Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism    

Abstract

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative realtime reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3#-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3#5#-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.Highbush blueberry (Vaccinium corymbosum; Ericaceae) is one of the most economically important fruit crops in North America. Blueberry fruit have been the focus of much recent attention due to numerous reports of their positive effects on human health. These benefits are generally attributed to high levels of polyphenolics, in particular the flavonoids (Rasmussen et al., 2005). Highbush blueberries have one of the highest in vitro antioxidant capacities of any fruit or vegetable (Prior and Gu, 2005;Wu et al., 2006). The major health benefits linked to the consumption of blueberries include a reduced risk for cardiovascular (Basu et al., 2010) and neurodegenerative (Neto, 2007) diseases. Furthermore, experiments on rodents suggest that blueberry extracts may also prevent cancer, slow tumor growth, and reverse cognitive and behavioral deficits related to stroke and aging (Lau et al., 2005;Gordillo et al., 2009).The three common types of flavonoids that accumulate in blueberry fruit are the flavonols, anthocya-

The MYB182 Protein Down-Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Poplar by Repressing Both Structural and Regulatory Flavonoid Genes

“…We had shown previously that the poplar ANR1 promoter is activated by MYB134 when cotransformed with the poplar bHLH131 cofactor into a transient expression system (Zifkin et al, 2012;Gesell et al, 2014). To show negative regulation by MYB182, a positive regulator such as MYB134 was used in combination with the MYB182 repressor construct, and the reduction in activation was measured.…”

“…Mutations for pMDC32-MYB182 mutant plasmids were performed by site-directed mutagenesis via overlap extension using PCR with mutated primers (Supplemental Table S2). Activator and cofactor plasmids (pMDC32-bHLH131, pMDC32-MYB134, and pMDC32-MYB115) and reporter plasmid pGREEN800LUC-ANR1 promoter constructs were described previously (Zifkin et al, 2012;Gesell et al, 2014;Hellens et al, 2005). The Arabidopsis (Arabidopsis thaliana) DFR promoter (1-kb upstream region) was amplified from Arabidopsis (ecotype Columbia) leaf genomic DNA extracted using the DNeasy Plant Mini Kit (Qiagen).…”

“…MYB134 is an activator of PA biosynthesis and stimulates the poplar ANR1 promoter in transient expression assays in Arabidopsis (Zifkin et al, 2012;Gesell et al, 2014). Transgenic poplar plants overexpressing MYB134 accumulate up to 50 times more PAs in their leaves, yet they have normal anthocyanin levels and only slightly elevated flavonol contents, suggesting that MYB134 regulates PAs specifically (Mellway et al, 2009).…”

Fingerprinting of Vaccinium corymbosum cultivars using DNA of fruits

“…Recently increased production and consumption of blueberries can be attributed to the health benefits ascribed to this fruit related to their rich quantities of polyphenolic compounds, in particular flavonoids. Some researchers consider blueberries as having one of the highest in vitro antioxidant capacities among fresh fruits (Prior, Gu 2005;Zifkin et al 2012).…”

“…Blueberry fruits and leaves are characterized by a high level of polyphenolic compounds (Prior, Gu 2005;Zifkin et al 2012). The composition and content of polyphenolic compounds in blueberries are influenced by the cultivar, the growing season and location (Howard et al 2003;Dragovic-Uzelac et al 2010).…”