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Journal of Integrative Agriculture
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Genome-wide identification and expression profiling of the PP2C family in alfalfa (Medicago sativa) and functional analysis of MsPP2C-A genes in response to alkali stress

Xiqiang Liu1#, Dengxia Yi 2#, Xiaoxia Li1#, Yaling Liu3,4, Yaqin Wei5, Meiying Guo5

1 Institute of Ecological Protection and Restoration, Chinese Academy of Forestry/Grassland Research Center, National Forestry and Grassland Administration, Beijing 100091, China

2 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

3 National Technology Innovation Center for Prataculture, Hohhot, Inner Mongolia 010070, China

4 Inner Mongolia M-Grass Ecology and Environment (Group) Co., Ltd., Hohhot, Inner Mongolia 010016, China

5 College of Grassland Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010010, China

 Highlights 

104 MsPP2Cs have been identified in alfalfa and systematically characterized.

MsPP2C-A members are significantly upregulated in response to alkali stress.

MsPP2C38 acts as an important regulator in alkali stress signal responses.

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摘要  

【目的】本研究旨在系统鉴定紫花苜蓿PP2C基因家族成员,阐明其进化特征、结构特点与表达模式,并探究MsPP2C在碱胁迫下的响应特征与功能。【方法】以‘中苜1号’基因组为参考,结合生物信息学方法鉴定MsPP2C家族成员,系统分析其进化发育、染色体分布、基因复制、基因结构及保守基序;通过150 mmol·L⁻¹碱性盐(Na₂CO₃+NaHCO₃)处理下获得的叶片转录组数据,筛选参与碱胁迫响应的MsPP2C基因,采用Mfuzz趋势聚类、KEGG功能富集、蛋白互作预测、RT‑qPCR验证及亚细胞定位试验,解析在碱胁迫响应中MsPP2C基因的表达模式与功能特征。【结果】共鉴定获得104个MsPP2C基因,分为13个亚家族,包含4对串联复制和13对片段复制基因,与蒺藜苜蓿、大豆具有较多共线性关系;基因结构与保守基序呈现明显的亚家族特异性。转录组分析筛选获得14个差异表达的MsPP2C基因,其中6个MsPP2C‑A亚家族成员(MsPP2C8MsPP2C38MsPP2C60MsPP2C62MsPP2C102MsPP2C103)在碱胁迫下快速上调并持续高表达,并且KEGG显著富集于植物激素信号转导与MAPK信号通路。蛋白互作网络显示MsPP2C38为核心调控因子,可与WRKY40、DREB2A等抗逆转录因子发生强互作;亚细胞定位实验证实MsPP2C8、MsPP2C38、MsPP2C62、MsPP2C103均定位于细胞核。【结论】本研究首次完成紫花苜蓿PP2C基因家族的系统解析,明确了MsPP2C‑A亚家族成员是调控紫花苜蓿响应碱胁迫的关键因子,揭示了MsPP2C38在抗逆信号网络中的核心作用,为紫花苜蓿耐碱性分子遗传改良提供了重要基因资源与理论依据。



Abstract  

Protein phosphatase type-2Cs (PP2Cs) are widely involved in regulating plant growth and development, cell division, and, importantly, responses to abiotic stresses through reversible protein phosphorylation. We investigated the PP2C gene family in alfalfa through genome-wide identification and expression profiling analysis. Overall, 104 MsPP2C members identified in the alfalfa genome were classified into 13 subfamilies. Phylogenetic relationships, chromosomal distributions, duplication events, gene structures, and conserved motifs of MsPP2Cs were systematically analyzed. Additionally, transcriptomic and real-time quantitative PCR analyses revealed 14 MsPP2C genes were significantly differentially expressed in alfalfa under alkali stress. Among these, MsPP2C8, MsPP2C38, MsPP2C60, MsPP2C62, MsPP2C102, and MsPP2C103 (subfamily A) were rapidly and markedly upregulated in response to alkali stress. KEGG enrichment analysis revealed these genes were involved in plant hormone signal transduction and MAPK signaling pathway. MsPP2C38 had a core regulatory role in a predicted protein-interaction network, and interacted strongly with the proteins WRKY40 and DREB2A. Subcellular localization assays indicated MsPP2C8, MsPP2C38, MsPP2C62, and MsPP2C103 to be localized in the nucleus. These findings improve our understanding of the PP2C family, and clarify the critical regulatory roles of subfamily A members in mediating salt–alkali stress responses and tolerance in alfalfa.

Keywords:  protein phosphatase type-2C       expression profiles        MsPP2C-A subfamily        alkali tolerance        signal response and regulation  
Accepted: Online: 30 May 2026  
Fund: 

This study was financially supported by the Biological Breeding-National Science and Technology Major Project (2022ZD04017), National Technology Innovation Center for Prataculture Special Fund for Innovation Platform Construction (CCPTZX2023W01), and Hohhot Key R&D Project (2023-JBGS-S-1-1).

Cite this article: 

Xiqiang Liu, Dengxia Yi , Xiaoxia Li, Yaling Liu, Yaqin Wei, Meiying Guo. 2026. Genome-wide identification and expression profiling of the PP2C family in alfalfa (Medicago sativa) and functional analysis of MsPP2C-A genes in response to alkali stress. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.05.039

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