Acetylation Reprogramming Of Fruit Quality Formation And Transcriptional Regulation Of Flesh Color Variation In Citrus Allotetraploid Somatic Hybrid Nh

Polyploidization is a major force in evolution and provides diverse genetic resources for future breeding.Polyploids are generally classified into two categories: autopolyploids,which involve the duplication of the same genome;and allopolyploids,which merge two diverged genomes into a common nucleus.Allopolyploidization,encompassing genome doubling and hybridization,endows allopolyploids with significant evolutionary advantages,such as robust growth vigor,increased biomass and enhanced environmental adaptability.Metabolites are crucial for plant growth,development and environmental responses and also serve as essential nutrients for humans.However,the impact of allopolyploidization on plant metabolism and its underlying molecular mechanisms remain elusive.Citrus allopolyploids can be employed for breeding disease-resistant and dwarfing rootstocks and they can also be used as parents in interploid crosses with diploid females to generate seedless triploids.Unraveling the metabolic reconstruction mechanisms of citrus allopolyploids holds great significance for enhancing the efficiency of cell engineering breeding,promoting genetic improvement in citrus,and enriching somatic cell interaction theories in higher plants.A citrus allotetraploid somatic hybrid named NH was generated through protoplast fusion between a diploid tangelo named ‘Nova’(Citrus clementina hort.× [C.paradisi Macfad.× C.reticulata Blanco])and a diploid pummelo named ‘HBP’(C.grandis Osbeck).To elucidate the molecular mechanisms underlying the formation of allopolyploid fruit metabolism,we analyzed the main fruit qualities and performed acetylome,transcriptome and molecular biology techniques on fruits from the allotetraploid NH and its diploid parents.1.Acetylome reprograming participates in the establishment of fruit metabolism during polyploidization in citrus.The allotetraploid NH was found to harbor nuclear genomes from both ‘Nova’ and ‘HBP’ and inherited its chloroplast and mitochondrial genomes from ‘HBP’.The fruit morphology of the allotetraploid is distinct from its parents.For instance,the levels of major organic acids were transgressive in NH allotetraploid and the flavonoid content in its fruit was close to the ‘Nova’ parent but significantly higher than the ‘HBP’ parent.However,the differences in gene and protein expression could not explain the accumulation pattern of organic acid and flavonol content in the NH allopolyploid fruit.Thus,we performed a quantitative acetylome analysis using TMT-(Tandem Mass Tag)labeling to decipher the metabolic characteristics of the allopolyploid NH.In total,4,115 peptides containing acetylated lysine residues were identified,corresponding to 4,175 specific Kac sites in 1,640 protein groups.Proteins contributing to primary metabolism,such as carbohydrate,energy,and amino acid metabolisms,were overrepresented among these acetylated proteins in citrus fruit.Moreover,more Kac sites and acetylated proteins in citrus fruit were involved in secondary metabolism than those reported in the acetylomes from the leaves of two model plants,Arabidopsis(Arabidopsis thaliana)and rice(Oryza sativa).Importantly,many of these acetylated proteins were associated with citrus fruit quality traits.In the allotetraploid,parental dominance(i.e.resemblance to one of the two parents)in specific fruit traits,such as fruit acidity and flavonol metabolism,was highly associated with parental Kac level dominance in pertinent enzymes due to Kac-mediated regulation of enzyme activity.Furthermore,protein Kac likely contributes to the discordance between transcriptomic and proteomic variations during allotetraploidization.Acetylome reprogramming can be partially explained by the expression pattern of several lysine deacetylases(KDACs).Overexpression of silent information regulator 2(Cg SRT2)and histone deacetylase 8(Cg HDA8)diverted metabolic flux from primary to secondary metabolism and partially restored a metabolic status to the allotetraploid,which expressed attenuated levels of Cg SRT2 and Cg HDA8.Additionally,KDAC inhibitor treatment greatly altered metabolism in citrus fruit.2.Transcriptional regulation in carotenoid formation of citrus allotetraploid fruit.We observed that the flesh color of ‘HBP’ and ‘Nova’ is pink and orange,respectively,while that of the allotetraploid NH is light yellow.Hence,the composition and content of carotenoids in the fruit of the allotetraploid NH and its two parents at different developmental stages were detected by HPLC.We found that the allotetraploid NH produced the same compounds as ‘Nova’ but at much lower levels than both parents.Transgressive concentration of abscisic acid(ABA)was observed in the allotetraploid,contrary to the trend of carotenoid content.RNA-seq analysis was used to investigate the differences in transcriptomes of the fruit from the allotetraploid NH and its parents at different developmental stages.We suggested that the decrease in carotenoid content in the fruit of allotetraploid NH may be attributed to the up-regulation of Cs NCED2,a key enzyme in carotenoid catalyzation and ABA biosynthesis.Sequencing analysis revealed that the CDS of Cs NCED2 in the allotetraploid NH was inherited from both parents,and only four amino acid differences were found in the protein sequence of Cs NCED2 between the parents.Mfuzz clustering analysis demonstrated that the expression pattern of Cs DRP3 was consistent with that of Cs NCED2.Cs DRP3 is a nuclear-cytoplasm co-localized DREB family gene,which directly binds to the promoter of Cs NCED2 and activates its expression.Overexpression of Cs DRP3 in citrus calli and tomato fruit significantly reduced carotenoid content and promoted ABA accumulation.These results suggest that Cs DRP3 negatively regulates carotenoid accumulation by promoting ABA synthesis.In summary,this study uncovered the acetylome reprogramming and transcriptional regulation in trait evolution during polyploidization.Furthermore,the study elucidated the roles of the “Cs DRP3-Cs NCED2” module in regulating carotenoid and ABA metabolism in fruit,providing a theoretical foundation and genetic resources for citrus fruit germplasm innovation and genetic improvement.