Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.)

Carotenoids directly influence citrus fruit color and nutritional value,which is critical to consumer acceptance.Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality.Despite the well-established carotenoid biosynthetic pathways,the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood.Our previous studies have reported that the Myc-type basic helix-loop-helix(bHLH)transcription factor(TF)regulates citrus proanthocyanidin biosynthesis.Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli,but its regulatory mechanism is still unclear.In the present study,we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR,CsHDS,CsHDR,CsPDS,CsLCYE,CsZEP,and CsNCED2,which was accompanied by changes in the contents of abscisic acid and gibberellin.The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR,CsHDS,and CsHDR,the keymetabolic enzymes of the methylerythritol 4-phosphate(MEP)pathway,thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes.In addition,CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes,CsPDS,CsLCYE,CsZEP,and CsNCED2,directly promoting carotenoid biosynthesis.This study reveals a novel network of carotenoid metabolism regulated by CsTT8.Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

Citrus β-carotene hydroxylase 2 (BCH2) participates in xanthophyll synthesis by catalyzing the hydroxylation of β-carotene and compensates for BCH1 in citrus carotenoid metabolism

Abstract As an essential horticultural crop,Citrus has carotenoid diversity,which affects its aesthetic and nutritional values.β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits,and non-heme di-iron carotene hydroxylase(BCH)enzymes are mainly responsible forβ,β-xanthophyll synthesis.Previous studies have focused on the hydroxylation of BCH1,but the role of its paralogous gene in citrus,BCH2,remains largely unknown.In this study,we revealed theβ-hydroxylation activity of citrus BCH2(CsBCH2)for the first time through the functional complementation assay using Escherichia coli,although CsBCH2 exhibited a lower activity in hydroxylatingβ-carotene intoβ-cryptoxanthin than citrus BCH1(CsBCH1).Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression.This study revealed the distinct expression patterns and functional characteristics of two paralogous genes,CsBCH1 and CsBCH2,and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis.The independent function of CsBCH2 and its cooperative function with CsBCH1 inβ-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering。

Natural Variation in CCD4 Promoter Underpins Species-Specific Evolution of Red Coloration in Citrus Peel

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference.Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments.In this study,using an integrated genetic approach we revealed that a 5;c/s-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C3 0 apocarotenoids responsible for red coloration of citrus peel.Functional analyses demonstrated that in addition the known role in synthesizing 3-citraurin,CCD4b is also responsible for the production of another important C3 0 apocarotenoid pigment,p-citraurinene.Furthermore,analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5'c/s-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C3 0 apocarotenoid-rich red-peeled accessions.Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids.Taken together,our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants,and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

Genome of Wild Mandarin and Domestication History of Mandarin

Mandarin （Citrus reticulata） is one of the most important citrus crops worldwide. Its domestication is believed to have occurred in South China, which has been one of the centers of mandarin cultivation for four millennia. We collected natural wild populations of mandarin around the Nanling region and cultivated landraces in the vicinity. We found that the citric acid level was dramatically reduced in cultivated mandarins. To understand genetic basis of mandarin domestication, we de novo assembled a draft genome of wild mandarin and analyzed a set of 104 citrus genomes. We found that the Mangshan mandarin is a primitive type and that two independent domestication events have occurred, resulting in two groups of cultivated mandarins （MD1 and MD2） in the North and South Nanling Mountains, respectively. Two bottlenecks and two expansions of effective population size were identified for the MD1 group of cultivated mandarins. However, in the MD2 group there was a long and continuous decrease in the population size. MD1 and MD2 mandarins showed different patterns of interspecific introgression from cultivated pummelo species. We identified a region of high divergence in an aconitate hydratase （ACO） gene involved in the regulation of citrate content, which was possibly under selection during the domestication of mandarin. This study provides concrete genetic evidence for the geographical origin of extant wild mandarin populations and sheds light on the domestication and evolutionary history of mandarin.

NEW INSIGHTS INTO THE PHYLOGENY AND SPECIATION OF KUMQUAT (FORTUNELLA SPP.) BASED ON CHLOROPLAST SNP, NUCLEAR SSR AND WHOLE-GENOME SEQUENCING

Kumquat(Fortunella spp.)is a fruit and ornamental crop worldwide due to the palatable taste and high ornamental value of its fruit.Although Fortunella is classified into the economically important true citrus fruit tree group together with Citrus and Poncirus,few studies have been focused on its evolutionary scenario.In this study,analysis of five chloroplast loci and 47 nuclear microsatellites(nSSR)loci from 38 kumquat and 10 citrus accessions revealed the independent phylogeny of Fortunella among citrus taxa,and that Fortunella mainly comprises two populations:CUL,cultivated Fortunella spp.(F.margarita,F.crassifolia and F.japonica);and HK,wild Hong Kong kumquat(Fortunella hindsii).Genomic analysis based on whole-genome SNPs indicated that the allele frequency of both pupations deviated from the neutral selection model,suggesting directional selection was a force driving their evolutions.CUL exhibited lower genomic diversity and higher linkage strength than HK,suggesting artificial selection involved in its origin.A high level of genetic differentiation(Fst=0.364)was detected and obviously asynchronous demographic changes were observed between CUL and HK.Based on these results,a new hypothesis for the speciation of Fortunella is proposed.