Uncovering Hierarchical Regulation among MYB-bHLH-WD40 Proteins and Manipulating Anthocyanin Pigmentation in Rice

Anthocyanins accumulate in various organs of rice, and the regulatory genes involved in pigmentation of specific organs, such as pericarp, hull, leaf, apiculus, and stigma have been elucidated. However, the corresponding gene for rice culm pigmentation has not been clarified. The well-known MYB-bHLH-WD40 (MBW) complex plays vital role in regulating the anthocyanin biosynthesis pathway in plants. However, the core members of MBW and the hierarchical regulation between these members are not fully elucidated in rice. Here, by map-based cloning, we identified the culm-specific pigmentation gene S1 whose alleles are also known for hull/pericarp pigmentation. We also clarified that one WD40 protein encoding gene, WA1, is indispensable for anthocyanin biosynthesis in rice. In the cascading regulation among MBW members, S1 (bHLH) acts as the master gene by activating the expression of C1 (MYB), and then C1 activates the expression of WA1 (WD40), which is unique in plant species. This enables MBW members to be coordinated in a common way to efficiently regulate anthocyanin biosynthesis genes. Based on these studies, we explored the minimal gene set required for anthocyanin biosynthesis in rice. These findings will help us design new rice varieties with anthocyanin accumulation in specific organs as needed.     

Overexpression of MdZAT5, an C2H2-Type Zinc Finger Protein,Regulates Anthocyanin Accumulation and Salt Stress Response in Apple Calli and Arabidopsis

Zinc finger proteins are widely involved and play an important role in plant growth and abiotic stress. In this research, MdZAT5, a gene encoding C2H2-type zinc finger protein, was cloned and investigated. The MdZAT5 was highly expressed in flower tissues by qRT-PCR analyses and GUS staining. Promoter analysis showed that MdZAT5 contained multiple response elements, and the expression levels of MdZAT5 were induced by various abiotic stress treatments. Overexpression of MdZAT5 in apple calli positively regulated anthocyanin accumulation by activating the expressions of anthocyanin biosynthesis-related genes. Overexpression of MdZAT5 in Arabidopsis also enhanced the accumulation of anthocyanin. In addition, MdZAT5 increased the sensitivity to salt stress in apple calli. Ectopic expression of MdZAT5 in Arabidopsis reduced the expression of salt-stress-related genes (AtNHX1 and AtABI1) and improved the sensitivity to salt stress. In conclusion, these results suggest that MdZAT5 plays a positive regulatory role in anthocyanin accumulation and negatively regulates salt resistance.     

Ethanol shock enhances the recovery of anthocyanin from lowbush blueberry

Deactivation of polyphenol oxidase (PPO) in natural products is essential for downstream processing of functional molecules used as food or food additives, particularly those served as antioxidants. In the present work, we identified two proteins with PPO activity from lowbush blueberry using ammonium sulphate precipitation and chromatography procedures. Deactivation of these proteins was studied using aqueous solutions of ethanol of different concentrations. The PPO activity was recovered after ethanol removal for the protein samples previously soaked in a low concentration ethanol solution. A complete and unrecoverable deactivation of the proteins was achieved using ethanol with concentration over 70% (v/v), as manifested by the significant changes in circular dichroism (CD) and fluorescence spectroscopy measurements. Based on these findings, we propose a new extraction process for blueberry anthocyanin, in which an ethanol shock, i.e. soaking blueberry fruit in a 70% (v/v) ethanol solution for 1 h, is implemented before subsequent procedures. This new process increases the anthocyanin yield by 55% in comparison to that without the ethanol shock.     

Identification of Anthocyanins-Related Glutathione S-Transferase (GST) Genes in the Genome of Cultivated Strawberry (Fragaria × ananassa)

Anthocyanins are responsible for the red color of strawberry, they are a subclass of flavonoids synthesized in cytosol and transferred to vacuole to form the visible color. Previous studies in model and ornamental plants indicated members of the glutathione S-transferase (GST) gene family were involved in vacuolar accumulation of anthocyanins. In the present study, a total of 130 FaGST genes were identified in the genome of cultivated strawberry (Fragaria × ananassa), which were unevenly distributed across the 28 chromosomes from the four subgenomes. Evolutionary analysis revealed the expansion of FaGST family was under stable selection and mainly drove by WGD/segmental duplication event. Classification and phylogenetic analysis indicated that all the FaGST genes were clarified into seven subclasses, among which FaGST1, FaGST37, and FaGST97 belonging to Phi class were closely related to FvRAP, an anthocyanin-related GST of wildwood strawberry, and this clade was clustered with other known anthocyanin-related GSTs. RNAseq-based expression analysis at different developmental stages of strawberry revealed that the expression of FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 was gradually increased during the fruit ripening, consistent with the anthocyanins accumulation. These expression patterns of those five FaGST genes were also significantly correlated with those of other anthocyanin biosynthetic genes such as FaCHI, FaCHS, and FaANS, as well as anthocyanin regulatory gene FaMYB10. These results indicated FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 may function in vacuolar anthocyanin accumulation in cultivated strawberry.     

Changes in the Content of Organic Acids and Expression Analysis of Citric Acid Accumulation-Related Genes during Fruit Development of Yellow (Passiflora edulis f. flavicarpa) and Purple (Passiflora edulis f. edulis) Passion Fruits

Organic acids are key components that determine the taste and flavor of fruits and play a vital role in maintaining fruit quality and nutritive value. In this study, the fruits of two cultivars of passion fruit Yellow (Passiflora edulis f. flavicarpa) and purple (Passiflora edulis f. edulis) were harvested at five different developmental stages (i.e., fruitlet, green, veraison, near-mature and mature stage) from an orchard located in subtropical region of Fujian Province, China. The contents of six organic acids were quantified using ultra-performance liquid chromatography (UPLC), activities of citric acid related enzymes were determined, and expression levels of genes involved in citric acid metabolism were measured by quantitative real-time PCR (qRT-PCR). The results revealed that citric acid was the predominant organic acid in both cultivars during fruit development. The highest citric acid contents were observed in both cultivars at green stage, which were reduced with fruit maturity. Correlation analysis showed that citrate synthase (CS), cytosolic aconitase (Cyt-ACO) and cytosolic isocitrate dehydrogenase (Cyt-IDH) may be involved in regulating citric acid biosynthesis. Meanwhile, the PeCS2, PeACO4, PeACO5 and PeIDH1 genes may play an important role in regulating the accumulation of citric acid. This study provides new insights for future elucidation of key mechanisms regulating organic acid biosynthesis in passion fruit.