Epigenetic Mechanism Of SlFolB Regulating Chloroplast Development In Tomato

The chloroplast is the main site of photosynthesis in plants and plays a key role in the yield and quality of crops.In our early work,we cloned the SlFolB gene in tomato and found that the leaves of the SlFolB gene edited lines showed significant yellowing and abnormal chloroplast development.Based on this study,the molecular mechanism of SlFolB in regulating chloroplast development in tomato was further investigated in depth.Phenotypic identification,spatio-temporal specific expression analysis,folate content determination,primary metabolism analysis,and combined analysis using transcriptome data and genome DNA methylation data were performed by silent and overexpression lines of SlFolB gene.The molecular mechanism of SlFolB regulates tomato chloroplast development through epigenetics,which provides an important candidate gene and theoretical basis for breeding high-yield and high-quality tomato varieties and crop genetic improvement.The main results are as follows:1.Sequence analysis showed that the length of SlFolB gene was 2622 bp,containing two exons,and the length of CDS sequence was 393 bp,encoding 130 amino acids,including a FOl B conserved domain,encoding dihydroneopterin aldolase.2.GUS staining and qRT-PCR results showed that SlFolB was expressed in root,stem,leaf,flower,and fruit in tomato,indicating that SlFolB plays a crucial role in the whole development of tomato.The subcellular localization results showed that SlFolB was localized in chloroplasts and nucleus,indicating that SlFolB may be involved in the interactions between the nuclear and chloroplast genomes and in chloroplast development.3.The chloroplast development phenotype was identified.Compared to wild-type tomato Alisa Craig,leaves of the RNAi lines showed significant yellowing and leaves of the overexpression lines showed no significant changes.Transmission electron microscopy results showed abnormal chloroplast development,a lower number of chloroplasts,and an increased number of plastid globules in the fenestrated tissue chloroplasts of leaves of the RNAi lines compared with wild-type tomato Alisa Craig,indicating that a serious defect in chloroplast development had arisen.In addition,chlorophyll content and chlorophyll fluorescence measurements showed that,compared with wild-type tomato Alisa Craig,the leaf chlorophyll content of the RNAi lines was significantly lower and photosynthetic efficiency was significantly reduced,while the starch grains in the chloroplasts of the overexpression lines were significantly larger.The above results indicate that SlFolB is essential for the normal development of tomato chloroplasts.4.The results of folate content determination(LC-MS)showed that folate synthesis was disrupted in the leaves of the RNAi lines compared to wild-type tomato Alisa Craig,where THF of tetrahydrofolate was significantly reduced and 5-methyltetrahydrofolate was not detected;In addition,dihydrofolate DHF,tetrahydrofolate THF and 5-formyltetrahydrofolate 5-CHO-THF were significantly lower in fruits of the RNAi lines compared to wild-type tomato Alisa Craig.In addition,5-formyltetrahydrofolate and SlFolB were involved in regulating folate synthesis,and folate 5-CHO-THF was significantly higher in fruits of wild-type AC and overexpression OE lines than wild-type AC.The above results indicate that SlFolB is involved in the synthesis and accumulation of folic acid in tomato leaves and fruits.5.The results of yield determination showed that overexpression of SlFolB significantly increased plant fruit yield and RNAi interference with SlFolB significantly decreased fruit yield compared to wild-type tomato Alisa Craig.The average yield per plant of the RNAi lines(0.48 kg)was significantly decreased by 59.4% and the average yield per plant of the overexpressing SlFolB plant(1.52 kg)was significantly increased by 27.5% compared to the average yield per plant of the wild-type tomato Alisa Craig(1.19 kg).6 Fruit primary metabolism(GC-MS)results showed that major soluble sugars were significantly reduced in leaves and fruits of the RNAi lines compared to wild-type tomato Alisa Craig,while no significant changes were observed in the overexpression line.7.KEGG enrichment analysis of transcriptome data identified a number of differentially expressed genes involved in the regulation of photosynthesis,photosynthesis associated nuclear genes,and porphyrin chlorophyll metabolism-related genes.Most differentially expressed genes were significantly reduced in the RNAi lines compared to the wild-type tomato Alisa Craig.The qRT-PCR results verified that the transcript levels of photosynthesis associated nuclear genes(LHCB1.1,LHCB1.2,LHCB2.4)and porphyrin chlorophyll metabolism-related genes(PORA)were significantly decreased,which was consistent with the results of transcriptome data.8.Genome-wide DNA methylation results showed that the RNAi5 lines had significantly lower DNA methylation levels compared to wild-type tomato Alisa Craig.Among them,DNA methylation levels were significantly increased in the promoter regions of photosynthesis associated nuclear genes(LHCB1.1,LHCB1.2,LHCB2.4)genes and in the coding regions of PORA genes related to porphyrin chlorophyll metabolism.The above results indicated that SlFolB finely regulated photosynthesis associated nuclear genes through epigenetics.SlFolB is involved in regulating tomato chloroplast development.