Exogenous Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression in Blueberry Leaves

Blueberry (Vaccinium corymbosum) leaves have a positive influence on health because of their phenolic contents, including anthocyanins. Phytohormone abscisic acid (ABA) promotes anthocyanin accumulation, but the underlying mechanisms are unclear in blueberry leaves. In this study, we found that exogenous ABA promotes anthocyanin accumulation in blueberry leaves and we explored the global molecular events involved in these physiological changes by treating in vitro-grown blueberry seedlings with ABA and performing transcriptome deep sequencing (RNA-seq). We identified 6390 differentially expressed genes (DEGs), with 2893 DEGs at 6 h and 4789 at 12 h of ABA treatment compared to the control. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis were significantly enriched at both stages of the ABA treatment. Analysis of DEGs in plant hormone signal transduction pathways revealed that exogenous ABA affected the expression of genes from other plant hormone signaling pathways, especially brassinosteroid, auxin, and gibberellin signaling. To elucidate the mechanism driving anthocyanin biosynthesis in blueberry in response to ABA treatment, we screened anthocyanin biosynthesis structural genes (ASG) from the phenylpropanoid and flavonoid biosynthetic pathways, MYB transcription factor genes from R2R3-MYB subgroups 5, 6, and 7 and ABRE-binding factor (ABF) genes from the ABA signal transduction pathway. Pearson’s correlation coefficient (r) analysis indicated that the ABFs, MYBs, and structural genes form a network to regulate ABA-induced anthocyanin biosynthesis and MYBA1 is likely to play an important role in this regulatory network. These findings lay the foundation for improving anthocyanin biosynthesis in blueberry leaves

Molecular cloning and functional analysis of a flavanone 3-hydroxylase gene from blueberry

<p><i>Vaccinium corymbosum</i> (blueberry) is touted as a superfood with numerous health benefits due to its high levels of flavonoids. Flavanone 3-hydroxylase (F3H) is a key regulatory enzyme of the flavonoid pathway. In this study, we cloned the full-length cDNA of <i>F3H</i> (designated <i>VcF3H</i>) from young blueberry leaves using rapid amplification of cDNA ends (RACE). The cDNA contained a 1080-bp open reading frame that encoded a 359-amino acid protein. The deduced VcF3H protein showed high similarities to other plant F3Hs. Conserved amino acid motifs required for ferrous iron binding (HXD) and 2-oxoglutarate binding (RXS) were identified in VcF3H, VcFLS (flavonol synthase), and VcANS (anthocyanidin synthase). Quantitative RT-PCR analysis demonstrated that <i>VcF3H</i> was expressed in all tissues tested, with particularly high expression in young leaves, fruits (pink and blue), and stems. Anthocyanins accumulated mainly in fruits, whereas flavonols were found mainly in leaves and stems. Furthermore, the expression pattern of <i>VcF3H</i> was similar to that of <i>VcCHS</i>, <i>VcDFR</i>, and <i>VcANS</i> in various tissues. Heterologous expression of <i>VcF3H</i> in <i>Arabidopsis thaliana</i> increased the anthocyanin content in leaves, but did not affect the flavonol content. Thus, <i>VcF3H</i> seems to be involved in anthocyanin synthesis in the flavonoid biosynthetic pathway when ectopically expressed in <i>Arabidopsis</i>.</p

Molecular Epidemiology and Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Cervids from Milu Park in Beijing, China

