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Analysis of VvBES1-1 Involvement in Flower Bud Differentiation of Red Globe Grape Based on Red and Blue Light Regulation TANG XueShen, DANG ShiZhuo, ZHOU Juan, LI JiaHao, LI MeiHua, HU Hao, ZHANG YaHong Scientia Agricultura Sinica    2025, 58 (8): 1650-1662.   DOI: 10.3864/j.issn.0578-1752.2025.08.015 Abstract （56）   HTML （1）    PDF （5271KB）（33）       Knowledge map    Save 【Objective】 BRI1-EMS-Suppressor 1 (BES1), a key transcription factor in brassinosteroid (BR) signaling, regulates plant photomorphogenesis and photoperiodic flowering. This study aimed to investigate the role of BES1 in flower bud differentiation of Vitis vinifera Red Globe under red and blue light regulation, thereby elucidating the BR-mediated mechanisms driven by light quality and providing insights into flowering regulation in other woody fruit trees.【Method】 Bioinformatics analysis was performed to characterize the protein structure and sequence alignment of VvBES1-1. Flower buds of Red Globe grape were collected under greenhouse natural light (CK, control) and red:blue=4:1 (R4B1) light treatment. qPCR was used to analyze the spatiotemporal expression patterns and tissue specificity of VvBES1-1 during flower bud differentiation. Subcellular localization was determined via tobacco (Nicotiana benthamiana) transient transformation. Protein-protein interactions were examined using bimolecular fluorescence complementation (BiFC), and transcriptional activation activity was assessed via yeast autoactivation assays. 【Result】VvBES1-1 contains a BES1_N domain and belongs to the BES1-S-type protein family, showing the closest phylogenetic relationship with Populus trichocarpa BES1. It is expressed throughout all developmental stages of Red Globe grapevines. During R4B1 treatment, the expression level of VvBES1-1 was significantly reduced compared to the control. Its expression peaked during the development of secondary inflorescence axes. Additionally, treatment with 10 mmol·L-¹ EBR enhanced VvBES1-1 expression in Red Globe grape flower buds. Yeast autoactivation assays demonstrated that VvBES1-1 possesses self-activation activity. Subcellular localization analysis revealed that VvBES1-1 is localized in the nucleus. Overexpression of VvBES1-1 in tobacco delayed flowering time, promoted stem elongation, and increased meristem number. Downregulation of VvBES1-1 expression integrated brassinosteroid (BR) and photoperiod signaling pathways to promote flower bud differentiation in Red Globe grapes. During flower bud differentiation, VvBES1-1 expression was significantly regulated by red and blue light. Under R4B1 treatment, its expression peaked after 6 hours of 10 mmol·L-¹ EBR exposure.【Conclusion】VvBES1-1 plays a critical role in flower bud differentiation of Red Globe grapes. It integrates BR and photoperiod signals to inhibit grape flower bud differentiation. However, under red and blue light conditions, the expression of VvBES1-1 is effectively downregulated, thereby promoting flower bud differentiation. This study provides insights into the regulatory mechanisms of light and phytohormones in grape reproductive development. Table and Figures | Reference | Related Articles | Metrics

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Effect of VdF3′5′H2 Overexpression on the Accumulation of Anthocyanin Composition in Spine Grape Cells GUO AoLin, LIN JunXuan, LAI GongTi, HE LiYuan, CHE JianMei, PAN Ruo, YANG FangXue, HUANG YuJi, CHEN GuiXin, LAI ChengChun Scientia Agricultura Sinica    2025, 58 (4): 802-818.   DOI: 10.3864/j.issn.0578-1752.2025.04.014 Abstract （81）   HTML （12）    PDF （12810KB）（59）       Knowledge map    Save 【Objective】This study aims to clone the key gene VdF3′5′H2 involved in anthocyanin biosynthesis and analyze its regulatory role of anthocyanin biosynthesis and component content in spine grape (Vitis davidii Foëx) cells, thereby providing technical support and a theoretical foundation for constructing spine grape cells with high-yield anthocyanin production and enabling targeted regulation of anthocyanin biosynthesis.【Method】Using spine grape cells as materials, the VdF3′5′H2 was cloned and a plant expression vector was constructed. This vector was subsequently transformed into spine grape cell, with positive cell lines were screened in antibiotic media and identified through fluorescence observation and PCR analysis. The phenotypic characteristics of the transgenic spine grape cells were analyzed, along with quantification of anthocyanin, flavonoid, and proanthocyanidin. Additionally, the metabolite profile of anthocyanins was detected by UPLC-MS/MS. The expression levels of genes associated with anthocyanin biosynthesis were assessed using real-time quantitative PCR (RT-qPCR), and a comprehensive analysis of gene expression and differential metabolites was carried out by the O2PLS method.