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。

Genome-wide Characterization of cis-acting Elements in the Promoters of Key Carotenoid Pathway Genes from the Main Species of Genus Citrus

Carotenoids are indispensable for both human health and plant survival.Citrus,is one of the fruit crops richest in carotenoid compounds,with approximately 115 kinds of carotenoids;tremendous diversity in carotenoids composition and concentration exists among various species,showing different colors from nearly white to crimson.The carotenoid biosynthetic pathway and the key carotenogenic genes have been identified in citrus;however,the underlying regulatory mechanisms remain unclear.In this study,among the main species of genus Citrus(primitive,wild,and cultivated),we detected carotenoids in flavedo using High-Performance Liquid Chromatography,and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes.Intriguingly,both carotenoid composition and content were generally increased during the evolution of citrus,and the corresponding variations in the promoters were identified,including the gain or loss of critical environmental stress-responsive elements and hormone-responsive elements,which are closely associated with carotenoid enhancement.In addition,pummelo has the most heat-responsive elements,but the Mangshan mandarin does not have this element in the promoters of PSY,which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation.Moreover,the abscisic acid-responsive motif was rich in almost all the seven species,but the ethylene-responsive motif was deficient,which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus.Overall,our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus,which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit 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.

Multiomics-based dissection of citrus flavonoid metabolism using a Citrus reticulata × Poncirus trifoliata population

Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes.Although citrus plants are among the most important fruit crops worldwide,the genetic basis of secondary metabolism in these plants is largely unknown.Here,we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues(young leaf,old leaf,mature pericarp,and mature pulp)of an F_(1) pseudo-testcross citrus population.We detected 80 flavonoids in this population and identified 138 quantitative trait loci(QTLs)for 57 flavonoids in these four tissues.Based on transcriptional profiling and functional annotation,twenty-one candidate genes were identified,and one gene encoding flavanone 3-hydroxylase(F3H)was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions.The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.

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.

Panicle-3D: A low-cost 3D-modeling method for rice panicles based on deep learning, shape from silhouette, and supervoxel clustering

Self-occlusions are common in rice canopy images and strongly influence the calculation accuracies of panicle traits. Such interference can be largely eliminated if panicles are phenotyped at the 3 D level.Research on 3 D panicle phenotyping has been limited. Given that existing 3 D modeling techniques do not focus on specified parts of a target object, an efficient method for panicle modeling of large numbers of rice plants is lacking. This paper presents an automatic and nondestructive method for 3 D panicle modeling. The proposed method integrates shoot rice reconstruction with shape from silhouette, 2 D panicle segmentation with a deep convolutional neural network, and 3 D panicle segmentation with ray tracing and supervoxel clustering. A multiview imaging system was built to acquire image sequences of rice canopies with an efficiency of approximately 4 min per rice plant. The execution time of panicle modeling per rice plant using 90 images was approximately 26 min. The outputs of the algorithm for a single rice plant are a shoot rice model, surface shoot rice model, panicle model, and surface panicle model, all represented by a list of spatial coordinates. The efficiency and performance were evaluated and compared with the classical structure-from-motion algorithm. The results demonstrated that the proposed method is well qualified to recover the 3 D shapes of rice panicles from multiview images and is readily adaptable to rice plants of diverse accessions and growth stages. The proposed algorithm is superior to the structure-from-motion method in terms of texture preservation and computational efficiency. The sample images and implementation of the algorithm are available online. This automatic, cost-efficient, and nondestructive method of 3 D panicle modeling may be applied to high-throughput 3 D phenotyping of large rice populations.

A comprehensive proteomic analysis of elaioplasts from citrus fruits reveals insights into elaioplast biogenesis and function

Elaioplasts of citrus peel are colorless plastids which accumulate significant amounts of terpenes.However,other functions of elaioplasts have not been fully characterized to date.Here,a LC–MS/MS shotgun technology was applied to identify the proteins from elaioplasts that were highly purified from young fruit peel of kumquat.A total of 655 putative plastid proteins were identified from elaioplasts according to sequence homology in silico and manual curation.Based on functional classification via Mapman,~50%of the identified proteins fall into six categories,including protein metabolism,transport,and lipid metabolism.Of note,elaioplasts contained ATP synthase and ADP,ATP carrier proteins at high abundance,indicating important roles for ATP generation and transport in elaioplast biogenesis.Additionally,a comparison of proteins between citrus chromoplast and elaioplast proteomes suggest a high level of functional conservation.However,some distinctive protein profiles were also observed in both types of plastids notably for isoprene biosynthesis in elaioplasts,and carotenoid metabolism in chromoplasts.In conclusion,this comprehensive proteomic study provides new insights into the major metabolic pathways and unique characteristics of elaioplasts and chromoplasts in citrus fruit.

A Novel Intelligent System for Dynamic Observation of Cotton Verticillium Wilt

Verticillium wilt is one of the most critical cotton diseases,which is widely distributed in cotton-producing countries.However,the conventional method of verticillium wilt investigation is still manual,which has the disadvantages of subjectivity and low efficiency.In this research,an intelligent vision-based system was proposed to dynamically observe cotton verticillium wilt with high accuracy and high throughput.Firstly,a 3-coordinate motion platform was designed with the movement range 6,100 mm×950 mm×500 mm,and a specific control unit was adopted to achieve accurate movement and automatic imaging.Secondly,the verticillium wilt recognition was established based on 6 deep learning models,in which the VarifocalNet(VFNet)model had the best performance with a mean average precision(mAP)of 0.932.Meanwhile,deformable convolution,deformable region of interest pooling,and soft non-maximum suppression optimization methods were adopted to improve VFNet,and the mAP of the VFNet-Improved model improved by 1.8%.The precision–recall curves showed that VFNet-Improved was superior to VFNet for each category and had a better improvement effect on the ill leaf category than fine leaf.The regression results showed that the system measurement based on VFNet-Improved achieved high consistency with manual measurements.Finally,the user software was designed based on VFNet-Improved,and the dynamic observation results proved that this system was able to accurately investigate cotton verticillium wilt and quantify the prevalence rate of different resistant varieties.In conclusion,this study has demonstrated a novel intelligent system for the dynamic observation of cotton verticillium wilt on the seedbed,which provides a feasible and effective tool for cotton breeding and disease resistance research.

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.