大豆苹果酸脱氢酶基因家族特征分析表明GmMDH2提高大豆耐盐能力

doi:10.1016/j.jia.2023.12.036

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (7): 2492-2510.DOI: 10.1016/j.jia.2023.12.036

大豆苹果酸脱氢酶基因家族特征分析表明GmMDH2提高大豆耐盐能力

收稿日期:2023-08-22 修回日期:2024-01-03 接受日期:2023-11-17 出版日期:2025-07-20 发布日期:2025-06-17

Genome-wide characterization of soybean malate dehydrogenase genes reveals a positive role for GmMDH2 in the salt stress response

Runnan Zhou^1*, Sihui Wang^1*, Peiyan Liu^1*, Yifan Cui¹, Zhenbang Hu², Chunyan Liu², Zhanguo Zhang², Mingliang Yang², Xin Li³, Xiaoxia Wu^1#, Qingshan Chen^2#, Ying Zhao^1#

¹Heilongjiang Green Food Science Research Institute, Harbin 150000, China
²National Key Laboratory of Smart Farm Technology and System/Key Laboratory of Soybean Biology, Ministry of Education/ College of Agriculture, Northeast Agricultural University, Harbin 150000, China
³Key Laboratory of Maize Genetics and Breeding, Heilongjiang Academy of Agricultural Sciences, Harbin 150000, China

Received:2023-08-22 Revised:2024-01-03 Accepted:2023-11-17 Online:2025-07-20 Published:2025-06-17
About author:Runnan Zhou, E-mail: runnanzhou@126.com; Sihui Wang, E-mail: 13074558253@163.com; Peiyan Liu, E-mail: liupeiyan823@163.com; #Correspondence Xiaoxia Wu, Tel: +86-451-55190481, E-mail: xxwu2012@126.com; Qingshan Chen, Tel: +86-451-55191259, E-mail: qshchen@126.com; Ying Zhao, E-mail: tianshi198937@126.com * These authors contributed equally to this study.
Supported by:
This study was financially supported by the Natural Science Foundation of Heilongjiang Province, China (TD2022C003 and YQ2022C010), the National Key R&D Program of China (2021YFD1201104-02-02 and 2021YFF1001202), and the National Natural Science Foundation of China (U20A2027, 31971899, 32272093, and 32272072).

摘要/Abstract

摘要：

苹果酸脱氢酶（MDH）是一种在植物体内广泛表达的酶。虽然MDH在植物生长发育、响应逆境过程中起着关键作用。但是在大豆中MDH基因研究的报道有限。我们通过全基因组分析鉴定了大豆MDH基因家族的17个成员，并分析了成员间的保守蛋白质基序。根据系统发育关系，将17个大豆MDH基因家族成员分为了五个亚群，通过共聚焦显微镜观察了6个MDH基因家族成员在拟南芥叶肉原生质体的亚细胞定位情况。通过启动子顺式作用元件和qRT-PCR分析发现，在非生物胁迫（干旱、盐碱）和激素处理下，MDH家族成员的转录水平显著提升，并表现出明显的时空特异性。在盐胁迫下， GmMDH2表达量明显提高，说明GmMDH2可能在大豆相应盐胁迫过程中发挥重要功能。GmMDH2的原核表达说明该重组酶具有NADP依赖性的MDH活性。进一步研究表明，过表达GmMDH2可以调节NADPH库的氧化还原状态和抗氧化活性，从而减少了转基因大豆中ROS的形成，显著提高了转基因大豆的耐盐性。基于单倍型分析表明，GmMDH2基因的变异与大豆的耐盐性密切相关。在GmMDH2基因启动子区域发现了一个可能与耐盐性相关的多态性位点。我们的研究不仅鉴定了大豆MDH基因家族成员，加深了我们对逆境反应机制的理解，同时也为大豆耐盐育种提供了一个潜在的候选基因。

Abstract:

