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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Proanthocyanidins (PAs; or condensed tannins) are a major class of flavonoids that contribute to citrus fruit quality. However, the molecular mechanism responsible for PA biosynthesis and accumulation in citrus remains unclear. Here, we identify a PH4–Noemi regulatory complex that regulates proanthocyanidin biosynthesis in citrus. Overexpression of PH4 or Noemi in citrus calli activated the expression of PA biosynthetic genes and significantly increased the PA content. Interestingly, Noemi was also shown to be up-regulated in CsPH4 -overexpressing lines compared with wild-type calli. Simultaneously, CsPH4 partially complemented the PA-deficient phenotype of the Arabidopsis tt2 mutant and promoted PA accumulation in the wild-type. Further analysis revealed that CsPH4 interacted with Noemi, and together these proteins synergistically activated the expression of PA biosynthetic genes by directly binding to the MYB-recognizing elements (MRE) of the promoters of these genes. Moreover, CsPH4 could directly bind to the promoter of Noemi and up-regulate the expression of this gene. These findings explain how the CsPH4–Noemi regulatory complex contributes to the activation of PA biosynthetic genes via a positive feedback loop and provide new insights into the molecular mechanisms underlying PA biosynthesis, which can be effectively employed for metabolic engineering to improve citrus fruit quality. [ABSTRACT FROM AUTHOR]