Enterocytozoon bieneusi is the most prevalent microsporidian species that can cause zoonotic diseases in humans and animals. Despite receiving increasing attention in relation to domestic animals, there has been limited information on the infection burden of E. bieneusi in cervids. Altogether, 215 fecal samples collected from four deer species in Beijing, China were examined by nested- Polymerase Chain Reaction (PCR)targeting the internal transcribed spacer (ITS) region. The overall prevalence of E. bieneusi in deer was 21.9% (47/215), with 30.0% (24/80) in Pere David’s deer, 27.3% (15/55) in fallow deer, 12.5% (5/40) in sika deer, and 7.5% (3/40) in Chinese water deer. Thirteen E. bieneusi genotypes were identified, including six known (HLJD-V, MWC_d1, BEB6, CGC2, JLD-XV, and HND-I) and seven novel genotypes (BJED-I to BJED-V, BJFD, and BJCWD). A phylogenetic analysis showed that 38.3% of the isolates belonged to zoonotic Group 1. In addition, E. bieneusi infection was first detected in fallow deer and Chinese water deer, which could act as potential zoonotic reservoirs. Our findings suggest that E. bieneusi circulates in deer and might be of importance to public health

Image_1_MYB pathways that regulate UV-B-induced anthocyanin biosynthesis in blueberry (Vaccinium corymbosum).jpeg

Ultraviolet-B (UV-B) promotes anthocyanin accumulation and improves fruit quality in plants. To explore the underlying network of MYB transcription factors that regulates UV-B-induced anthocyanin biosynthesis in blueberry (Vaccinium corymbosum), we analyzed the response of MYB transcription factor genes to UV-B treatment. Transcriptome sequencing analysis revealed that VcMYBA2 and VcMYB114 expression were upregulated and were positively correlated with the expression of anthocyanin structural genes under UV-B radiation according to weighted gene co-expression network analysis (WGCNA) data. The VcUVR8-VcCOP1-VcHY5 pathway perceives UV-B signals and promotes the expression of anthocyanin structural genes by upregulating VcMYBA2 and VcMYB114 or by regulating the VcBBXs-VcMYB pathway, ultimately promoting anthocyanin accumulation. By contrast, VcMYB4a and VcUSP1 were downregulated under UV-B treatment, and VcMYB4a expression was negatively correlated with that of anthocyanin biosynthesis genes in response to UV-B. Analysis of VcMYB4a-overexpressing and wild-type blueberry calli exposed to UV-B radiation revealed that VcMYB4a represses UV-B-induced anthocyanin accumulation. Yeast one-hybrid and dual luciferase assays showed that the universal stress protein VcUSP1 directly bound to the promoter of VcMYB4a. These results suggest that the VcUSP1-VcMYB4a pathway negatively regulates UV-B-induced anthocyanin biosynthesis and provide insight into UV-B-induced anthocyanin biosynthesis.</p

DataSheet_7_UV-B induces the expression of flavonoid biosynthetic pathways in blueberry (Vaccinium corymbosum) calli.xls

Ultraviolet-B (UV-B) radiation is an environmental signal that affects the accumulation of secondary metabolites in plants. In particular, UV-B promotes flavonoid biosynthesis, leading to improved fruit quality. To explore the underlying molecular mechanism, we exposed blueberry (Vaccinium corymbosum) calli to UV-B radiation and performed a transcriptome deep sequencing (RNA-seq) analysis to identify differentially expressed genes (DEGs). We detected 16,899 DEGs among different treatments, with the largest number seen after 24 h of UV-B exposure relative to controls. Functional annotation and enrichment analysis showed a significant enrichment for DEGs in pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis. In agreement with the transcriptome data, flavonol, anthocyanin and proanthocyanidin accumulated upon UV-B radiation, and most DEGs mapping to the phenylpropanoid and flavonoid biosynthetic pathways using the KEGG mapper tool were upregulated under UV-B radiation. We also performed a weighted gene co-expression network analysis (WGCNA) to explore the relationship among genes involved in plant hormone signal transduction, encoding transcription factors or participating in flavonoid biosynthesis. The transcription factors VcMYBPA1, MYBPA2.1, MYB114, MYBA2, MYBF, and MYB102 are likely activators, whereas MYB20, VcMYB14, MYB44, and VcMYB4a are inhibitors of the flavonoid biosynthetic pathway, as evidenced by the direction of correlation between the expression of these MYBs and flavonoid biosynthesis-related genes. The transcription factors bHLH74 and bHLH25 might interact with MYB repressors or directly inhibited the expression of flavonoid biosynthetic genes to control flavonoid accumulation. We also observed the downregulation of several genes belonging to the auxin, gibberellin and brassinosteroid biosynthetic pathways, suggesting that MYB inhibitors or activators are directly or indirectly regulated to promote flavonoid biosynthesis under UV-B radiation.</p