Malate dehydrogenase (MDH) is a widely expressed enzyme that plays a key role in plant growth, development, and stress responses. However, information on MDH genes in the soybean genome is limited. Seventeen members of the soybean MDH family were identified by genome-wide analysis, and the genes were analyzed for the presence of conserved protein motifs. The genes were divided into five clusters according to their phylogenetic relationships. The intracellular localizations of six GmMDHs were determined by confocal microscopy of Arabidopsis mesophyll protoplasts. Transcripts of GmMDHs were significantly increased by abiotic stress (drought, salt, and alkalinity) and hormone treatments, as shown by an analysis of cis-regulatory elements and quantitative real-time polymerase chain reaction (qRT-PCR). The GmMDHs displayed unique expression patterns in various soybean tissues. Notably, the expression levels of a chloroplast isoform (GmMDH2) were unusually high under salt stress, presumably indicating a critical role in soybean responses to salinity. Expression of GmMDH2 in Escherichia coli showed that the recombinant enzyme has nicotinamide adenine dinucleotide phosphate (NADP)-dependent MDH activity. The redox states of the NADP (reduced form) (NADPH) pool and antioxidant activities were shown to be modulated by GmMDH2 gene overexpression, which in turn reduced reactive oxygen species (ROS) formation in transgenic soybean, significantly enhancing the salt stress resistance. Gene-based association analysis showed that variations in GmMDH2 were strongly linked to seedling salt tolerance. A polymorphism potentially associated with salt tolerance was discovered in the promoter region of GmMDH2. These findings not only improve our understanding of the stress response mechanism by identifying and characterizing the MDH gene family throughout the soybean genome but they also identified a potential candidate gene for the future enhancement of salt tolerance in soybean.

Key words: soybean (Glycine max (Linn.) Merr.) , malate dehydrogenase , expression profile , salt stress

Runnan Zhou, Sihui Wang, Peiyan Liu, Yifan Cui, Zhenbang Hu, Chunyan Liu, Zhanguo Zhang, Mingliang Yang, Xin Li, Xiaoxia Wu, Qingshan Chen, Ying Zhao. 大豆苹果酸脱氢酶基因家族特征分析表明GmMDH2提高大豆耐盐能力[J]. Journal of Integrative Agriculture, 2025, 24(7): 2492-2510.

Runnan Zhou, Sihui Wang, Peiyan Liu, Yifan Cui, Zhenbang Hu, Chunyan Liu, Zhanguo Zhang, Mingliang Yang, Xin Li, Xiaoxia Wu, Qingshan Chen, Ying Zhao. Genome-wide characterization of soybean malate dehydrogenase genes reveals a positive role for GmMDH2 in the salt stress response[J]. Journal of Integrative Agriculture, 2025, 24(7): 2492-2510.

参考文献

Abbai R, Singh V K, Nachimuthu V V, Sinha P, Selvaraj R, Vipparla A K, Singh A K, Singh U M, Varshney R K, Kumar A. 2019. Haplotype analysis of key genes governing grain yield and quality traits across 3K RG panel reveals scope for the development of tailor-made rice with enhanced genetic gains. Plant Biotechnology Journal, 17, 1612–1622.

Berkemeyer M, Scheibe R, Ocheretina O. 1998. A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L. Journal of Biological Chemistry, 273, 27927–27933.

Bustin S A, Benes V, Garson J A, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl M W, Shipley G L, Vandesompele J, Wittwer C T. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611–622.

Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. TBtools: An integrative Toolkit developed for interactive analyses of big biological data. Molecular Plant, 13, 1194–1202.

Chen J, Zhang P, Zhao Y, Zhao J, Wu X, Zhang R, Cha R, Yao Q, Gao Y. 2022. Nitroreductase-instructed supramolecular assemblies for microbiome regulation to enhance colorectal cancer treatments. Science Advances, 8, 2789.

Chen X, Wang Y, Wang J N, Cao Q C, Sun R X, Zhu H J, Zhang Y R, Ji J D, Liu Q H. 2022. m(6)A modification of circSPECC1 suppresses RPE oxidative damage and maintains retinal homeostasis. Cell Reports, 41, 111671.

Chen Y, Fu Z, Zhang H, Tian R, Yang H, Sun C, Wang L, Zhang W, Guo Z, Zhang X, Tang J. 2020. Cytosolic malate dehydrogenase 4 modulates cellular energetics and storage reserve accumulation in maize endosperm. Plant Biotechnol Journal, 18, 2420–2435.

Crecelius F, Streb P, Feierabend J. 2003. Malate metabolism and reactions of oxidoreduction in cold-hardened winter rye (Secale cereale L.) leaves. Journal of Experimental Botany, 54, 1075–1083.

Cushman J C. 1993. Molecular cloning and expression of chloroplast NADP-malate dehydrogenase during crassulacean acid metabolism induction by salt stress. Photosynthesis Research, 35, 15–27.

