Functional Analysis Of Sweet Potato IbCCD4 And IbNCED3 In Carotenoid Metabolism

Sweet potato(Ipomoea batatas [L.] Lam)is one of the most nutritious tuber crops in the world,rich in vitamins,carotenoids and anthocyanins,which have antioxidant function to health benefits.The flesh color of sweet potato tuberous root is mainly white,yellow,orange and purple.Orange-fleshed sweet potatoes are rich in carotenoids,which have the ability of scavenging reactive oxygen radicals,slowing aging,and preventing chronic degenerative diseases.While some studies have resolved the accumulation patterns and metabolic pathways of carotenoids in above-ground organs such as flowers and fruits,few research has been done on the analysis of expression profiling and function of genes related to carotenoid metabolic pathways in the tuberous root of sweet potato.In this paper,we used targeted metabolomics to study the carotenoid content and fraction dynamics of three sweet potato varieties with white,yellow and orange-fleshed tuberous root(‘Xushu18’,‘Xinxiang’ and ‘Sushu8’)at six developmental stages.The expression level of genes related to carotenoid metabolism was analyzed by quantitative real-time PCR(q RT-PCR),and the IbCCD4 and IbNCED3 genes with high correlation with carotenoid metabolism in sweet potato tuberous root were screened by correlation analysis.IbCCD4 and IbNCED3 were cloned,then overexpression vectors of IbCCD4 and IbNCED3 were constructed and the functions of IbCCD4 and IbNCED3 in response to salt stress were dissected in transgenic Arabidopsis,respectively.CRISPR/Cas9 gene editing vectors of IbCCD4 and IbNCED3 were constructed and transgenic sweet potato plant were obtained.A c DNA yeast library of sweet potato tuberous root was constructed.The promoter sequence of IbCCD4 and IbNCED3 were cloned and the bait vectors were constructed,and the proteins interacting with them were screened in the sweet potato tuber c DNA yeast library by yeast hybridization technique.The main results were as follows.1.carotenoid targeted metabolomics assayThe highest total carotenoid content was detected in orange-fleshed sweet potato ‘Sushu8’,which mainly accumulated β-carotene and β-cryptoxanthin.The yellow-fleshed sweet potato ‘Xinxiang’ mainly accumulated β-cryptoxanthin and zeaxanthin.However,the white-fleshed sweet potato ‘Xushu18’ had the lowest total carotenoid content and mainly accumulated β-cryptoxanthin.2.Correlation Analysis of gene expression level involved in carotenoid metabolic pathway and the content of carotenoid componentThe expression level of IbCCD4 in ‘Xinxiang’ and ‘Xushu18’ was higher than that in ‘Sushu8’.The content of β-carotene,β-cryptoxanthin and the total carotenoid in ‘Sushu8’ were significantly positively correlated with the expression level of IbPSY and IbNCED3.It indicates that IbCCD4 and IbNCED3 have high correlation with carotenoid metabolism in sweet potato tuberous root.3.Cloning and functional analysis of IbCCD4The full-length genome sequence and ORF sequence of IbCCD4 were cloned.The IbCCD4 protein is most closely related to It CCD4 of Ipomoea triloba,a closely related wild species of sweet potato.The expression level of IbCCD4 was higher in the leaves than that in the tuberous roots.Subcellular localization of IbCCD4 was in chloroplasts.Under normal growth conditions,there were no significant differences in total carotenoid content and expression level of genes involved in carotenoid metabolism pathway.Under Na Cl stress,the root length of IbCCD4 overexpressing transgenic Arabidopsis was significantly shorter,and its rosette leaves showed significantly increased the content of anthocyanin and carotenoid,and increased expression level of most genes involved in the carotenoid metabolic pathway,and decreased expression level of resistancerelated genes and the activities of POD,SOD,and CAT.The MDA content was significantly higher than that in wide type.This suggested that IbCCD4 negatively regulated salt tolerance in Arabidopsis.Compared with the wild type,the tuberous root colour of the IbCCD4 overexpressing transgenic sweet potato lines turned to white,and the total carotenoid content in the leaves and the tuberous root was significantly lower with the similar component.The edit types of base deletion and substitution were detected in CRISPR/Cas9-IbCCD4 gene editing sweet potato.4.Cloning and functional analysis of IbNCED3The full-length genome sequence and ORF sequence of IbNCED3 were cloned.IbNCED3 is most closely related to In NCED3 of Ipomoea nil.The expression level of IbNCED3 was higher at some developmental stages such as 60 DAP in leaf of ‘Sushu8’,60 DAP and 120 DAP in tuberous root of ‘Sushu8’,and 60 DAP in tuberous root of ‘Xinxiang’.Subcellular localization of IbNCED3 was in chloroplasts.Under normal growth conditions,compared with wild-type Arabidopsis,overexpression of IbNCED3 transgenic Arabidopsis seeds delayed germination by 5 ~7 d,slowed plant growth,significantly increased ABA(abscisic acid)content in rosette leaves,and accumulated large amounts of ABA-GE(abscisic acid glucosyl ester).However,there was no significant difference of the total carotenoid content and the expression level of most genes involved in the carotenoid metabolic pathway between overexpressing IbNCED3 transgenic Arabidopsis and wild type.Under Na Cl stress,IbNCED3 overexpressing transgenic Arabidopsis showed significantly increased root growth,reduced the carotenoid content and the expression level of most genes involved in the carotenoid metabolic pathway,and increased the expression level of resistance-related genes compared with wild-type Arabidopsis.The IbNCED3 overexpression transgenic sweet potato regeneration lines grew slowly.The edit types of base deletion,substitution,and insertion were detected in CRISPR/Cas9-IbNCED3 gene editing sweet potato.5.Construction of a yeast c DNA library of sweet potato tuberous rootA c DNA library from sweet potato tuberous root was constructed using the Gateway method.6.Screening proteins interacted with promoter sequence of IbCCD4 and IbNCED3Forty interacted proteins with IbCCD4 promotor were obtained.Three interacted proteins with IbNCED3 promotor were obtained.The results provide the molecular targets for improving carotenoid quality in sweet potato tuberous root,and provide new ideas for breeding carotenoid-rich and stress-resistant sweet potato varieties.The results of this study enrich the understanding of the carotenoid metabolism mechanisms in underground organs of plants.