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Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for the coloration and nutritional value of fruits. However, the synergistic regulation of these 2 processes at the transcriptional level remains largely unknown. In this study, we identified a WRKY transcription factor, CrWRKY42, from the transcriptome data of the yellowish bud mutant "Jinlegan" ([Citrus unshiu × C. sinensis] × C. reticulata) tangor and its wild-type "Shiranui" tangor, which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 directly bound to the promoter of β-carotene hydroxylase 1 (CrBCH1) and activated its expression. The overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene β-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could bind to the promoters of NONYELLOW COLORING (CrNYC) and STAY-GREEN (CrSGR) and activate their expression, thus promoting chlorophyll degradation. The overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expression of genes involved in both pathways. Our data revealed that CrWRKY42 acts as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening., Competing Interests: Conflict of interest statement. There are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis -regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

A Citrus sinensis R2R3 MYB transcription factor (CsMYB96) has previously been shown to be strongly associated with the expression of many genes related to wax biosynthesis in the fruit. In this study, CsMYB96 was found to alleviate water loss by simultaneously regulating the expression of genes encoding plasma membrane intrinsic proteins (CsPIPs) and wax-related genes. Expression profiling indicated that CsPIP1;1 and CsPIP2;4 had high expression that was representative of other aquaporins, and they were down-regulated in the peel of post-harvest citrus fruit. CsPIP2;4 was further characterized as the predominant CsPIP, with high expression and high-water channel activity. Transient overexpression of CsPIP2;4 accelerated water loss in citrus fruit. In silico analysis further indicated that the expression of CsMYB96 had a significant negative correlation with that of CsPIPs. In vivo and in vitro experiments confirmed that CsMYB96 was able to directly repress the expression of CsPIPs. In addition, CsMYB96 was able to activate wax-related genes and promote wax biosynthesis for defense against water loss. Transient and stable overexpression of CsMYB96 reduced water loss from both citrus fruit and Arabidopsis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H 2 O 2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H 2 O 2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including β-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene β-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis., (� The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chromoplast-specific lycopene β-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Anthocyanins are preferentially accumulated in certain tissues of particular species of citrus. A R2R3-MYB transcription factor (named Ruby1) has been well documented as an activator of citrus anthocyanin biosynthesis. In this study, we characterized CsMYB3, a transcriptional repressor that regulates anthocyanin biosynthesis in citrus. CsMYB3 was expressed in anthocyanin-pigmented tissues, and the expression was closely associated with that of Ruby1, which is a key anthocyanin activator. Overexpression of CsMYB3 in Arabidopsis resulted in a decrease in anthocyanins under nitrogen stress. Overexpression of CsMYB3 in the background of CsRuby1-overexpressing strawberry and Arabidopsis reduced the anthocyanin accumulation level. Transient promoter activation assays revealed that CsMYB3 could repress the activation capacity of the complex formed by CsRuby1/CsbHLH1 for the anthocyanin biosynthetic genes. Moreover, CsMYB3 could be transcriptionally activated by CsRuby1 via promoter binding, thus forming an 'activator-and-repressor' loop to regulate anthocyanin biosynthesis in citrus. This study shows that CsMYB3 plays a repressor role in the regulation of anthocyanin biosynthesis and proposes an 'activator-and-repressor' loop model constituted by CsRuby1 and CsMYB3 in the regulation of anthocyanin biosynthesis in citrus., (© The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Although remarkable progress has been made toward understanding carotenoid biosynthesis, the mechanisms that regulate the transcription of carotenogenic genes remain poorly understood. Lycopene β-cyclases (LCYb) are critical enzymes located at the branch point of the carotenoid biosynthetic pathway. Here, we used the promoter sequence of LCYb1 as bait in a yeast one-hybrid screen for promoter-binding proteins from sweet orange ( Citrus sinensis ). This screen identified a MADS transcription factor, CsMADS6, that was coordinately expressed with fruit development and coloration. Acting as a nucleus-localized transcriptional activator, CsMADS6 directly bound the promoter of LCYb1 and activated its expression. Overexpression of CsMADS6 in citrus calli increased carotenoid contents and induced the expression of LCYb1 and other carotenogenic genes, including phytoene synthase ( PSY ), phytoene desaturase ( PDS ), and carotenoid cleavage dioxygenase1 ( CCD1 ). CsMADS6 up-regulated the expression of PSY , PDS , and CCD1 by directly binding to their promoters, which suggested the multitargeted regulation of carotenoid metabolism by CsMADS6. In addition, the ectopic expression of CsMADS6 in tomato ( Solanum lycopersicum ) affected carotenoid contents and the expression of carotenogenic genes. The sepals of CsMADS6 -overexpressing tomato lines exhibited dramatic changes in carotenoid profiles, accompanied by changes in plastid ultrastructure. Global transcriptome analysis of transgenic sepals revealed that CsMADS6 regulates a series of pathways that promote increases in flux through the carotenoid pathway. Overall, these findings establish that CsMADS6 directly regulates LCYb1 and other carotenogenic genes to coordinately and positively modulate carotenoid metabolism in plants, which may provide strategies to improve the nutritional quality of crops., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α - and β-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and β-branch carotenoids was identified using Green Ougan (MT), a stay-green mutant of Citrus reticulata cv Suavissima. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of β-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and β-carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of CrMYB68 in MT. Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate CrBCH2 and CrNCED5. Moreover, transient overexpression experiments using leaves of Nicotiana benthamiana indicated that CrMYB68 can also negatively regulate NbBCH2 and NbNCED5. To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Flavonols and hydroxycinnamic acids are important phenylpropanoid metabolites in plants. In this study, we isolated and characterized a citrus R2R3-MYB transcription factor CsMYBF1, encoding a protein belonging to the flavonol-specific MYB subgroup. Ectopic expression of CsMYBF1 in tomato led to an up-regulation of a series of genes involved in primary metabolism and the phenylpropanoid pathway, and induced a strong accumulation of hydroxycinnamic acid compounds but not the flavonols. The RNAi suppression of CsMYBF1 in citrus callus caused a down-regulation of many phenylpropanoid pathway genes and reduced the contents of hydroxycinnamic acids and flavonols. Transactivation assays indicated that CsMYBF1 activated several promoters of phenylpropanoid pathway genes in tomato and citrus. Interestingly, CsMYBF1 could activate the CHS gene promoter in citrus, but not in tomato. Further examinations revealed that the MYBPLANT cis-elements were essential for CsMYBF1 in activating phenylpropanoid pathway genes. In summary, our data indicated that CsMYBF1 possessed the function in controlling the flavonol and hydroxycinnamic acid biosynthesis, and the regulatory differences in the target metabolite accumulation between two species may be due to the differential activation of CHS promoters by CsMYBF1. Therefore, CsMYBF1 constitutes an important gene source for the engineering of specific phenylpropanoid components.

