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.

Transcriptome and co-expression network analyses provide insights into fruit shading that enhances carotenoid accumulation in pomelo(Citrus grandis)

Carotenoids are indispensable for human health,and citrus fruit are a crucial source of dietary carotenoids.Bagging,an important orchard practice to enhance fruit economic value,is widely used in many horticultural crops,including citrus fruit.The bagged‘Majiayou’pomelo(Citrus grandis)produces vivid deeper red pulp,a fantastic agronomic trait,but the underlying molecular regulatory mechanism remains largely unexplored.Here,the enhancement of carotenoids,especially lycopene,was confirmed by HPLC analysis of carotenoids in the pulp of bagged fruit and controls.qRT-PCR analysis of the 14 carotenoid pathway genes further revealed that upregulated PSY and downregulated CCD1 expression in bagged fruit could directly enhance the accumulation of carotenoids.In addition,RNA-seq analysis identified 311 differentially expressed genes(DEGs)in the bagged fruit and controls in five developmental stages.Weighted gene co-expression network analysis(WGCNA)identified 13 critical candidate genes among the DEGs,which are closely associated with lycopene accumulation.The underlying regulatory mechanism of these candidate genes on the transcription of carotenoid pathway genes in the bagged fruit was discussed.Considering that the candidate genes were involved in the corresponding metabolic pathways,the increase in sucrose content and decrease in ABA in bagged fruit were also identified,implying that these candidate genes may be indirectly related to carotenoid enhancement in pulp by regulating phytohormones,primary metabolism,and stress responses.The results provide new insights into the potential regulatory mechanism of lycopene enhancement in the pulp of bagged‘Majiayou’pomelo,facilitating breeding and orchard management efforts to improve the nutritional quality and esthetic value of citrus,and perhaps other fruit crops.

Down regulated expression of S_(2)-RNase attenuates self-incompatibility in “Guiyou No.1”pummelo

Self-incompatibility(SI)substantially restricts the yield and quality of citrus.Therefore,breeding and analyzing selfcompatible germplasm is of great theoretical and practical signi ficance for citrus.Here,we focus on the mechanism of a self-compatibility mutation in‘Guiyou No.1'pummelo(Citrus maxima),which is a spontaneous mutant of‘Shatian’pummelo(Citrus maxima,self-incompatibility).The rate of fruit set and the growth of pollen tubes in the pistil con firmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of‘Guiyou No.1'.Segregation ratios of the S genotype in progeny,expression analysis,and western blotting validated that the reduced levels of S_(2)-RNase mRNA contribute to the loss of SI in‘Guiyou No.1'.Furthermore,we report a phased assembly of the‘Guiyou No.1'pummelo genome and obtained two complete and well-annotated S haplotypes.Coupled with an analysis of SV variations,methylation levels,and gene expression,we identi fied a candidate gene(CgHB40),that may in fluence the regulation of the S/^RNase promoter.Our data provide evidence that a mutation that affects the pistilled to the loss of SI in‘Guiyou No.1'by in fluencing a poorly understood mechanism that affects transcriptional regulation.This work signi ficantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes.

Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating b-cryptoxanthin in Citrus

Carotenoids in plant foods provide health benefits by functioning as provitamin A.One ofthe vital provitamin A carotenoids,b-cryptoxanthin,is typically plentiful in citrus fruit.However,little is known about the genetic basis of b-cryptoxanthin accumulation in citrus.Here,we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrus and determine the taxonomic profile of b-cryptoxanthin.We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of b-cryptoxanthin accumulation through a genome-wide association study.We identified a causal gene,CitCYP97B,which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined.We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the b-ring of b-cryptoxanthin in a heterologous expression system.In planta experiments provided further evidence that CitCYP97B negatively regulates b-cryptoxanthin content.Using the sequenced Citrus accessions,we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B,thereby altering b-cryptoxanthin accumulation in fruit.Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrus types during citrus evolution.Overall,these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of b-cryptoxanthin-biofortified products.

A rare inter-haplotypic recombination at the S-locus contributed to the loss of self-incompatibility in trifoliate orange

Dear Editor,In trans-specific S-haplotypes,crossover recombination at the S-locus,which controls self-incompatibility,has been thought to be highly suppressed.Here,we discovered a previously unreported super S haplotype,derived from a rare inter-haplotypic recombination event,that contained two complete suites of functional S-RNase and SLF genes and could break the SI response in trifoliate orange through“self-recognition”within the same pollen.

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.

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.

The abscisic acid-responsive transcriptional regulatory module CsERF110-CsERF53 orchestrates citrus fruit coloration

Carotenoid biosynthesis is closely associated with abscisic acid(ABA)during the ripening process of non-climacteric fruits,but the regulatory mechanism that links ABA signaling to carotenoid metabolism remains largely unclear.Here,we identified two master regulators of ABA-mediated citrus fruit coloration,CsERF110 and CsERF53,which activate the expression of carotenoid metabolism genes(CsGGPPS,CsPSY,CsPDS,CsCRTISO,CsLCYB2,CsLCYE,CsHYD,CsZEP,and CsNCED2)to facilitate carotenoid accumulation.Further investigations showed that CsERF110 not only activates the expression of CsERF53 by binding to its promoter but also interacts with CsERF53 to form the transcriptional regulatory module CsERF110-CsERF53.We also discovered a positive feedback regulatory loop between the ABA signal and carotenoid metabolism regulated by the transcriptional regulatory module CsERF110-CsERF53.Our results reveal that the CsERF110-CsERF53 module responds to ABA signaling,thereby orchestrating citrus fruit coloration.Considering the importance of carotenoid content for citrus and many other carotenoid-rich crops,the revelation of molecular mechanisms that underlie ABA-mediated carotenoid biosynthesis in plants will facilitate the development of transgenic/gene-editing approaches,further contributing to improving the quality of citrus and other carotenoid-rich crops.