Cushman J C, Bohnert H J. 1997. Molecular genetics of crassulacean acid metabolism. Plant Physiology, 113, 667–676.

Dai H, Wu H, Amanguli M, Wang L, Maimaiti A, Zhang J. 2014. Analysis of salt-tolerance and determination of salt-tolerant evaluation indicators in cotton seedlings of different genotypes. Scientia Agricultura Sinica, 47, 1290–1300. (in Chinese)

Ding Y, Ma Q H. 2004. Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat. Biochimie, 86, 509–518.

Foyer C H, Noctor G. 2005. Redox homeostasis and antioxidant signaling: A metabolic interface between stress perception and physiological responses. The Plant Cell, 17, 1866–1875.

Fryer M J, Oxborough K, Mullineaux P M, Baker N R. 2002. Imaging of photo-oxidative stress responses in leaves. Journal of Experimental Botany, 53, 1249–1254.

Giannopolitis C N, Ries S K. 1977. Superoxide dismutase. I. occurrence in higher plants. Journal of Plant Physiology, 59, 309–314.

Gietl C. 1992. Malate dehydrogenase isoenzymes: cellular locations and role in the flow of metabolites between the cytoplasm and cell organelles. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1100, 217–234.

Gui J G, Rui H H, Xin S L, Xiu J Y. 2006. Analysis of the principal components and the subordinate function of alfalfa drought resistance. Acta Agrestia Sinica, 14, 142–146.

Guo B J, Sun H W, Qi J, Huang X Y, Hong Y, Hou J, Lü C, Wang Y L, Wang F F, Zhu J, Guo G G, Xu R G. 2023. A single nucleotide substitution in the MATE transporter gene regulates plastochron and the many noded dwarf phenotype in barley (Hordeum vulgare L.). Journal of Integrative Agriculture, 22, 2295–2305.

Havaux M, Ksas B, Szewczyk A, Rumeau D, Franck F, Caffarri S, Triantaphylidès C. 2009. Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress. BMC Plant Biology, 9, 130.

Hodges D M, DeLong J M, Forney C F, Prange R K. 1999. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207, 604–611.

Hossain M S, Dietz K J. 2016. Tuning of redox regulatory mechanisms, reactive oxygen species and redox homeostasis under salinity stress. Frontiers in Plant Science, 7, 548.

Imran M, Liu J Y. 2016. Genome-wide identification and expression analysis of the malate dehydrogenase gene family in Gossypium arboreum. Pakistan Journal of Botany, 48, 1081–1090.

Imran M, Munir M Z, Ialhi S, Abbas F, Younus M, Ahmad S, Naeem M K, Waseem M, Iqbal A, Gul S, Widemann E, Shafiq S. 2022. Identification and characterization of malate dehydrogenases in tomato (Solanum lycopersicum L.). International Journal of Molecular Sciences, 23, 10028.

Imran M, Tang K, Liu J Y. 2016. Comparative genome-wide analysis of the malate dehydrogenase gene families in cotton. PLoS ONE, 11, e0166341.

Imran M, Zhang B, Tang K, Liu J. 2017. Molecular characterization of a cytosolic malate dehydrogenase gene (GhcMDH1) from cotton. Chemical Research in Chinese Universities, 33, 87–93.

Kandoi D, Mohanty S, Tripathy B C. 2018. Overexpression of plastidic maize NADP-malate dehydrogenase (ZmNADP-MDH) in Arabidopsis thaliana confers tolerance to salt stress. Protoplasma, 255, 547–563.

Kumar R G, Shah K, Dubey R S. 2000. Salinity induced behavioural changes in malate dehydrogenase and glutamate dehydrogenase activities in rice seedlings of differing salt tolerance. Plant Science, 156, 23–34.

Li N, Sun M H, Jiang Z S, Shu H R, Zhang S Z. 2016. Genome-wide analysis of the synonymous codon usage patterns in apple. Journal of Integrative Agriculture, 15, 983–991.

Librado P, Rozas J J B. 2009. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452.

Liu W, Xie Y, Ma J, Luo X, Nie P, Zuo Z, Lahrmann U, Zhao Q, Zheng Y, Zhao Y. 2015. IBS: An illustrator for the presentation and visualization of biological sequences. Bioinformatics, 31, 3359–3361.

