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.

In plants, microRNAs (miRNAs) regulate gene expression mainly at the post-transcriptional level. Previous studies have demonstrated that miRNA-mediated gene silencing pathways play vital roles in plant development. Here, we used a high-throughput sequencing approach to characterize the miRNAs and their targeted transcripts in the leaf, flower and fruit of sweet orange. A total of 183 known miRNAs and 38 novel miRNAs were identified. An in-house script was used to identify all potential secondary siRNAs derived from miRNA-targeted transcripts using sRNA and degradome sequencing data. Genome mapping revealed that these miRNAs were evenly distributed across the genome with several small clusters, and 69 pre-miRNAs were co-localized with simple sequence repeats (SSRs). Noticeably, the loop size of pre-miR396c was influenced by the repeat number of CUU unit. The expression pattern of miRNAs among different tissues and developmental stages were further investigated by both qRT-PCR and RNA gel blotting. Interestingly, Csi-miR164 was highly expressed in fruit ripening stage, and was validated to target a NAC transcription factor. This study depicts a global picture of miRNAs and their target genes in the genome of sweet orange, and focused on the comparison among leaf, flower and fruit tissues. This study provides a global view of miRNAs and their target genes in different tissue of sweet orange, and focused on the identification of miRNA involved in the regulation of fruit ripening. The results of this study lay a foundation for unraveling key regulators of orange fruit development and ripening on post-transcriptional level.

Dwarfing is a pivotal agronomic trait affecting both yield and quality. Citrus species exhibit substantial variation in plant height, among which internode length is a core element. However, the molecular mechanism governing internode elongation remains unclear. Here, we unveiled that the transcriptional cascade consisting of B‐BOX DOMAIN PROTEIN 22 (BBX22) and ELONGATED HYPOCOTYL 5 (HY5) finely tunes plant height and internode elongation in citrus. Loss‐of‐function mutations of BBX22 in an early‐flowering citrus (Citrus hindsii “SJG”) promoted internode elongation and reduced pigment accumulation, whereas ectopic expression of BBX22 in SJG, sweet orange (C. sinensis), pomelo (C. maxima) or heterologous expression of BBX22 in tomato (Solanum lycopersicum) significantly decreased internode length. Furthermore, exogenous application of gibberellin A3 (GA₃) rescued the shortened internode and dwarf phenotype caused by BBX22 overexpression. Additional experiments revealed that BBX22 played a dual role in regulation internode elongation and pigmentation in citrus. On the one hand, it directly bound to and activated the expression of HY5, GA metabolism gene (GA2 OXIDASE 8, GA2ox8), carotenoid biosynthesis gene (PHYTOENE SYNTHASE 1, PSY1) and anthocyanin regulatory gene (Ruby1, a MYB DOMAIN PROTEIN). On the other hand, it acted as a cofactor of HY5, enhancing the ability of HY5 to regulate target genes expression. Together, our results reveal the critical role of the transcriptional cascade consisting of BBX22 and HY5 in controlling internode elongation and pigment accumulation in citrus. Unraveling the crosstalk regulatory mechanism between internode elongation and fruit pigmentation provides key genes for breeding of novel types with both dwarf and health‐beneficial fortification in citrus.

Deciphering the genetic basis of organoleptic traits is critical for improving the quality of fruits, which greatly shapes their appeal to consumers. Here, we characterize the citrus R3-MYB transcription factor TRIPTYCHON-LIKE (CitTRL), which is closely associated with the levels of citric acid, proanthocyanidins (PAs), and anthocyanins. Overexpression of CitTRL lowered acidity levels and PA contents in citrus calli as well as anthocyanin and PA contents in Arabidopsis leaves and seeds. CitTRL interacts with the two basic helix–loop–helix (bHLH) proteins CitbHLH1 and ANTHOCYANIN 1 (CitAN1) to regulate fruit quality. We show that CitTRL competes with the R2R3-MYB CitRuby1 for binding to CitbHLH1 or CitAN1, thereby repressing their activation of anthocyanin structural genes. CitTRL also competes with a second R2R3-MYB, CitPH4, for binding to CitAN1, thus altering the expression of the vacuolar proton-pump gene PH5 and Leucoanthocyanidin reductase, responsible for vacuolar acidification and proanthocyanidins biosynthesis, respectively. Moreover, CitPH4 activates CitTRL transcription, thus forming an activator–repressor loop to prevent the overaccumulation of citric acid and PAs. Overall, this study demonstrates that CitTRL acts as a repressor of the accumulation of citric acid, PAs, and anthocyanins by a cross-regulation mechanism. Our results provide an opportunity to simultaneously manipulate these key traits as a means to produce citrus fruits that are both visually and organoleptically appealing.