MYB transcription factor represents one of the largest gene families in plant genomes. Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide, and recently the genome has been sequenced. This provides an opportunity to investigate the organization and evolutionary characteristics of sweet orange MYB genes from whole genome view. In the present study, we identified 100 R2R3-MYB genes in the sweet orange genome. A comprehensive analysis of this gene family was performed, including the phylogeny, gene structure, chromosomal localization and expression pattern analyses. The 100 genes were divided into 29 subfamilies based on the sequence similarity and phylogeny, and the classification was also well supported by the highly conserved exon/intron structures and motif composition. The phylogenomic comparison of MYB gene family among sweet orange and related plant species, Arabidopsis, cacao and papaya suggested the existence of functional divergence during evolution. Expression profiling indicated that sweet orange R2R3-MYB genes exhibited distinct temporal and spatial expression patterns. Our analysis suggested that the sweet orange MYB genes may play important roles in different plant biological processes, some of which may be potentially involved in citrus fruit quality. These results will be useful for future functional analysis of the MYB gene family in sweet orange.

Apomixis, or asexual seed formation, is prevalent in Citrinae via a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in Citrinae , a chromosome-level genome of the Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinae accessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinae diversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella , we found that apomixis of some varieties originated via introgression. In apomictic Fortunella , the locus associated with apomixis contains the FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus and Fortunella , due to either two or three miniature inverted transposon element (MITE) insertions. A transcription factor, FhARID , encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties, which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinae and has potential ramifications for citrus breeding. [ABSTRACT FROM AUTHOR]

Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5. [ABSTRACT FROM AUTHOR]

