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Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.).

ABSTRACT: Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis-elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC8708680 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionBlueberry (Vaccinium corymbosum L.) is a popular fruit with an abundance of anthocyanins in its leaves and fruits. Light is one of the pivotal environmental elements that affects plant growth and development, but the regulatory mechanism between light quality and anthocyanin formation is poorly understood.MethodsAn integrated transcriptome and metabolome analysis was performed to investigate the effects of white (control), blue (B), red (R), and red/blue (60R/40B) light on blueberry growth and reveal the potential pathway controlling anthocyanin biosynthesis in blueberry leaves.ResultsThe anthocyanin content was significantly improved by the blue and red/blue light when compared with white light, whereas there was a significant reduction in the photosynthesis under the blue light, showing an inverse trend to that of anthocyanin accumulation. Transcriptomic analysis resulted in the assembly of 134,709 unigenes. Of these, 22 were differentially expressed genes (DEGs) that participate in the anthocyanin biosynthesis pathway, with the majority being significantly up-regulated under the blue light. Most of the photosynthesis-related genes that were down-regulated were expressed during anthocyanin accumulation. Targeted metabolome profiling identified 44 metabolites associated with anthocyanin biosynthesis. The contents of most of these metabolites were higher under blue light than the other light conditions, which was consistent with the transcriptome results. The integrated transcriptome and metabolome analysis suggested that, under blue light, leucoanthocyanidin dioxygenase (LDOX), O-methyltransferase (OMT), and UDP-glucose flavonoid glucosyltransferase (UFGT) were the most significantly expressed, and they promoted the synthesis of cyanidin (Cy), malvidin (Mv), and pelargonidin (Pg) anthocyanidins, respectively. The expression levels of dihydroflavonol 4-reductase (DFR) and OMT, as well as the accumulation of delphinidin (Dp), peonidin (Pn), and petunidin (Pt), were significantly increased by the red/blue light.DiscussionThe blue and red/blue lights promoted anthocyanin biosynthesis via inducing the expression of key structural genes and accumulation of metabolites involved in anthocyanin synthesis pathway. Moreover, there was a possible feedback regulating correlation between anthocyanin biosynthesis and photosynthesis under different light qualities in blueberry leaves. This study would provide a theoretical basis for elucidating the underlying regulatory mechanism of anthocyanin biosynthesis of V. corymbosum.

Project description:BackgroundTo investigate hypoglycemic activity and elucidate the active composition of the fruit blueberry (Vaccinium corymbosum).MethodsMethanol extracts of blueberry (MEB) were separated using a D101 macroporous resin column to yield quinic acid derivative (Fr.1)- and flavonoid (Fr.2)-rich fractions. The effects of the blueberry extracts on mRNA expression of GLUT-2 (glucose transporter type 2) and PPARγ (peroxisome proliferator-activated receptor-γ), as well as on the activities of PPRE (peroxisome proliferator response element) and NF-κB were analyzed in LO2 normal liver cells. Real-time PCR was used to detect the expression of GLUT-2, PPARγ, TNF-α, IL-1β, and IL-6 mRNA. The PPRE and NF-κB activities were detected by a luciferase reporter assay. Western blotting was used to detect the levels of PPARγ, GLUT-2, and p65. The active compositions were isolated using various chromatography columns, and were analyzed by NMR.ResultsmRNA and protein expression of GLUT-2 and PPARγ were significantly increased upon treatment with 400 μg/mL extracts of blueberry (P<0.05). The PPRE activity was also significantly increased in a dose-dependent manner upon administration of MEB (P<0.05). Furthermore, the NF-κB activity induced by lipopolysaccharides was inhibited by MEB (P<0.05). No fraction separated from MEB exhibited PPRE activation or NF-κB inhibition activity. Blueberry extract may execute its hypoglycemic activity by stimulating expression of GLUT-2 and PPARγ, and by inhibiting the inflammatory pathway. Together, quinic acid derivatives and flavonoids may result in a synergistic effect. Fourteen phenolic acids, including eight flavonoids, four quinic acid derivatives, and two other phenolic acids, were isolated and identified, and caffeoylquinic acid derivatives and quercetin glycosides were found to be the major constituents of blueberry.ConclusionBlueberry may have hypoglycemic activity that functions through synergistic effects with caffeoylquinic acid derivatives and quercetin glycosides.

Project description:BackgroundPropagation of cuttings is frequently used in various plant species, including blueberry, which shows special root characteristics that may hinder adventitious root (AR) formation. AR formation is influenced by various factors, and auxin is considered to play a central role; however, little is known of the related regulatory mechanisms. In this study, a comparative transcriptome analysis of green cuttings treated with or without indole-butyric acid (IBA) was performed via RNA_seq to identify candidate genes associated with IBA-induced AR formation.ResultsRooting phenotypes, especially the rooting rate, were significantly promoted by exogenous auxin in the IBA application. Blueberry AR formation was an auxin-induced process, during which adventitious root primordium initiation (rpi) began at 14 days after cutting (DAC), root primordium (rp) was developed at 21 DAC, mature AR was observed at 28 DAC and finally outgrowth from the stem occurred at 35 DAC. Higher IAA levels and lower ABA and zeatin contents might facilitate AR formation and development. A time series transcriptome analysis identified 14,970 differentially expressed genes (DEGs) during AR formation, of which there were 7467 upregulated and 7503 downregulated genes. Of these, approximately 35 candidate DEGs involved in the auxin-induced pathway and AR formation were further identified, including 10 auxin respective genes (ARFs and SAURs), 13 transcription factors (LOB domain-containing protein (LBDs)), 6 auxin transporters (AUX22, LAX3/5 and PIN-like 6 (PIL6s)) and 6 rooting-associated genes (root meristem growth factor 9 (RGF9), lateral root primordium 1 (LRP1s), and dormancy-associated protein homologue 3 (DRMH3)). All these identified DEGs were highly upregulated in certain stages during AR formation, indicating their potential roles in blueberry AR formation.ConclusionsThe transcriptome profiling results indicated candidate genes or major regulatory factors that influence adventitious root formation in blueberry and provided a comprehensive understanding of the rooting mechanism underlying the auxin-induced AR formation from blueberry green cuttings.

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Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.).

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