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 0 cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C 30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing b-citraurin, CCD4b is also responsible for the production of another important C 30 apocarotenoid pigment, b-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 0 cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C 30 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.
Carotenoids are indispensable for both human health and plant survival. Citrus, known for being rich in carotenoids, is a vital source of dietary carotenoids in the human diet. Ethylene can accelerate carotenoid accumulation in citrus, but the underlying transcriptional regulation mechanisms remain unclear. 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, but its regulatory metabolism is largely unknown. Here a group I ethylene response factor CsERF061 was identified 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. Additionally, overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, increased the number of chromoplasts needed to sequester the elevated levels of carotenoids, and also accompanied by the alteration of abscisic acid and gibberellin contents. Electrophoretic mobility shift assay and dual-luciferase assay verified that CsERF061 activates the promoters of the other nine key carotenoid pathway genes, including 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 is useful for manipulating the carotenoid accumulation in citrus, as well as other plants.
SUMMARY 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 (H2O2), 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 H2O2 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.
Carotenoids are natural pigments that influence the color of citrus fruit. The red-colored carotenoid β-citraurin is responsible for the peel color in ‘Newhall’ orange (Citrus sinensis). Although jasmonates are known to regulate the biosynthesis and accumulation of carotenoids, their effects on β-citraurin biosynthesis in citrus fruit remain unclear. Here, we determined that treatment with methyl jasmonate (MeJA) significantly promotes fruit coloration and β-citraurin production in ‘Newhall’ orange. A MeJA treatment induced the expression of CsMYC2, which encodes a transcription factor that serves as a master regulator of jasmonate responses. CsMYC2 bound the promoter of the gene that encodes carotenoid cleavage dioxygenase 4b (CsCCD4b), the key gene for β-citraurin biosynthesis, and the promoters of genes that encode phytoene synthase (CsPSY), lycopene β-cyclase (CsLCYb), and β-carotene hydroxylase (CsBCH) and induced their expression. In addition, CsMYC2 promoted CsMPK6 expression. Notably, we found that CsMPK6 interacted with CsMYC2 and that this interaction decreased the stability and DNA-binding activity of CsMYC2. Thus, we conclude that negative feedback regulation attenuates JA signaling during the jasmonate-induced coloration of citrus fruit. Together, our findings indicate that jasmonates induce β-citraurin biosynthesis in citrus by activating a CsMPK6-CsMYC2 cascade, thereby affecting fruit coloration.
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