Fruit Ripening Molecular Mechanism Of‘liuyuezaoyou' Pummelo

‘Guanximiyou'(Citrus maxima‘Guanximiyou')is the largest pummelo variety cultivated in China.The yield of‘Guanximiyou'in Fujian alone accounts for about 2/3 of the total yield of pummelo in China and around 1/3 in the world.However,the mature period of‘Guanximiyou'ranges from October to December every year,which is very concentrated,resulting in a long shelf-life vacancy.Therefore,the cultivation of new pummel varieties with different mature periods is very important for the market demand.Recently,a‘Guanximiyou'early-ripening bud mutant was found in Pinghe county,Fujian Province.After many years of field observation,the character of‘Guanximiyou'mutant was found to be very stable.Given that the fruit of the mutant ripens in the lunar June,it was named as‘Liuyuezao'.At present,the fruit ripening mechanism of citrus is still unclear.Thus,it is important to study the mechanism of fruit ripening.In this study,by using‘Guanximiyou'(WT)and its early-maturing variety‘Liuyuezao'(MT)as materials,the difference of the two materials during fruit ripening was studied and compared by means of multi-omics(including whole genome resequencing,transcriptome,whole genome DNA methylation,etc.).The main findings are as follows:1.Development of SSR markers for WT and MT.SSR molecular markers for the early screening of WT and MT were screened and identified based on high-throughput sequencing data.Transcriptome sequencing and whole-genome resequencing were performed for the assembly of transcriptome and plastid genome and for the identification of SSRs.A total of 41 polymorphic SSRs were screened out and corresponding primers were designed,among which 4 pairs of SSR primers were verified to be very effective for the early screening of the early ripening mutants through polyacrylamide gel detection.2.Analysis of genetic difference between WT and MT.The WT and MT whole genome resequencing data were aligned to the C.maxima reference genome.Through the whole genome variation analysis,a large number of SNPs and In Dels were found.Variant genes with SNPs or In Dels were subjected to GO,COG and KEGG enrichment analysis.We identified 48 variant genes categorized to the bscisic acid(ABA),auxin(IAA)and ethylene(C₂H₄)metabolism and signaling pathway,indicating that the endogenous hormones may be closely related to the early ripening mutation of pummelo.3.Endogenous hormone contents and fruit quality comparisons of WT and MT fruits during ripening.By using the WT fruits harvested from the young fruit to the commercial maturity period(S1–S9)and the MT harvested from the young fruit to the commercial mature period(S1–S6)and an extra fully mature period(S7)as materials,contents of endogenous hormones,soluble sugars,organic acids and peel thickness were measured and compared.Results showed that the ABA varied greatly between the two varieties.MT fruits showed a single peak pattern of ABA accumulation with the peak at the S4 stage,which was significantly higher than that of WT at all stages.Meanwhile,the peel thickness of MT is smaller than WT since the S4 period,indicating that ABA may be the key factor that contribute to the early maturity of honey pomelo,and the S4 period is the watershed point of the fruit ripening for the two varieties.In addition,the citrate metabolism of MT also varied greatly,which is different from the accumulation patterns of WT and traditional citrus.The citrate content in WT and traditional citrus fruits usually showed first rises then falls pattern during the ripening process.However,the citrate content in MT fruits gradually accumulates as ripening,which might contribute to the short ripening time of MT fruit.4.Transcriptome differences between WT and MT during fruit ripening.For the deeper understanding of the dynamic differences between WT and MT at the transcriptional level,RNA-seq technology was applied to compare the pulp transcriptome differences of the two varieties at S1–S6(from young fruit to commercial maturity stage).Totally,we identified 2099 DEGs at all the six stages,and there are 212,407,259,893,887,and 503 DEGs at the 6 stages,respectively.At S4 period,the number of DEGs between WT and MT peaked,which was consistent with physiological data.The KEGG pathway enrichment analysis revealed that DEGs belonging to 33 different pathways enriched,of which a large number of DEGs were categorized to ABA,IAA,BR and hormone signal transduction pathways.Moreover,many starch and sucrose,glycolysis,phenylpropane and other pathways related DEGs