Identification of Malate Dehydrogenase MDH Gene Family and Functional Characterization of VvMDH10 in Grape

doi:10.13560/j.cnki.biotech.bull.1985.2025-0661

Abstract

Abstract:

Objective To investigate the gene structure and expression of malate dehydrogenase (MDH) in grapevine (Vitis vinifera), and to provide insights into the biological functions of VvMDH genes and to support further breeding of high-quality grape varieties. Method Bioinformatics approaches were employed to identify 11 MDH genes in grapevine, followed by analyses of their protein physicochemical properties, gene homology, chromosomal locations, conserved motifs, gene structures, and promoter regions. Real-time quantitative PCR (RT-qPCR) was used to validate the expression patterns of VvMDH gene family members under drought and saline-alkali stress conditions. Arabidopsis plants heterologously overexpressing VvMDH10 were obtained using the floral dip method for drought phenotype analysis, to investigate the effect of VvMDH10 on plant drought tolerance. Result A total of 11 VvMDH gene family members were identified, unevenly distributed across 9 chromosomes. Physicochemical analysis revealed that most VvMDH proteins are alkaline. Subcellular localization predictions indicated VvMDH is located in the cytosol, chloroplasts, and mitochondria. Phylogenetic analysis classified the VvMDH members into 5 subfamilies, showing high conservation in clustering patterns with homologous proteins from Arabidopsis. Members of the same subfamily exhibited similar gene structures and motif distributions. Promoter cis-acting element analysis demonstrated that VvMDH genes are enriched with light-responsive, hormone-responsive, and stress-responsive elements (e.g., drought stress), suggesting their potential regulation by various phytohormones or stresses. Transcriptome data revealed tissue-specific expression patterns of VvMDH genes. RT-qPCR results indicated that drought and saline-alkali treatments could induce the expression of multiple VvMDH genes, with VvMDH10 expression significantly upregulated especially under drought stress. Detached Arabidopsis thaliana leaves heterologously overexpressing VvMDH10 exhibited a significantly lower water loss rate compared to wild-type plants. Under drought stress, the overexpressing lines showed significantly higher biomass and activities of antioxidant-related enzymes, significantly lower malondialdehyde (MDA) content compared to the wild type. Conclusion The VvMDH10 gene can positively regulate plant drought tolerance and can serve as a genetic resource for molecular breeding of drought-resistant grapevines.

Key words: Marselan grape, malate dehydrogenase (MDH), VvMDH10, drought resistance, salt-alkali tolerance, genome-wide identification, overexpression

GAO Liang, CUI Xin-yuan, LIU Ming-yuan, HAN Lei. Identification of Malate Dehydrogenase MDH Gene Family and Functional Characterization of VvMDH10 in Grape[J]. Biotechnology Bulletin, 2026, 42(10): 142-152.

Figures/Tables 9

Fig. 1 Chromosome localization of VvMDH genes in grape

Fig. 2 Phylogenetic tree of MDH proteins in Vitis vinifera (Vv), Arabidopsis thaliana (At), Oryza sativa (Os), Solanum lycopersicum (Sl) and Populus trichocarpa (Pt)

Fig. 3 Phylogenetic tree (A), gene structure (B), and protein conserved motif (C) of VvMDH gene family

Fig. 4 Analysis of cis-acting elements in the promoter region of VvMDH

Fig. 5 Expression analysis of VvMDH in different tissues of grape

Fig. 6 Relative expression of VvMDH genes under drought treatmentsDifferent lower letters indicate significant differences (P<0.05). The same below

Fig. 7 Relative expression of VvMDH genes under salt-alkali treatments

Fig. 8 Relative expression of VvMDH10 in transgenic A. thaliana

Fig. 9 Comparisons of phenotype and physiological indices in VvMDH10-overexpressing Arabidopsis under drought stressArabidopsis thaliana wild-type and overexpression lines were grown in pots containing peat soil for 4 weeks. The entire pots were fully watered first, then watering was stopped. The phenotypes of the plants before and after drought treatment were observed, and MDA content and activities of various enzymes were determined. Then the Arabidopsis thaliana wild-type and overexpression lines were grown in pots containing peat soil for 4 weeks, and their entire rosette leaves were cut separately, placed in petri dishes, and put into an illumination incubator. The fresh weight was measured at regular intervals. A: Plant phenotype. B: Shoot fresh weight. C: SOD activity. D: MDA content. E: POD activity. F: CAT activity. G: Leaf water loss phenotype. H: Statistical analysis of leaf water loss rate. Data in Figures B-F and H are presented as mean ± standard deviation

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