Ma B, Yuan Y, Gao M, Xing L, Li C, Li M, Ma F. 2018. Genome-wide identification, classification, molecular evolution and expression analysis of malate dehydrogenases in apple. International Journal of Molecular Sciences, 19, 3312.

Mao H, Li S, Chen B, Jian C, Mei F, Zhang Y, Li F, Chen N, Li T, Du L, Ding L, Wang Z, Cheng X, Wang X, Kang Z. 2022. Variation in cis-regulation of a NAC transcription factor contributes to drought tolerance in wheat. Molecular Plant, 15, 276–292.

Nakano Y, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology, 22, 867–880.

Nan N, Wang J, Shi Y, Qian Y, Jiang L, Huang S, Liu Y, Wu Y, Liu B, Xu Z Y. 2020. Rice plastidial NAD-dependent malate dehydrogenase 1 negatively regulates salt stress response by reducing the vitamin B6 content. Plant Biotechnol Journal, 18, 172–184.

Noctor G. 2006. Metabolic signalling in defence and stress: the central roles of soluble redox couples. Plant, Cell and Environment, 29, 409–425.

Noctor G, Foyer C H. 2016. Intracellular redox compartmentation and ROS-related communication in regulation and signaling. Plant Physiology, 171, 1581–1592.

Queval G, Noctor G. 2007. A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: application to redox profiling during Arabidopsis rosette development. Analytical Biochemistry, 363, 58–69.

Raghavendra A S, Padmasree K. 2003. Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation. Trends in Plant Science, 8, 546–553.

Raschke M, Boycheva S, Crèvecoeur M, Nunes N A, Witt S, Fernie A R, Amrhein N, Fitzpatrick T B. 2011. Enhanced levels of vitamin B6 increase aerial organ size and positively affect stress tolerance in Arabidopsis. The Plant Journal, 66, 414–432.

Reng W, Riessland R, Scheibe R, Jaenicke R J E J o B. 1993. Cloning, site-specific mutagenesis, expression and characterization of full-length chloroplast NADP-malate dehydrogenase from Pisum sativum. European Journal of Biochemistry, 217, 189–197.

Scheibe R, Dietz K J. 2012. Reduction-oxidation network for flexible adjustment of cellular metabolism in photoautotrophic cells. Plant Cell & Environment, 35, 202–216.

Scheibe R. 2004. Malate valves to balance cellular energy supply. Physiologia Plantarum, 120, 21–26.

Schieber M, Chandel N S. 2014. ROS function in redox signaling and oxidative stress. Current Biology, 24, 453–462.

Selinski J, König N, Wellmeyer B, Hanke G T, Linke V, Neuhaus H E, Scheibe R. 2014. The plastid-localized NAD-dependent malate dehydrogenase is crucial for energy homeostasis in developing Arabidopsis thaliana seeds. Molecular Plant, 7, 170–186.

Sew Y S, Ströher E, Fenske R, Millar A H. 2016. Loss of mitochondrial malate dehydrogenase activity alters seed metabolism lmpairing seed maturation and post-germination growth in Arabidopsis. Plant Physiology, 171, 849–863.

Song J, Zou X, Liu P, Cardoso J A, Schultze K R, Liu G, Luo L, Chen Z. 2022. Differential expressions and enzymatic properties of malate dehydrogenases in response to nutrient and metal stresses in Stylosanthes guianensis. Plant Physiology and Biochemistry, 170, 325–337.

Sweetman C, Deluc L G, Cramer G R, Ford C M, Soole K L. 2009. Regulation of malate metabolism in grape berry and other developing fruits. Phytochemistry, 70, 1329–1344.

Tambasco S M, Titiz O, Raschle T, Forster G, Amrhein N, Fitzpatrick T B. 2005. Vitamin B6 biosynthesis in higher plants. Proceedings of the National Academy of Sciences of the United States, 102, 13687–13692.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.

Tang W, Ye J, Yao X, Zhao P, Xuan W, Tian Y, Zhang Y, Xu S, An H, Chen G, Yu J, Wu W, Ge Y, Liu X, Li J, Zhang H, Zhao Y, Yang B, Jiang X, Peng C, et al. 2019. Genome-wide associated study identifies NAC42-activated nitrate transporter conferring high nitrogen use efficiency in rice. Nature Communications, 10, 5279.

