Research On The Regulation Mechanism Of Proanthocyanidin Accumulation And ABA Metabolism In Citrus

Color is one of the important evaluation indexes of citrus fruit quality and directly determines the economic benefits.As the main pigments in citrus,carotenoids,anthocyanins and proanthocyanidins(PAs),are important target traits for citrus breeding.The plant hormone abscisic acid(ABA),as a derivative of oxidative cleavage of carotenoids,is closely related to the accumulation of carotenoid in citrus fruits.In addition,ABA also plays an important role in regulating citrus fruit ripening and senescence.The current research on citrus color is mainly focused on the biosynthesis and transcriptional regulation of carotenoids and anthocyanins.However,the accumulation of citrus PAs and the relationship between ABA and carotenoid accumulation are still very limited.Abundant citrus germplasm resources provide ideal materials for the study of citrus color,especially for the study of the relationship between these two pigments.Hong Anliu sweet orange(Citrus sinensis Osbeck cv.Hong Anliu)is a color mutant,which shows a phenotype of lack of PAs in the endothelium,a large accumulation of lycopene in the flesh,and obvious decrease in ABA content.In this study,the regulatory mechanism underlying PA accumulation in citrus was systematically analyzed using the material of Hong Anliu sweet orange.Moreover,using Hong Anliu sweet orange,this study identified the regulatory factor that regulates citrus ABA metabolism,and initially established a molecular regulatory network that ABA affects carotenoid accumulation and promotes fruit ripening and senescence.The main results were as follows:1.Regulatory mechanism of citrus PA accumulation.The accumulation of PAs in the seeds of sweet orange varieties Anliu,Hong Anliu and Succari was significantly different.Quantitative determination showed that the PA accumulation in the endothelium of Hong Anliu and Succari were absent,while the accumulation of PA in the endothelium of Anliu was normal.Through transcriptome analysis of their seeds and pulps,transcription factors(TFs)CsPH4 and Noemi,which potentially regulated the accumulation of citrus PAs,were obtained.Sequence analysis showed that CsPH4 and Noemi belonged to R2R3-MYB and b HLH proteins,respectively.Subcellular localization and transcriptional activity analysis suggested that CsPH4 and Noemi were nucleus-localized transcriptional activators.The CsPH4-overexpressing and Noemi-overexpressing citrus calli were successfully obtained.Phenotypic analysis showed that the PA content of CsPH4-overexpressing or Noemi-overexpressing citrus calli were significantly increased.qRT-PCR analysis indicated that PA biosynthetic genes were significantly upregulated in CsPH4-overexpressing or Noemi-overexpressing citrus calli,and Noemi was also significantly upregulated in CsPH4-overexpressing citrus calli.Overexpression of CsPH4 in Arabidopsis(Arabidopsis thaliana)showed that CsPH4 promoted PA accumulation in the wild-type and partially complemented the PA-deficient phenotype in tt2 mutant.The results of yeast two-hybrid analysis,bimolecular fluorescence complementation assay,and GST pull down revealed that CsPH4 interacted with Noemi to form a CsPH4-Noemi complex.Dual luciferase assays and tobacco transient expression assays indicated that the CsPH4-Noemi complex activated the expression of PA biosynthetic genes(DFR,ANS,ANR and LAR).Simultaneously,the CsPH4-Noemi complex also activated the expression of Noemi.Furthermore,electrophoretic mobility shift assays demonstrated that CsPH4 directly bound to MRE site on DFR,ANR,LAR and Noemi promoters.These findings revealed the molecular mechanism underlying the CsPH4-Noemi complex regulating the accumulation of citrus PAs via a positive feedback loop,which can be effectively employed for metabolic engineering to improve citrus fruit quality.2.Regulatory mechanism of citrus fruit ABA metabolism and senescence.In this study,the candidate TF CsHB5,which was potentially involved in regulating the metabolism of citrus carotenoids,was obtained through transcriptome data.Sequence analysis showed that CsHB5 belonged to the HD-ZIP I family.Subcellular localization and transcriptional activity analysis indicated that CsHB5 acted as a nucleus-localized transcriptional activator.qRT-PCR analysis indicated that the expression of CsHB5 was drastically induced by water loss,but repressed by low temperature.The CsHB5-overexpressing citrus calli were successfully obtained.Phenotypic analysis showed that the ABA and reactive oxygen species(ROS)content of CsHB5-overexpressing citrus calli were significantly increased.qRT-PCR analysis indicated that ABA metabolism-related genes(BCH1,NCED2,CYP707A1 and AAO3)were significantly upregulated in CsHB5-overexpressing citrus calli.Silencing the expression of CsHB5 in citrus calli downregulated the expression of ABA metabolism-related genes(BCH1,NCED2,CYP707A1 and AAO3),but had no significant effect on the content of ABA and ROS.Heterogenous overexpression of CsHB5 in tomato(Solanum lycopersicum)and Arabidopsis significantly promoted leaf yellowing and senescence.Meanwhile,the levels of ABA in CsHB5-overexpressing tomato were significantly increased,while lycopene content were decreased.Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes,including chlorophyll degradation,nutrient compound biosynthesis and transport,as well as ABA and ROS signaling transduction.The results of yeast one-hybrid assays,electrophoretic mobility shift assays,and dual luciferase assays indicated that CsHB5 directly bound to the promoters of BCH1 and NCED2 and activated their transcriptions.These findings revealed that CsHB5 participated in citrus senescence by directly controlling ABA accumulation.This work deepens our understanding of the complex regulatory network of fruit senescence and provides an insight into the regulatory mechanism underlying ABA-mediated senescence of non-climacteric fruits.