Mining And Functional Analysis Of NAC Transcription Factors Related To Juice Sacs Granulation Of ’Sanhongmiyou’

’Sanhongmiyou’ [Citrus grandis Osbeck] is a citrus plant in the rue family,which is a new variety bred from the bud of ‘Guanximiyou’.It is named for its red pericarp,red pulp,and red sarcocarp.Its flavor is sweet and sour and has high nutritional value.Juice sacs granulation is prone to occur during the growth and development and post-harvest storage of the ’San hong’ fruit,which greatly affects its edibility and commercial value.A large number of existing studies have shown that as the degree of juice sacs granulation increases,the content of lignin also increases.The production of lignin is one of the main factors of grapefruit juice cell granulation,but the specific molecular mechanism of its occurrence so far still uncertain.This study intends to study the mechanism of juice granulation from the perspective of transcription factors related to lignin metabolism.Using the juice sacs of the ’San hong’ fruit,combined with the dynamic changes of lignin content during growth and development and the established transcriptome database,the key transcription factors involved in the synthesis of lignin were identified and screened.The research on the regulation of cytokine transcription lays the foundation and provides an important way to study the mechanism of juice sac granulation.The main findings are as follows:1.The lignin content of pomelo juice sac increased 157-212 d after flowering,and the lignified cell wall became thicker.With the increase of the number of days after anthesis,the fruit weight and transverse and longitudinal diameters of the ’San hong’ fruit gradually increase;the juice sac lignin content also shows an overall upward trend,the lignin content of mesial column at 212 d reached 14.84%;the fruit juice sac was stained with Weisner reagent.It was observed that the cell wall was stained light red in the early stage of granulation,and then the staining degree gradually deepened and gradually stained to dark red in the late stage,indicating that the the lignified cell wall of ’San hong’ juice sacs thickened during the growth process.2.Screening of key NAC transcription factors for lignin synthesis in pummelo juice sac.The study conducted transcriptome sequencing by selecting the mesial column,distal column at 157 d,180 d,and 212 d.According to the transcription levels of all genes,3473 DEGs were screened,mainly enriched in phenylpropane and the biosynthesis of secondary metabolites.On the one hand,the NAC transcription factors related to lignin synthesis were screened out by analyzing the trend of different genes in different parts and combining KO and GO annotations.On the other hand,through the correlation analysis between WGCNA and lignin content,1287 related genes were obtained,and the key NAC transcription factors were screened out using DEGs in the lignin synthesis pathway as nodes.3.Identification of NAC gene family members and clone expression analysis.There are 105 NAC genes in the juice sac of the pummelo,and the genes are distributed on 9 chromosomes.The tandem duplication of the NAC gene in pummelo has a higher probability of occurrence,resulting in a larger number of members,and there is a highly conserved collinearity with Arabidopsis and Eriobotrya japonica fragment.The NAC genes cloned from the juice sac of the ’San hong’ fruit were named Cg NAC012,Cg NAC043,Cg NAC076,Cg NAC073-1,Cg NAC073-2,and were found to be nuclear localization proteins by subcellular analysis.Through q PCR analysis,it was found that the expression trends of these genes in the mesial column,distal column and granulated juice sac 157-212 d were basically similar,the expression level gradually increased from 157-212 d and reaches the maximum.The expression level of mesial column and granulated juice sac was higher than that of distal column.Further analysis of the juice sacs in the mature stage of’Huangjinmiyou’ showed that the expression level of Cg NAC076 in granulated juice sacs was not significantly different from that of ungranulated juice sacs,while Cg NAC012,Cg NAC043,Cg NAC073-1,Cg NAC073-2 were in the granulated juice sacs.The expression level in juice sacs was significantly higher than that in ungranulated juice sacs.4.Cg NAC043 is involved in the transcriptional regulation of lignin.Through clustering of transcription factors related to lignin biosynthesis that have been reported in other species,it was found that Cg NAC043 is highly conserved.Through the yeast two-hybrid system,it was found that Cg NAC043 has transcriptional activation activity in Y2 H yeast,and the activation activity is located at the C-terminus.Transient transformation of Cg NAC043 into the mesocarp of pummelo was found to promote the expression of downstream Cg MYB58.The promoter lengths of Cg MYB46,Cg C3 H,Cg CCo AOMT1,Cg4 CL,Cg CCR genes cloned from pummelo juice sacs were 1827,2095,1961,1726,1949 bp,respectively.The results of dual luciferase showed that Cg NAC043 could not only activate the promoter of Cg MYB46 with 16.1 fold;it also could directly activate the promoters of Cg C3 H and Cg CCo AOMT1 with 8.9 and 4.3 fold respectively.5.Cg NAC073-2 promotes the biosynthesis of lignin.The cloned Cg NAC073-2promoter is 1961 bp.Through dual luciferase analysis,found that Cg NAC012 and Cg NAC043 could activate the promoter of Cg NAC073-2 by 5.5 and 15.1 fold respectively.Through the yeast two-hybrid system,it was found that Cg NAC073-2has transcription activities in Y2 H yeast,located at the C-terminus.The p1301-Cg NAC073-2 plant expression vector was constructed and transfer to tobacco.It was found the plant of the over-expressing Cg NAC073-2 is generally higher than that of WT,the leaves are large,the root is more vigorous,and the stem is thicker.Through q PCR analysis,it was found that the gene expression of strain OE#2 was the most significant.Among them,the expression levels of Nt PAL,Nt C4 H,Nt CCR,and Nt COMT in leaves were significantly higher,which were 2.01,2.88,3.47,1.62 fold that of WT,respectively.The expression levels of Nt PAL,Nt C4 H,Nt CAD,Nt CCR,Nt COMT,Nt CCOAOMT,and Nt MYB86 in the stem were significantly higher,which were 1.78,2.20,1.80,3.25,5.35,1.92,16.70 fold that of WT,respectively.Weisner staining on the stem of strain OE#2,and it was found that the secondary wall was significantly thicker than that of WT.6.The content of lignin intermediate product in transgenic Cg NAC073-2 tobacco is obviously accumulated.LC-MS/MS was used to determine the content of lignin synthesis intermediates in roots,stems and leaves of OE and WT tobacco.The contents of coniferaldehyde and cinnamic acid in OE leaves were significantly higher than those of WT,which were 8.14 and 8.95 folds,respectively.The root content of4-coumaric acid,ferulic acid,coniferaldehyde and coumarin aldehyde in OE were significantly higher than that in WT,which were 2.5,6,3.95,and 2.77 folds,respectively.The content of erucic acid,coffee alcohol,and caffealdehyde in OE stems was significantly higher than that of WT,which were 2.84,6.37,41.51 folds,respectively.The above results indicate that with the increase in the degree of granulation of’San hong’ juice sacs at different developmental stages,Cg NAC043,Cg NAC073-2activate and promote the expression of downstream transcription factors and genes which participated in the lignin synthesis.It lays the foundation for the research of lignin synthesis and provides a theoretical basis for the molecular breeding of ’San hong’ in the future.