Genome-Wide Analysis of the Universal Stress Protein Gene Family in Blueberry and Their Transcriptional Responses to UV-B Irradiation and Abscisic Acid.

Universal stress proteins (USPs) play essential roles in plant development, hormonal regulation, and abiotic stress responses. However, the characteristics and functional divergence of USP family members have not been studied in blueberry (Vaccinium corymbosum). In this study, we identified 72 VcUSP genes from the Genome Database for Vaccinium. These VcUSPs could be divided into five groups based on their phylogenetic relationships. VcUSPs from groups Ⅰ, Ⅳ, and Ⅴ each possess one UspA domain; group Ⅰ proteins also contain an ATP-binding site that is not present in group Ⅳ and Ⅴ proteins. Groups Ⅱ and Ⅲ include more complex proteins possessing one to three UspA domains and UspE or UspF domains. Prediction of cis-regulatory elements in the upstream sequences of VcUSP genes indicated that their protein products are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of RNA deep sequencing data showed that 21 and 7 VcUSP genes were differentially expressed in response to UV-B radiation and exogenous abscisic acid (ABA) treatments, respectively. VcUSP41 and VcUSP68 expressions responded to both treatments, and their encoded proteins may integrate the UV-B and ABA signaling pathways. Weighted gene co-expression network analysis revealed that VcUSP22, VcUSP26, VcUSP67, VcUSP68, and VcUSP41 were co-expressed with many transcription factor genes, most of which encode members of the MYB, WRKY, zinc finger, bHLH, and AP2 families, and may be involved in plant hormone signal transduction, circadian rhythms, the MAPK signaling pathway, and UV-B-induced flavonoid biosynthesis under UV-B and exogenous ABA treatments. Our study provides a useful reference for the further functional analysis of VcUSP genes and blueberry molecular breeding.

Genome-wide analysis of blueberry B-box family genes and identification of members activated by abiotic stress.

BACKGROUND: B-box (BBX) proteins play important roles in regulating plant growth, development, and abiotic stress responses. BBX family genes have been identified and functionally characterized in many plant species, but little is known about the BBX family in blueberry (Vaccinium corymbosum). RESULT: In this study, we identified 23 VcBBX genes from the Genome Database for Vaccinium (GDV). These VcBBXs can be divided into five clades based on gene structures and conserved domains in their encoded proteins. The prediction of cis-acting elements in the upstream sequences of VcBBX genes and protein-protein interactions indicated that VcBBX proteins are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of transcriptome deep sequencing (RNA-seq) data showed that VcBBX genes exhibited organ-specific expression pattern and 11 VcBBX genes respond to ultraviolet B (UV-B) radiation. The co-expression analysis revealed that the encoded 11 VcBBX proteins act as bridges integrating UV-B and phytohormone signaling pathways in blueberry under UV-B radiation. Reverse-transcription quantitative PCR (RT-qPCR) analysis showed that most VcBBX genes respond to drought, salt, and cold stress. Among VcBBX proteins, VcBBX24 is highly expressed in all the organs, not only responds to abiotic stress, but it also interacts with proteins in UV-B and phytohormone signaling pathways, as revealed by computational analysis and co-expression analysis, and might be an important regulator integrating abiotic stress and phytohormone signaling networks. CONCLUSIONS: Twenty-three VcBBX genes were identified in blueberry, in which, 11 VcBBX genes respond to UV-B radiation, and act as bridges integrating UV-B and phytohormone signaling pathways according to RNA-seq data. The expression patterns under abiotic stress suggested that the functional roles of most VcBBX genes respose to drought, salt, and cold stress. Our study provides a useful reference for functional analysis of VcBBX genes and for improving abiotic stress tolerance in blueberry.

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

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.

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

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.

Conservation and Diversity of miR166 Family Members From Highbush Blueberry (Vaccinium corymbosum) and Their Potential Functions in Abiotic Stress.

MicroRNA166 (miR166) is highly conserved and has diverse functions across plant species. The highbush blueberry (Vaccinium corymbosum) genome is thought to harbor 10 miRNA166 loci (Vco-miR166), but the extent of their evolutionary conservation or functional diversification remains unknown. In this study, we identified six additional Vco-miR166 loci based on conserved features of the miR166 family. Phylogenetic analyses showed that mature Vco-miR166s and their precursor cluster in several clades are evolutionary conserved with diverse species. The cis-regulatory elements in the Vco-miR166 promoters indicated functions related to different phytohormones and defense responses. We also identified putative targets of vco-miR166s, which targeted the same gene families, suggesting the functional conservation and diversification of Vco-miR166 family members. Furthermore, we examined the accumulation patterns of six mature Vco-miR166s in response to abiotic stresses by stem-loop reverse RT-qPCR, which revealed their upregulation under freezing, cold, and heat stress, while they were downregulated by drought compared to control growth conditions. However, Vco-miR166 members showed different expression patterns when exposed to salt stress. These results showed that conserved Vco-miR166 family members display functional diversification but also coordinately influence plant responses to abiotic stress.

Arabidopsis CIB3 regulates photoperiodic flowering in an FKF1-dependent way.

Arabidopsis cryptochrome 2 (CRY2) and FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) are blue light receptors mediating light regulation of growth and development, such as photoperiodic flowering. CRY2 interacts with a basic helix-loop-helix transcription factor CIB1 in response to blue light to activate the transcription of the flowering integrator gene FLOWERING LOCUS T (FT). CIB1, CIB2, CIB4, and CIB5 function redundantly to promote flowering in a CRY2-dependent way and form various heterodimers to bind to the noncanonical E-box sequence in the FT promoter. However, the function of CIB3 has not been described. We discovered that CIB3 promotes photoperiodic flowering independently of CRY2. Moreover, CIB3 does not interact with CRY2 but interacts with CIB1 and functions synergistically with CIB1 to promote the transcription of the GI gene. FKF1 is required for CIB3 to promote flowering and enhances the CIB1-CIB3 interaction in response to blue light.