Color Variation Of Navel Orange Flavedo And Regulatory Analysis Of Lycopene ?-cyclase

Chlorophyll degradation and carotenoid accumulation are indispensable for most horticultural fruit crops,making significant contributions to the fruit's pigment quality.The diversity of chlorophyll and carotenoid content in the fruit makes it appear green,red,orange,yellow,or even brown.Although our knowledge of chlorophyll degradation and carotenoid biosynthesis has recently increased,little is known about the mechanism that co-regulate these two pathways during fruit ripening.Here,a novel brown navel orange mutant‘Zong Cheng'was studied,and its phenotype and main fruit qualities were analyzed.The mutation site of‘Zong Cheng'was identified by integrating multi-omics and multiple genetic resources,and the molecular mechanism of retained chlorophyll and enhanced carotenoid in flavedo was analyzed through biochemical methods.In addition,we explored the enzyme activity diversity of chromoplast-specific lycopene?-cyclase?LCYb2?alleles,which is closely associated with the formation of citrus red traits.Moreover,the critical transcription factors that directly regulating CsLCYb2 were screened,and their functions were analyzed.The main findings were as follows:1.The traits and mutation mechanism of a brown citrus mutant‘Zong Cheng'‘Zong Cheng'?MT?is a novel brown navel orange mutant,which was discovered in a‘Lane Late Navel Orange'?WT?orchard in Zigui County?Hubei Province,China?.The fruit quality analysis revealed that the peel color,firmness,and aroma were different between WT and MT,while the other qualities were the same.The retained chlorophyll retained and increased carotenoid content in flavedo of MT during ripening indicated that the metabolic basis of brown fruit formation is the simultaneous accumulation of chlorophyll and carotenoids in flavedo.Microscopy and electron microscope observations showed that the chloroplast degradation in flavedo of MT was hindered after the breaker stage,and many chloroplasts even retained at the ripening stage.The GS-MS results of primary metabolites and volatiles revealed that many metabolites are significantly different between MT and WT at the ripening stage.The fruit quality of MT was generally worse than that of WT during storage.Further Penicillium italicum inoculation revealed that the resistance of MT fruits is weaker than that of WT.Using the strategy of integrating multiple-omics and multiple genetic resources,the mutant gene of MT was identified as being STAY-GREEN?SGR?.Sequencing discovered two SGR alleles?CsSGRa and CsSGRb?in citrus.Compared with CsSGRa,the translation of CsSGRb was terminated prematurely,which is caused by alternative splicing from sequence variation,and finally resulting in protein truncation.In MT,two consecutive bases in the CDS of the CsSGRa gene were replaced and formed a stop code,which results in a short protein(hereafter named CsSGRa^STOP),while CsSGRb was normal.Transient expression in tobacco leaves uncovered that only CsSGRa could degrade chlorophyll,while both CsSGRa ^STOPand CsSGRb lost this function,which explained the damage of chlorophyll degradation in MT.In addition,using engineered bacteria and transgenic citrus calli,we found both CsSGRa and CsSGRb could inhibit carotenoid biosynthesis,while CsSGRa^STOPlost this function.Bi FC and Y2H experiments further confirmed that CsSGRa and CsSGRb directly interact with CsPSY1 and inhibit carotenoid biosynthesis,while CsSGRa ^STOPcan not interact with CsPSY1.These results explained the increased carotenoids in MT.Consequently,the mutation of CsSGRa is responsible for retained chlorophyll and enhanced carotenoid in flavedo of MT,which causes the brown phenotype.2.The function of CsLCYb2 and its interacting transcription factors in fruit color formationThe chromoplast-specific LCYb2 is a key carotenoid biosynthetic enzyme.Two citrus LCYb2 alleles?CsLCYb2a and CsLCYb2b?were overexpressed in tomato to produced golden tomato fruits with massive accumulation of?-carotene.Overexpression of the allele CsLCYb2a in tomato induced increased?-carotene by 9.3-fold?1.5 mg/g dry weight?and undetected lycopene;while overexpression of the allele CsLCYb2b induced increased?-carotene and decreased lycopene,which implied the functional divergence of CsLCYb2 alleles.In addition,the CsLCYb2 promoter was used to screen yeast one-hybrid library,and two critical candidate transcription factors CsERF061 and CsMADS3 were discovered.CsMADS3 belongs to the MADS-Box family.Gene expression and protein abundance analysis showed that the expression level of CsMADS3 was closely associated with fruit ripening and coloring.CsMADS3 is a nucleus-localized transcription activator,which can directly bind to and activate the CsLCYb2 promoter.Overexpression of CsMADS3 in citrus callus promoted the accumulation of carotenoids,up-regulated most genes in the carotenoid biosynthetic pathway,and also changed chromoplast and phytohormone content.Overexpression of CsMADS3 in tomato fruits accelerated fruit degreening and enhanced the accumulation of carotenoids,accompanied by the up-regulation of key genes in chlorophyll degradation and carotenoid biosynthesis pathways,and the change of chromoplast and phytohormone.Biochemical experiments further confirmed that CsMADS3 could bind to and activate the promoters of SGR and the other carotenoid pathway genes,including PSY,PDS,CRTISO,BCH,ZEP,NCED,CCD1,and CCD4,indicating the multi-target regulation of CsMADS3.CsERF061 belongs to the group I ethylene response factor family.Its expression was induced by ethylene and was highly correlated with fruit ripening and coloring.CsERF061 is a nuclear-localized transcription activator that can directly bind to and activate the CsLCYb2 promoter.Overexpression of the CsERF061 gene in citrus callus and tomato fruit induced the significantly increased carotenoid content and up-regulated expression of a series of key carotenoid pathway genes,accompanied by alterations in chromoplast and phytohormone content.Biochemical experiments confirmed that CsERF061 also activated the promoters of the other carotenoid pathway genes,including PSY,PDS,CRTISO,LCYb1,BCH,ZEP,NCED,CCD1,and CCD4,indicating the multi-target regulation of CsERF061.In conclusion,our study clarified the molecular mechanism of the mutation of'Zong Orange',and revealed the critical role of SGR in regulating citrus fruit ripening,and also discovered the functional diversity of SGR alleles and their unique and fine regulation mechanism in chlorophyll degradation and carotenoid accumulation.In addition,the enzyme activity diversity of CsLCYb2 alleles was clarified,and the function of CsMADS3 on chlorophyll degradation and carotenoid biosynthesis during fruit ripening was revealed,and the function of CsERF061 in the regulation of carotenoid metabolism mediated by ethylene was also discovered.This study contributes to the further analysis of the coordinated regulation mechanism of chlorophyll and carotenoid metabolism in citrus fruits.