An R2R3‐MYB transcription factor represses the transformation of α‐ and β‐branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate

Abstract

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.

ZmPBF and ZmGAMYB transcription factors independently transactivate the promoter of the maize (Zea mays) β‐carotene hydroxylase 2 gene

“…Very little is known about the regulation of these genes in cereals, or indeed in other plants. A few transcription factors have been shown to influence carotenogenic genes in dicots, including Arabidopsis RAP2.2 and PIF1 (Welsch et al ., ; Toledo‐Ortiz et al ., ), tomato ( Solanum lycopersicum ) RIN and BBX20 (Martel et al ., ; Xiong et al ., ), and citrus CrMYB68 and CsMADS6 (Zhu et al ., ; Lu et al ., ). RAP2.2, a member of the AP2 gene family, binds to the Arabidopsis PSY promoter and represses PSY and PDS (encoding phytoene desaturase) in root‐derived callus, thus reducing the carotenoid content (Welsch et al ., ).…”

A kiwifruit (Actinidia deliciosa) R2R3‐MYB transcription factor modulates chlorophyll and carotenoid accumulation

“…Homologues of AtMYB78 have been described as playing a role during response to heat and drought stress in soybean and sorghum (Pereira et al, 2011;Johnson et al, 2014). In the phylogeny we also included the two recently published MYBs implicated in carotenoid pathway regulation, ElRCP1 and CrMYB68 (Sagawa et al, 2016;Zhu et al, 2017). These fall into separate clades and do not cluster either together or near AdMYB7.…”

“…The second recent paper to describe a role for MYBs in carotenoid accumulation presents a very different mode of action. From studies using a Citrus reticulata stay-green mutant, Green Ougan (MT), and the revertant, de-greening wild-type-like Green Ougan (MT-WT), the authors were able to identify an MYB TF, CrMYB68, which appeared to be negatively correlated with the activity of carotenoid pathway genes b-carotene hydroxylase 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) (Zhu et al, 2017). These two papers, while offering very different roles for MYBs, show that MYB TFs are involved in carotenoid regulation.…”

Transcriptome analysis provides new insight into the molecular mechanisms in regulating carotenoid accumulation in pericarp of fruits from two pummelo cultivars Citrus grandis (L.) Osbeck

“…Reduced expression of Carotenoid Pigmentation 1 (an R2R3-MYB transcription factor) was associated with improved carotenoid biosynthesis in Mimulus lewisii [83]. Another R2R3-MYB transcriptional factor (CrMYB68) in a staygreen mutant of Citrus reticulata cv Suavissima directly and negatively regulated CrBCH2 and 9-cisepoxycarotenoid dioxygenase 5, as a consequence, the transformation of α-carotene and β-cartoene and the ABA biosynthesis were delayed [84]. In this study, one MYB44 transcription factor and two MYB related proteins were distinctly down regulated in 'Huangbao' in comparison with 'Guanxi'.…”