homologous protein FLN1 in rice.1.In order to further clarify the role of FLN2 and FLN1 in regulating chloroplast development and related regulatory network,we constructed the whole genome yeast library of rice by using rice leaf cDNA.OsHSA1BD and OsFLN1BD were constructed as bait vectors and screened the whole genome yeast library of rice.The yeast two-hybrid screen identified 5 unique OsFLN1 interacting proteins and21 unique HSA1 interacting proteins,and OsFLN1 and HSA1 both have interaction with LOC_Os08g29110 for many times.2.We verified the specificity of the interaction between OsFLN1 or HSA1/Os FLN2 and their interaction proteins by constructing the target genes-AD vectors and retransforming the plasmids into a yeast reporter strain.The result revealed that there are 3 proteins that are interact with OsFLN1 and 9proteins that are interact with HSA1.OsFLN1 and HSA1 both have strong interaction with LOC_Os08g29110.3.We also verified the specificity of the interaction between OsFLN1 or HSA1 and LOC_Os08g29110 by doing Pull-down,split firefly luciferase complementation（SFLC）assay and bimolecular fluorescence complementation experiments（BIFC）.All these results also supported OsFLN1 and HSA1 both have strong interaction with LOC_Os08g29110 in vitro and vivo.4.Gene annotation and protein sequence analysis showed that LOC_Os08g29110 encode a rice thioredoxin protein OsTRXz.In order to explore the function of OsTRXz,we first of all constructed OsTRXz-GFP fusion protein.The subcellular localization result showed that the complete OsTRXz protein is located in chloroplast.N terminal of OsTRXz has a 56aa that can located protein into chloroplast.,but the defeicent protein with deleted the N terminal signal peptideΔCTP-TRXz can not be transported to the chloroplast.5.Besides the results in molecular level,we wanted to have more evidences on the genetic level and at the same time analyze the biological functions of the interaction genes.So we made OsTRXz loss-of-function mutations by using CRISPR/Cas9.We obtained 41 independent transgenic mutant lines,including 8 independent T₀ transgenic lines harbored homozygous mutations in OsTRXz and 33independent transgenic lines harbored heterozygous mutations in OsTRXz.And all homozygous mutants exhibited a severe albino phenotype in the seedlings and seedling lethality.33 heterozygous mutations showed normal green seedlings in T₀ lines.T₁ plants derived from heterozygous mutant transgenic T₀lines showed a WT-to-mutant phenotypic segregation ratio of 3:1,and the mutant plants also showed albino seedlings and lethality.These results indicate that relative to the wild type,the OsTRXz mutation is a recessive mutation,and the loss of function of OsTRXz will cause the inhibition of the biosynthesis of the rice chloroplasts.6.In agreement with the severe albino phenotypes of trxz mutants,low amounts of Chl a,b and carotenoid（Car）were detected in each line.Within the few chloroplasts contained in trxz mesophyll cells,thylakoids displayed an abnormal structure with fewer lamellae,thus indicating that chloroplast morphology was affected in trxz plants7.Quantitative RT-PCR and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in trxz mutants.The above results show that OsTRXz,OsFLN1 and Os HSA1 are involved in the formation of PEP complex,which is essential in the process of chloroplast gene transcription and chloroplast synthesis in rice.On the basis of the results of this paper,we can find the interantion network through the verification and rescreening of other interaction proteins,which will lay the foundation for further clarifying the molecular regulatory network of the rice chloroplasts development.