In plant, a few 22-nt mi RNAs direct cleavages of their targets and trigger the biogenesis of phased small interfering RNAs (phasi RNAs) in plant. In this study, we characterized a mi RNA triggering phasi RNAs generation, miR3954, and explored its downstream target genes and potential function. Our results demonstrated that miR3954 showed specific expression in the flowers of citrus species, and it targeted a NAC transcription factor ( Cs7 g22460) and two non-coding RNA transcripts (lnc RNAs, Cs1 g09600 and Cs1 g09635). The production of phasi RNAs was detected from transcripts targeted by miR3954, and was further verified in both sequencing data and transient expression experiments. Phasi RNAs derived from the two lnc RNAs targeted not only miR3954-targeted NAC gene but also additional NAC homologous genes. No homologous genes of these two lnc RNAs were found in plants other than citrus species, implying that this miR3954-lnc RNAs-phasi RNAs- NAC pathway is likely citrus-specific. Transgenic analysis indicated that the miR3954-overexpressing lines showed decreased transcripts of lnc RNA, elevated abundance of phasi RNAs and reduced expression of NAC genes. Interestingly, the overexpression of miR3954 leads to early flowering in citrus plants. In summary, our results illustrated a model of the regulatory network of miR3954-lnc RNA-phasi RNAs- NAC, which may be functionally involved in flowering in citrus. [ABSTRACT FROM AUTHOR]

The phytoene synthase ( PSY) gene product is involved in the carotenoid biosynthesis pathway. In this study, the promoter of the sweet orange gene CsPSY of sweet orange 'Cara Cara' ( Citrus sinensis Osbeck [ Cs]) was isolated and characterized. Its sequence included a number of regulatory elements predicted to be responsive to light and other environmental cues. The 926-bp region upstream of CsPSY was fused to the β-glucuronidase ( GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants showed that the CsPSY promoter drove expression in the young seedlings, the leaves, the roots and in parts of the flower, in a developmentally regulated fashion. A promoter deletion analysis revealed that the region −479 to −306 nt positively regulated expression, the region −306 to −93 nt was associated with negative regulation of expression and the region −46 to +21 nt maintained basal promoter activity. In the presence of sucrose, gene expression was regulated via a cis-acting promoter element(s). Two highly repetitive sequences (Sp1 and A-box), located in the region from −479 to −306 nt, may act as light-responsive elements regulating CsPSY transcription during photomorphogenesis. [ABSTRACT FROM AUTHOR]

ABSTRACT Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2‐type zinc‐finger transcription factor, CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α‐branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB, CitHYD, and CitNCED2, promoting β‐branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one‐hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4‐mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4‐mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops. [ABSTRACT FROM AUTHOR]

As is well known, postharvest abscisic acid (ABA) treatment of citrus fruits can significantly enhance fruit coloration by promoting the accumulation of carotenoids, however, the underlying molecular mechanism is still unclear. In this study, we further confirmed the effect of ABA treatment in promoting citrus fruit coloration by activating the expression of 4 key carotenoid metabolism genes (CsPSY , CsLCYB , CsHYD , and CsNCED2) during postharvest storage. A novel basic helix-loop-helix (bHLH) transcription factor, CsbHLH6, was identified using the promoters of 3 key ABA-induced carotenoid metabolism genes (CsLCYB , CsHYD , and CsNCED2) by yeast one-hybrid screening. Overexpression of CsbHLH6 enhanced carotenoid accumulation in citrus fruits and calli. Interference with CsbHLH6 expression in citrus calli and fruits indicated that CsbHLH6 was essential for ABA-induced carotenoid biosynthesis and fruit coloration. As a nucleus-localized transcriptional activator, CsbHLH6 directly bound to the promoters of 6 key carotenoid metabolism genes (CsPSY , CsLCYB , CsLCYE , CsHYD , CsZEP , and CsNCED2), subsequently activating their expression in vitro and in vivo. Furthermore, ABA signaling significantly raised CsbHLH6 promoter activity, thus enhancing the regulation of carotenoid accumulation by CsbHLH6. In summary, our study reveals the molecular mechanism by which CsbHLH6 regulates ABA-induced citrus fruit coloration during postharvest storage, which is crucial for improving the postharvest appearance quality of citrus fruits. • Abscisic acid (ABA) treatment promotes fruit coloration during postharvest storage. • CsbHLH6 plays an essential role in postharvest ABA-mediated fruit coloration. • CsbHLH6 activates the expression of 6 key ABA-induced carotenoid biosynthesis genes. • ABA signaling greatly enhances the regulation of carotenoid accumulation by CsbHLH6. [ABSTRACT FROM AUTHOR]