【Result】The open reading frame (ORF) of VdF3′5′H2 gene is 1 527 bp, encoding a protein comprising 508 amino acid residues. The VdF3′5′H2 protein exhibits high homology with its counterparts in related plants within the same family, containing the CYP75 conserved domain, a heme-binding site, and three characteristic conserved motifs. Phylogenetic analysis revealed that F3′H and F3′5′H cluster within the same branch, implying an evolutionary relationship from F3′H to F3′5′H, with VdF3′5′H2 positioned at a more ancestral state compared to VdF3′5′H1. Furthermore, subcellular localization indicated that the VdF3′5′H2 protein resides in the cytoplasm. The contents of anthocyanins, flavonoids and proanthocyanidins in two transgenic spine grape cells overexpression VdF3′5′H2 showed a significant increase compared to the wild type control. The content ratio of delphinidin-anthocyanins in the two transgenic cells increased to 7.82% (T6) and 14.32% (T10), respectively, Petunidin-anthocyanins increaseed to 7.30% and 10.16%, respectively, while the content ratio of malvidin- anthocyanins increased to 58.08% and 42.30%, respectively. In contrast, the content ratios of the three types of anthocyanins in the wild-type cells were 1.92%, 1.48%, and 8.49%, respectively. Additionally, the content ratios of cyanidin- and paeonidin-anthocyanins in the two transgenic cells decreased significantly. Overexpression of VdF3′5′H2 led to the downregulation of PAL, CHS, CHI and F3H expression levels, while upregulating the expression levels of VdF3′5′H1, LDOX and UFGT genes. The comprehensive analysis integrating gene expression data with differential metabolites showed that the overexpression of VdF3′5′H2 modulated the transcriptional regulation of genes involved in anthocyanin biosynthesis, thereby influencing both the synthesis pathways and accumulation patterns of various types of anthocyanins.【Conclusion】The overexpression of VdF3′5′H2 gene significantly enhanced the biosynthesis and accumulation of anthocyanins in spine grape cells, by regulating gene expression and altering the metabolic flux of the anthocyanin biosynthesis pathway, there by modulating the composition and content ratio of antocyanins in spine grape cells. Table and Figures | Reference | Related Articles | Metrics

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Effect of FGF5 and FGF21 on Proliferation of Dermal Papilla Cells in Cashmere Goat WANG Niu, SHI XinRan, ZHANG WeiDong, WANG Xin Scientia Agricultura Sinica    2025, 58 (4): 819-830.   DOI: 10.3864/j.issn.0578-1752.2025.04.015 Abstract （61）   HTML （6）    PDF （6578KB）（71）       Knowledge map    Save 【Background】The hair follicle cycle in goat is divided into three phases: the growth phase (anagen), the regression phase (catagen), and the resting phase (telogen). Fibroblast growth factor 5 (FGF5) and fibroblast growth factor 21 (FGF21) are important regulators in goat hair cycle. However, the precise localization of FGF5 and FGF21 in goat skin tissue and their regulatory mechanisms remain unclear during hair follicle cycle transition. Dermal papilla cells (DPCs) located in the dermal papilla region, are essential for hair follicle development. When DPCs lose their function, hair follicle remains in the telogen, leading to hair loss.【Objective】Therefore, this study aimed to identify the expression and localization of FGF5 and FGF21 in Cashmere goat skin tissue, understand their roles in DPCs, and to analyze their regulatory mechanisms. This would enrich the knowledge of fibroblast growth factor family proteins in hair follicle cycle regulation and provide a theoretical basis for further elucidating the molecular mechanisms of Cashmere goat hair follicle cycle transition. 【Method】Real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the expression levels of FGF5, FGF21 and their receptor 1 (FGFR1) in the skin tissue of Cashmere goats at anagen and telogen. Single-cell transcriptome sequencing data were analyzed to determine the expression and localization of FGF5, FGF21 and FGFR1, which were validated through immunofluorescence methods. Adenovirus was explored to overexpress FGF5 and FGF21 in DPCs. The effects on DPCs proliferation were measured using MTT, EdU and flow cytometry. The expressions of FGFR1 and proliferation-related genes were detected using RT-qPCR, immunofluorescence and western blotting methods. RT-qPCR, immunofluorescence and western blotting were used to investigate the effect of FGF5 and FGF21 on the proliferation of DPCs. 【Result】FGF5, FGF21 and FGFR1 exhibited higher expression levels in anagen than that in telogen, all of which were located within DPCs. Both FGF5 and FGF21 restrained DPCs viability, which significantly reduced the number of EdU-positive cells. FGF5 decreased the percentage of DPCs in G2 and S phases, while FGF21 specifically affected the G2 phase. The expression of FGFR1 was significantly increased following the overexpression of FGF5 and FGF21. Moreover, the expressions of cell proliferation markers KI67 and PCNA were also significantly inhibited with the overexpression of FGF5 and FGF21. Additionally, the expression of β-catenin, a key component of Wnt/β-catenin signaling pathway, was reduced. Thereby the activity of transcription factors TCF3 and JUN was inhibited, which were the downstream effectors of Wnt/β-catenin signaling pathway. Thus the inhibition further led to the decreased expression of proliferation-related genes MYC and CYCLIND1. 