Transcription Factor VviMYB86 Oppositely Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Grape Berries

Proanthocyanidins (PAs) and anthocyanins are two vital groups of flavonoid compounds for grape berries and red wines. Several transcription factors (TFs) have been identified to be involved in regulating PA and anthocyanin biosynthesis in grape berries. However, research on TFs with different regulatory mechanisms for these two biosynthesis branches in grapes remains limited. In this study, we identified an R2R3-MYB TF, VviMYB86, whose spatiotemporal gene expression pattern in grape berries coincided well with PA accumulation but contrasted with anthocyanin synthesis. Both in vivo and in vitro experiments verified that VviMYB86 positively regulated PA biosynthesis, primarily by upregulating the expression of the two leucoanthocyanidin reductase (LAR) genes in the Arabidopsis protoplast system, as well as in VviMYB86-overexpressing grape callus cultured under 24 h of darkness. Moreover, VviMYB86 was observed to repress the anthocyanin biosynthesis branch in grapes by downregulating the transcript levels of VviANS and VviUFGT. Overall, VviMYB86 is indicated to have a broad effect on flavonoid synthesis in grape berries. The results of this study will help elucidate the regulatory mechanism governing the expression of the two LAR genes in grape berries and provide new insights into the regulation of PA and anthocyanin biosynthesis in grape berries.

VviWRKY40, a WRKY Transcription Factor, Regulates Glycosylated Monoterpenoid Production by VviGT14 in Grape Berry

Glycosylated volatile precursors are important, particularly in wine grape berries, as they contribute to the final aroma in wines by releasing volatile aglycones during yeast fermentation and wine storage. Previous study demonstrated that VviGT14 was functioned as a critical monoterpene glucosyltransferase in grape berry, while the transcriptional regulation mechanism of VviGT14 was still unknown. Here we identified VviWRKY40 as a binding factor of VviGT14 promoter by both DNA pull-down and yeast one-hybrid screening, followed by a series of in vitro verification. VviWRKY40 expression pattern negatively correlated with that of VviGT14 in grape berries. And the suppressor role of VviWRKY40 was further confirmed by using the dual luciferase assay with Arabidopsis protoplast and grape cell suspension system. Furthermore, the grape suspension cell ABA treatment study showed that ABA downregulated VviWRKY40 transcript level but promoted that of VviGT14, indicating that VviWRKY40 was at the downstream of ABA signal transduction network to regulate monoterpenoid glycosylation. These data extend our knowledge of transcriptional regulation of VviGT14, and provide new targets for grape breeding to alter monoterpenoid composition.

ACTIN7 Is Required for Perinuclear Clustering of Chloroplasts during Arabidopsis Protoplast Culture

In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed ACTIN (ACT) genes. Of the eight expressed isoforms, ACT2, ACT7, and ACT8 are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in act7 mutants, but not act2-1 or act8-2. ACT7 expression was upregulated during protoplast culture whereas ACT2 and ACT8 expression did not substantially change. In act2-1, ACT7 expression increased in response to loss of ACT2, whereas in act7-1, neither ACT2 nor ACT8 expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in act7-1 was only two-thirds that of wild-type or act2-1 after 96 h culture. Over-expression of ACT2 and ACT8 under control of ACT7 regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in act7 mutants, rather than strong functional specialization of the ACT7 isoform.

Combining a Simple Method for DNA/RNA/Protein Co-Purification and Arabidopsis Protoplast Assay to Facilitate Viroid Research

Plant–viroid interactions represent a valuable model for delineating structure–function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.