were also identified.The enrichment of these DEGs indicates that the phytohormone metabolism and metabolic processes of the two varieties differed significantly,and the ripening speed of MT at S4–S6 periods was faster than WT.5.Expression patterns of DEGs and screening of candidate key genes.Combined transcriptome and physiological analysis revealed that the expression of a large number of DEGs were highly consistent with ABA,IAA,sugar and acid metabolism,and cell softening pathways.We found that the ABA content change pattern difference between WT and MT might be mainly regulated by the degradation pathway gene CgCYP707A4,whose expression is low in MT and high in WT.And it was found that the expression pattern of citrate synthase gene CgCS1 was highly consistent with the citric acid content changes,indicating that it may be the key factor that lead to the citric acid metabolism difference between MT and WT.Moreover,among the 18 fruit softening related DEGs,11 DEGs(4 PGs,3 XTHs,1 PME,3 PLs)that play roles in promoting fruit softening showed higher transcription levels in MT than WT,and 2 fruit softening suppression related DEGs(Cg PMEi2,Cg PMEi3)showed lower expression in MT than in WT.It can explain well why the MT pulp ripens faster than WT pulp.6.Functional verification of CgCS1 gene.In order to verify the function of CgCS1,the gene sequence was successfully cloned from MT,and the recombinant plasmid p GWB502-CgCS1 was constructed and transformed into Shatian pummelo.Two transgenic plants(OE-176 and OE-141),whose CgCS1expression level was respectively 15 and 3 times higher than the nontransgenic controls(CK),were obtained.It was found that the leaf organic acid contents of OE-176 and OE-141 were both higher than the control,and OE-176 showed the highest level,indicating that CgCS1 can regulate the synthesis of citric acid and may be a key gene that affects the change of MT citric acid metabolism.In the future study,the citric acid contents in the transgenic fruits should be determined to better clarify the gene's function.7.Functional verification of CgCYP707A4 gene.A strawberry transient expression system was established,and CgCYP707A4 gene was overexpressed in strawberry.It was found that the transient overexpression of CgCYP707A4 reduced the ABA content and delayed the color change of strawberry fruit,indicating that CgCYP707A4 negatively regulates fruit ripening.VIGS was further applied to silence the homologous gene of CgCYP707A4 in pepper(Ca CYP707A1).It was found that both Ca NCED1 and Ca NCED3 were down-regulated,indicating that after the silence of Ca CYP707A,ABA degradation slowed down and more ABA was accumulated.Therefore,the ABA synthesis genes Ca NCED1 and Ca NCED3 were down-regulated to maintain ABA balance.Moreover,among the ABA receptor genes,9 Ca PYLs were down-regulated except Ca PYL7(extremely significantly up-regulated),indicating that Ca PYL7is the main ABA signals receptor gene in response to the accumulation of ABA.8.DNA hypomethylation promotes fruit ripening of pummelo.DNA methylation is considered to be another important factor that regulates fruit ripening after endogenous hormones and transcription factors.Whole-genome DNA methylation sequencing and analysis were carried out using the MT and WT fruits at S3–S6 periods.It was found that the number of the three types of hypo-DMR(CG,CHG and CHH)is lower than that of hyper-DMR,and this change is determined by CHH.We further used methylation inhibitor(5-Azacytidine)to treat the young fruit of pummelo fruit.It was found that after 5-Azacytidine treatment,it only takes 4 days for the peel to mature and change color.Therefore,it was concluded that DNA hypomethylation promotes the ripening of pummelo fruits.Physiological data analysis revealed that the color change of the peel was largely caused by the degradation of chlorophyll and partially caused by the degradation of carotenoids.In order to further explore the molecular regulation mechanism of DNA demethylation on peel maturation,a transcriptome analysis was performed.It was found that the decrease of chlorophyll was caused by the down-regulation of expression of chlorophyll synthesis pathway genes(POR,CAO)and degradation pathway genes(The up-regulated expression of CLHs and RCCR work together to promote the degradation of chlorophyll a and chlorophyll b).Simultaneously,the expression of carotenoid metabolism genes(ZDS,Ctr Z,etc.)is down-regulated,which promotes the decrease of the corresponding carotenoid content,and in turn leads to the carotene content decreases.