Table_1_MYB pathways that regulate UV-B-induced anthocyanin biosynthesis in blueberry (Vaccinium corymbosum).xls

Ultraviolet-B (UV-B) promotes anthocyanin accumulation and improves fruit quality in plants. To explore the underlying network of MYB transcription factors that regulates UV-B-induced anthocyanin biosynthesis in blueberry (Vaccinium corymbosum), we analyzed the response of MYB transcription factor genes to UV-B treatment. Transcriptome sequencing analysis revealed that VcMYBA2 and VcMYB114 expression were upregulated and were positively correlated with the expression of anthocyanin structural genes under UV-B radiation according to weighted gene co-expression network analysis (WGCNA) data. The VcUVR8-VcCOP1-VcHY5 pathway perceives UV-B signals and promotes the expression of anthocyanin structural genes by upregulating VcMYBA2 and VcMYB114 or by regulating the VcBBXs-VcMYB pathway, ultimately promoting anthocyanin accumulation. By contrast, VcMYB4a and VcUSP1 were downregulated under UV-B treatment, and VcMYB4a expression was negatively correlated with that of anthocyanin biosynthesis genes in response to UV-B. Analysis of VcMYB4a-overexpressing and wild-type blueberry calli exposed to UV-B radiation revealed that VcMYB4a represses UV-B-induced anthocyanin accumulation. Yeast one-hybrid and dual luciferase assays showed that the universal stress protein VcUSP1 directly bound to the promoter of VcMYB4a. These results suggest that the VcUSP1-VcMYB4a pathway negatively regulates UV-B-induced anthocyanin biosynthesis and provide insight into UV-B-induced anthocyanin biosynthesis.</p

DataSheet_4_UV-B induces the expression of flavonoid biosynthetic pathways in blueberry (Vaccinium corymbosum) calli.pdf

Ultraviolet-B (UV-B) radiation is an environmental signal that affects the accumulation of secondary metabolites in plants. In particular, UV-B promotes flavonoid biosynthesis, leading to improved fruit quality. To explore the underlying molecular mechanism, we exposed blueberry (Vaccinium corymbosum) calli to UV-B radiation and performed a transcriptome deep sequencing (RNA-seq) analysis to identify differentially expressed genes (DEGs). We detected 16,899 DEGs among different treatments, with the largest number seen after 24 h of UV-B exposure relative to controls. Functional annotation and enrichment analysis showed a significant enrichment for DEGs in pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis. In agreement with the transcriptome data, flavonol, anthocyanin and proanthocyanidin accumulated upon UV-B radiation, and most DEGs mapping to the phenylpropanoid and flavonoid biosynthetic pathways using the KEGG mapper tool were upregulated under UV-B radiation. We also performed a weighted gene co-expression network analysis (WGCNA) to explore the relationship among genes involved in plant hormone signal transduction, encoding transcription factors or participating in flavonoid biosynthesis. The transcription factors VcMYBPA1, MYBPA2.1, MYB114, MYBA2, MYBF, and MYB102 are likely activators, whereas MYB20, VcMYB14, MYB44, and VcMYB4a are inhibitors of the flavonoid biosynthetic pathway, as evidenced by the direction of correlation between the expression of these MYBs and flavonoid biosynthesis-related genes. The transcription factors bHLH74 and bHLH25 might interact with MYB repressors or directly inhibited the expression of flavonoid biosynthetic genes to control flavonoid accumulation. We also observed the downregulation of several genes belonging to the auxin, gibberellin and brassinosteroid biosynthetic pathways, suggesting that MYB inhibitors or activators are directly or indirectly regulated to promote flavonoid biosynthesis under UV-B radiation.</p

