Single-Molecule Real-Time and Illumina Sequencing to Analyze Transcriptional Regulation of Flavonoid Synthesis in Blueberry

Abstract

Blueberries (Vaccinium corymbosum) contain large amounts of flavonoids, which play important roles in the plant’s ability to resist stress and can also have beneficial effects on human health when the fruits are eaten. However, the molecular mechanisms that regulate flavonoid synthesis in blueberries are still unclear. In this study, we combined two different transcriptome sequencing platforms, single-molecule real-time (SMRT) and Illumina sequencing, to elucidate the flavonoid synthetic pathways in blueberries. We analyzed transcript quantity, length, and the number of annotated genes. We mined genes associated with flavonoid synthesis (such as anthocyanins, flavonols, and proanthocyanidins) and employed fluorescence quantitative PCR to analyze the expression of these genes and their correlation with flavonoid synthesis. We discovered one R2R3 MYB transcription factor from the sequencing library, VcMYB1, that can positively regulate anthocyanin synthesis in blueberries. VcMYB1 is mainly expressed in colored (mature) fruits. Experiments showed that overexpression and transient expression of VcMYB1 promoted anthocyanin synthesis in Arabidopsis, tobacco (Nicotiana benthamiana) plants and green blueberry fruits. Yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay, and transient expression experiments showed that VcMYB1 binds to the MYB binding site on the promoter of the structural gene for anthocyanin synthesis, VcMYB1 to positively regulate the transcription of VcDFR, thereby promoting anthocyanin synthesis. We also performed an in-depth investigation of transcriptional regulation of anthocyanin synthesis. This study provides background information and data for studying the synthetic pathways of flavonoids and other secondary metabolites in blueberries.

Genome-wide identification and comprehensive analysis reveal potential roles of long non-coding RNAs in fruit development of southern highbush blueberry (Vaccinium corymbosum L.)

“…The LAR gene, which plays a positive role in proanthocyanin accumulation, had a high expression level at the early stages. Still, its expression was not detected in the ripening stage, which is consistent with previous studies ( Zifkin et al., 2011 ; Colle et al., 2019 ; Tang et al., 2021 ). When the expressions of FLS and LAR were inhibited during fruit ripening, the flavonol and proanthocyanidin syntheses were prevented, while anthocyanin biosynthesis was indirectly promoted.…”

“…We discovered that the accumulation of flavonols was strongly correlated with the flavonol synthase gene FLS and its cis -regulatory lncRNA. Another study ( Tang et al., 2021 ) also found that when blueberries ripened, their flavonol synthesis and FLS expression both dropped. The LAR gene, which plays a positive role in proanthocyanin accumulation, had a high expression level at the early stages.…”

Sucrose non-fermenting1-related protein kinase VcSnRK2.3 promotes anthocyanin biosynthesis in association with VcMYB1 in blueberry

“…Blueberry transcriptome data obtained from Illumina and SMRT sequencing were used to identify the gene family members of SnRK2 in blueberry (Tang et al, 2021). The excavated VcSnRK2 protein sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/ BLAST/) in the National Center for Biotechnology Information (NCBI) database.…”

“…Blueberry fruit injection assays were performed with reference to previous studies (An et al, 2019;Tang et al, 2021). The open reading frame of the SnRK2.3 gene was cloned into the pGreenII62-SK vector to obtain the VcSnRK2.3-pGreenII62-SK vector.…”

“…In apple, MdMYB1 and its alleles play important roles in anthocyanin biosynthesis (Ban et al, 2007;Espley et al, 2007). In recent studies, VcMYB1 was shown to function as a regulator of anthocyanin biosynthesis, and VcMYB1 positively regulates anthocyanin synthesis and accumulation by binding to the VcDFR and VcUFGT anthocyanin biosynthesis genes' promoters (Tang et al, 2021;. In addition, it has been demonstrated that the transcription factors MYBA and MYBPA coregulate anthocyanin biosynthesis in blueberries (Karppinen et al, 2021;Lafferty et al, 2022).…”