【Conclusion】FGF5 and FGF21 played a critical role as regulatory factors in controlling the biological function of DPCs. This study provided the first evidences that FGF21, similar to FGF5, restrained the proliferation of DPCs by suppressing the Wnt/β-catenin signaling pathway. These findings contributes to our understanding of how fibroblast growth factors influence hair follicle cycle transition, particularly in the context of Cashmere goat hair growth and cashmere production. Table and Figures | Reference | Related Articles | Metrics

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Screening of Interaction Proteins with AhSAP1 in Peanut Using the Yeast Two-Hybrid System ZHU YanTing, DANG Hao, NIU SiJie, LIN JingYi, YANG Hua, YANG Qiang, ZHANG Chong, CAI TieCheng, ZHUANG WeiJian, CHEN Hua Scientia Agricultura Sinica    2024, 57 (21): 4376-4390.   DOI: 10.3864/j.issn.0578-1752.2024.21.016 Abstract （131）   HTML （11）    PDF （3959KB）（135）       Knowledge map    Save 【Objective】Seed size directly affects the yield of peanut. The key transcription factor AhSAP1, which regulates peanut seed size, was obtained by QTL mapping in the early stage, but the molecular mechanism of AHSAP1 regulating peanut seed size remains unclear. In this paper, a peanut embryo yeast two-hybrid cDNA library was constructed and AhSAP1 was used as bait to screen interacting proteins, and the spatial and temporal expression characteristics of candidate interacting protein genes were analyzed. It laid the foundation for further study on the molecular mechanism of AhSAP1 regulating peanut kernel development. 【Method】The peanut embryo Escherichia coli cDNA library was constructed and identified by SMART (switching mechanism at 5′ end of the RNA transcript) method. The decoy vector pGBKT7-AhSAP1 was constructed and its toxicity and self-activation to yeast cells were evaluated. The peanut embryo cDNA library plasmid and the bait plasmid pGBKT7-AhSAP1 were co-transformed into Y2H Gold yeast strains, and the positive colonies with good growth and blue color were screened and sequenced to obtain the candidate interacting protein gene sequences and predict the biological functions. The expression characteristics of candidate interacting protein genes in different tissues and organs of peanut, induced by exogenous plant hormones and induced by low calcium stress were determined by RNA-seq. According to the functional annotation results, the candidate factors that may be involved in plant seed development were selected, their CDS full-length sequences were amplified, and the target vector pGADT7 was constructed, and point-to-point yeast two-hybrid interaction was verified with pGBKT7-AhSAP1 co-transformed yeast cells. 【Result】The titer of peanut embryo Escherichia coli secondary cDNA library was 1.05×108 cfu/mL, the recombination rate was 98%, the average insert fragment size was more than 1 000 bp, and the library quality was high. The yeast two-hybrid decoy vector pGBKT7-AhSAP1 was successfully constructed, which had no self-activation in yeast cells and no toxicity to yeast. Sixty-eight yeast-positive clones were screened, and 60 candidate interacting proteins were obtained by sequence similarity comparison and removal of duplicating, which were mainly involved in energy production and metabolism, translation, ribosome structure and biological development, transcription, signal transduction mechanism, post-translational modification, inorganic ion transport and metabolism, chromatin structure, etc. Twelve candidate interacting proteins were selected for one-to-one yeast two-hybrid verification with AhSAP1, and 8 candidate interacting proteins were found to interact with AhSAP1. 【Conclusion】The mixed cDNA library of peanut embryo development at different stages was successfully constructed, and 60 candidate interacting proteins with AhSAP1 were screened. The candidate interacting proteins were mainly involved in energy production and metabolism, translation, ribosome structure and biogenesis, transcription, signal transduction mechanism, post-translational modification, inorganic ion transport and metabolism, chromatin structure, etc. These candidates interacting protein genes were expressed in root, stem, leaf, inflorescence, fruit needle, pericarp, seed coat and embryo, and the interaction between 8 candidate interacting proteins and AhSAP1 was confirmed. Table and Figures | Reference | Related Articles | Metrics

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