DataSheet_5_UV-B induces the expression of flavonoid biosynthetic pathways in blueberry (Vaccinium corymbosum) calli.pdf

Ultraviolet-B (UV-B) radiation is an environmental signal that affects the accumulation of secondary metabolites in plants. In particular, UV-B promotes flavonoid biosynthesis, leading to improved fruit quality. To explore the underlying molecular mechanism, we exposed blueberry (Vaccinium corymbosum) calli to UV-B radiation and performed a transcriptome deep sequencing (RNA-seq) analysis to identify differentially expressed genes (DEGs). We detected 16,899 DEGs among different treatments, with the largest number seen after 24 h of UV-B exposure relative to controls. Functional annotation and enrichment analysis showed a significant enrichment for DEGs in pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis. In agreement with the transcriptome data, flavonol, anthocyanin and proanthocyanidin accumulated upon UV-B radiation, and most DEGs mapping to the phenylpropanoid and flavonoid biosynthetic pathways using the KEGG mapper tool were upregulated under UV-B radiation. We also performed a weighted gene co-expression network analysis (WGCNA) to explore the relationship among genes involved in plant hormone signal transduction, encoding transcription factors or participating in flavonoid biosynthesis. The transcription factors VcMYBPA1, MYBPA2.1, MYB114, MYBA2, MYBF, and MYB102 are likely activators, whereas MYB20, VcMYB14, MYB44, and VcMYB4a are inhibitors of the flavonoid biosynthetic pathway, as evidenced by the direction of correlation between the expression of these MYBs and flavonoid biosynthesis-related genes. The transcription factors bHLH74 and bHLH25 might interact with MYB repressors or directly inhibited the expression of flavonoid biosynthetic genes to control flavonoid accumulation. We also observed the downregulation of several genes belonging to the auxin, gibberellin and brassinosteroid biosynthetic pathways, suggesting that MYB inhibitors or activators are directly or indirectly regulated to promote flavonoid biosynthesis under UV-B radiation.</p

DataSheet_3_UV-B induces the expression of flavonoid biosynthetic pathways in blueberry (Vaccinium corymbosum) calli.pdf

Ultraviolet-B (UV-B) radiation is an environmental signal that affects the accumulation of secondary metabolites in plants. In particular, UV-B promotes flavonoid biosynthesis, leading to improved fruit quality. To explore the underlying molecular mechanism, we exposed blueberry (Vaccinium corymbosum) calli to UV-B radiation and performed a transcriptome deep sequencing (RNA-seq) analysis to identify differentially expressed genes (DEGs). We detected 16,899 DEGs among different treatments, with the largest number seen after 24 h of UV-B exposure relative to controls. Functional annotation and enrichment analysis showed a significant enrichment for DEGs in pathways related to plant hormone signal transduction and phenylpropanoid and flavonoid biosynthesis. In agreement with the transcriptome data, flavonol, anthocyanin and proanthocyanidin accumulated upon UV-B radiation, and most DEGs mapping to the phenylpropanoid and flavonoid biosynthetic pathways using the KEGG mapper tool were upregulated under UV-B radiation. We also performed a weighted gene co-expression network analysis (WGCNA) to explore the relationship among genes involved in plant hormone signal transduction, encoding transcription factors or participating in flavonoid biosynthesis. The transcription factors VcMYBPA1, MYBPA2.1, MYB114, MYBA2, MYBF, and MYB102 are likely activators, whereas MYB20, VcMYB14, MYB44, and VcMYB4a are inhibitors of the flavonoid biosynthetic pathway, as evidenced by the direction of correlation between the expression of these MYBs and flavonoid biosynthesis-related genes. The transcription factors bHLH74 and bHLH25 might interact with MYB repressors or directly inhibited the expression of flavonoid biosynthetic genes to control flavonoid accumulation. We also observed the downregulation of several genes belonging to the auxin, gibberellin and brassinosteroid biosynthetic pathways, suggesting that MYB inhibitors or activators are directly or indirectly regulated to promote flavonoid biosynthesis under UV-B radiation.</p