Taniguchi M, Miyake H. 2012. Redox-shuttling between chloroplast and cytosol: Integration of intra-chloroplast and extra-chloroplast metabolism. Current Opinion in Plant Biology, 15, 252–260.

Teng X, Zhong M, Zhu X, Wang C, Ren Y, Wang Y, Zhang H, Jiang L, Wang D, Hao Y, Wu M, Zhu J, Zhang X, Guo X, Wang Y, Wan J. 2019. FLOURY ENDOSPERM16 encoding a NAD-dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice. Plant Biotechnol Journal, 17, 1914–1927.

Tesfaye M, Temple S J, Allan D L, Vance C P, Samac D A. 2001. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiology, 127, 1836–1844.

Tóth K, Batek J, Stacey G. 2016. Generation of soybean (Glycine max) transient transgenic roots. Current Protocols in Plant Biology, 1, 1–13.

Tsuchida H, Tamai T, Fukayama H, Agarie S, Nomura M, Onodera H, Ono K, Nishizawa Y, Lee B H, Hirose S, Toki S, Ku M S, Matsuoka M, Miyao M. 2001. High level expression of C4-specific NADP-malic enzyme in leaves and impairment of photoautotrophic growth in a C3 plant, rice. Plant Cell Physiology, 42, 138–145.

Velikova V, Yordanov I, Edreva A. 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Science, 151, 59–66.

Wang Q J, Sun H, Dong Q L, Sun T Y, Jin Z X, Hao Y J, Yao Y X. 2016. The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants. Plant Biotechnol Journal, 14, 1986–1997.

Wang Z A, Li Q, Ge X Y, Yang C L, Luo X L, Zhang A H, Xiao J L, Tian Y C, Xia G X, Chen X Y, Li F G, Wu J H. 2015. The mitochondrial malate dehydrogenase 1 gene GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton. Scientific Reports, 5, 10343.

Yao Y X, Dong Q L, Zhai H, You C X, Hao Y J. 2011. The functions of an apple cytosolic malate dehydrogenase gene in growth and tolerance to cold and salt stresses. Plant Physiology and Biochemistry, 49, 257–264.

Yoo S D, Cho Y H, Sheen J. 2007. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nature Protocols, 2, 1565.

Zhang R, Hussain S, Wang Y, Liu Y, Li Q, Chen Y, Wei H, Gao P, Dai Q. 2021. Comprehensive evaluation of salt tolerance in rice (Oryza sativa L.) germplasm at the germination stage. Agronomy, 11, 1569.

Zhang Y, Wang Y, Sun X, Yuan J, Zhao Z, Gao J, Wen X, Tang F, Kang M, Abliz B, Zhang Z, Zhang H, Wang F, Li Z. 2022. Genome-wide identification of MDH family genes and their association with salt tolerance in rice. Plants (Basel), 11, 1498.

Zhang Z P, Li Z, He F, Lü J J, Xie B, Yi X Y, Li J M, Li J, Song J H, Pu Z E, Ma J, Peng Y Y, Chen G Y, Wei Y M, Zheng Y L, Li W. 2023. Genome-wide association and linkage mapping strategies reveal the genetic loci and candidate genes of important agronomic traits in Sichuan wheat. Journal of Integrative Agriculture, 22, 3380–3393.

Zhao Y, Cao P, Cui Y, Liu D, Li J, Zhao Y, Yang S, Zhang B, Zhou R, Sun M, Guo X, Yang M, Xin D, Zhang Z, Li X, Lv C, Liu C, Qi Z, Xu J, Wu X, Chen Q. 2021. Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD+-dependent glycerol–3-phosphate dehydrogenase in soybean. Journal of Integrative Plant Biology, 63, 1036–1053.

Zhao Y, Liu M, Wang F, Ding D, Zhao C J, He L, Li Z T, Xu J Y. 2019. The role of AtGPDHc2 in regulating cellular redox homeostasis of Arabidopsis under salt stress. Journal of Integrative Agriculture, 18, 1266–1279.

Zhao Y N, Yu H, Zhou J M, Smith S M, Li J Y. 2020. Malate circulation: Linking chloroplast metabolism to mitochondrial ROS. Trends in Plant Science, 25, 446–454.

大豆苹果酸脱氢酶基因家族特征分析表明GmMDH2提高大豆耐盐能力

Genome-wide characterization of soybean malate dehydrogenase genes reveals a positive role for GmMDH2 in the salt stress response

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics