Cucumber NF-YC2/9 Directly Affect CsTIC21 Regulation Chloroplasts Photomorphogenesis

Cucumber(Cucumis sativus L.)have high economic and nutritional value,is an important vegetable crop cultivated worldwide,and is one of the most consumed melon crops worldwide.Cucumber with large and thin leaves is an important plant for studying chloroplast development and photosynthesis.Chloroplasts are photosynthetic organelles of plants,which can convert light energy into chemical energy and store it in the organic matter produced to maintain plant growth.Chloroplast structure morphogenesis is a prerequisite for plant photosynthesis and affects the normal growth and development of plants.Light signals can control gene transcription,chlorophyll synthesis and protein degradation in chloroplasts,thereby further influencing chloroplast development.TOC-TIC(Translocon at the Outer Chloroplast envelope-Translocon at the Inner Chloroplast envelope)system is able to transport nuclear coding proteins synthesized in the cytoplasm to the chloroplast,which is the key to ensure the morphological development of the chloroplast.In this study,through functional verification of cucumber leaf color mutant candidate gene CsTIC21,combined with upstream regulatory transcription factors,light signal regulation of cucumber chloroplast morphological and structural development key components were studied.The main results are as follows:1.The mutation of cucumber TIC21 gene is the cause of albino phenotype of cucumber albino seedling(as)mutant.The phenotype of cucumber leaf color mutant as was observed and found that the cotyledon showed obvious yellow phenotype in seedling stage and was lethal in the late growth stage.Mutants exhibit longer hypocotyls and shorter roots with fewer lateral roots compared to controls.Previous studies had shown that as may be caused by mutation in the cucumber TIC21 gene(CsTIC21).Analysis of the conserved domain of TIC21 in different species revealed that the mutation site of Cstic21 was located in the fourth transmembrane domain of the coding protein,resulting in the defective gene coding protein.Analysis of CsTIC21 transcription levels in controls and mutants showed that CsTIC21 expression was detected in different periods and organs,with the highest expression level in true leaves.CsTIC21 was functionally validated using virus-mediated gene silencing(VIGS)and CRISPR/Cas9-mediated gene editing systems,and the phenotypes of TRSV-CsTIC21 silenced plants and knockout lines were consistent with the mutants.The chlorophyll content of the knockout lines were significantly reduced,the chloroplast structure was deformed,and the normal plastid morphology could hardly be observed.2.Cucumber NF-YC2/9 are involved in chloroplasts photomorphogenesis induced by CsTIC21.CsTIC21 showed lower transcription levels in yellowed seedlings grown under dark conditions,and CsTIC21 expression was up-regulated after light restoration.At the same time,Cstic21 mutations showed a phenotype similar to yellowed seedlings,suggesting that light signals may initiate chloroplast morphogenesis in cucumber seedlings by inducing CsTIC21 expression.Upstream transcription factor screening tests were conducted using the predicted photoresponsive elements in the CsTIC21 gene promoter,and NF-YC family members were found to be involved in transcriptional regulation of the CsTIC21 gene.The genome databases of Arabidopsis thaliana and cucumber were searched and compared to identify 7 members of the cucumber NF-YC gene family,and the cucumber NF-YC gene was renamed according to the homologous NF-YC gene names of Arabidopsis thaliana.VIGS technique was used to silence all 7 NF-YC family genes in cucumber,and the chlorophyll content and photosynthetic capacity of the silenced plants were determined.Leaf yellows were observed in plants silenced by TRSV-CsNF-YC2,TRSV-CsNF-YC9,TRSV-CsNF-YC11-1 and TRSV-CsNF-YC11-2.The transcription of CsNF-YC2 and CsNF-YC9 can be positively regulated by the light signal.3.CsNF-YC2 and CsNF-YC9 directly bind to the promoter elements of CsTIC21.We found that CsTIC21 expression was significantly down-regulated in TRSV-CsNF-YC2/-YC9 plants.Dual luciferase reporter system(DLR)analysis showed that ectopic expression of CsNF-YC2 and CsNF-YC9 significantly increased CsTIC21 promoter driven luciferase gene activity.Yeast single hybridization(Y1H),chromatin immunocoprecipitation-q PCR(Ch IP-q PCR)assay,and gel electrophoresis migration assay(EMSA)demonstrated that CsNF-YC2 and CsNF-YC9 specifically bind to the CCAAT-box element on the CsTIC21 promoter.These results indicated that CsNF-YC2 and CsNF-YC9 regulate chloroplast development in cucumber by positively regulating the expression of CsTIC21.4.CsNF-YC2 and CsNF-YC9 synergistically promote the transcriptional activity of CsTIC21.We demonstrated protein-level interactions between CsNF-YC2 and CsNF-YC9 in vitro and in vivo by yeast two-hybrid assay(Y2H),luciferase complementary imaging assay(Bi FC)and Pull down.At the same time,the dual luciferase reporting system showed that co-injection of CsNF-YC2/-YC9/pro CsTIC21 significantly increased the activity of pro CsTIC21 compared with CsNF-YC2/pro CsTIC21 and CsNF-YC9/pro CsTIC21 alone.These evidences further confirm that cucumber NF-YC is involved in photosignal-mediated regulation of chloroplast TIC21 protein transport channels.In summary,we used VIGS gene silencing and CRISPR/Cas9-mediated knockout tests to verify the correlation between TIC21 gene function and albino phenotype in cucumber.Two transcription factors NF-YC2 and NF-YC9 related to chloroplast development in cucumber were identified,revealing the functions of the homologous genes of NF-YC2 and NF-YC9 in cucumber.CsNF-YC2 and CsNF-YC9 were found to play a role in the light-mediated TIC pathway by directly inducing CsTIC21 expression.Our results suggest that NF-YC2/9-TIC21 modulates light-induced chloroplast morphogenesis in cucumber,providing a new way to study the mechanism of light-regulated chloroplast development in plants.