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Background <p>Alfalfa (<i>Medicago sativa</i> L.) is globally important forage crop whose survival and productivity at higher latitudes depend on cold-stress tolerance. However, the molecular mechanisms of how alfalfa defends against freezing stress remain unclear.</p> Results <p>In this study, the candidate gene <i>MsCBF4</i> was cloned from the cold-tolerant alfalfa genotype “Dongnong NO.1”. <i>MsCBF4</i> belongs to the APETALA2/Ethylene Response Factor (AP2/ERF) transcription factor family and functions as a transcriptional activator via a C-terminal activation domain. The encoded protein localized to the nucleus. <i>MsCBF4</i> is induced by cold and freezing conditions and is expressed in roots, stems, leaves, flowers, and pods, with the highest expression levels in roots and leaves. Overexpression of <i>MsCBF4</i> in <i>Arabidopsis thaliana</i> seedlings significantly enhanced root length, lateral root number, and fresh weight under cold stress. Lines overexpressing <i>MsCBF4</i> demonstrated enhanced frost tolerance at both the seedling and mature stages, likely through improved physiological scavenging of reactive oxygen species (ROSs), thereby limiting membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance were upregulated in transgenic <i>Arabidopsis</i>. The <i>MsCBF4</i> promoter exhibits transcriptional activity and can be directly activated by cold responses. Yeast one-hybrid (Y1H) and dual-luciferase reporter (Dual-LUC) assays showed that <i>MsERF6</i> specifically binds to the <i>MsCBF4</i> promoter and positively regulates its expression.</p> Conclusion <p><i>MsCBF4</i> enhances frost tolerance in <i>Arabidopsis</i> by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes.</p>

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The alfalfa AP2/ERF transcription factor MsCBF4 enhances frost tolerance in Arabidopsis thaliana

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月24日

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  • Nai-Peng Ren,
  • Hui Zang,
  • Yi-Fu Xuan,
  • Jie-Lin Liu,
  • Xiang-Ping Liu,
  • Guo-Liang Li

摘要

Background

Alfalfa (Medicago sativa L.) is globally important forage crop whose survival and productivity at higher latitudes depend on cold-stress tolerance. However, the molecular mechanisms of how alfalfa defends against freezing stress remain unclear.

Results

In this study, the candidate gene MsCBF4 was cloned from the cold-tolerant alfalfa genotype “Dongnong NO.1”. MsCBF4 belongs to the APETALA2/Ethylene Response Factor (AP2/ERF) transcription factor family and functions as a transcriptional activator via a C-terminal activation domain. The encoded protein localized to the nucleus. MsCBF4 is induced by cold and freezing conditions and is expressed in roots, stems, leaves, flowers, and pods, with the highest expression levels in roots and leaves. Overexpression of MsCBF4 in Arabidopsis thaliana seedlings significantly enhanced root length, lateral root number, and fresh weight under cold stress. Lines overexpressing MsCBF4 demonstrated enhanced frost tolerance at both the seedling and mature stages, likely through improved physiological scavenging of reactive oxygen species (ROSs), thereby limiting membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance were upregulated in transgenic Arabidopsis. The MsCBF4 promoter exhibits transcriptional activity and can be directly activated by cold responses. Yeast one-hybrid (Y1H) and dual-luciferase reporter (Dual-LUC) assays showed that MsERF6 specifically binds to the MsCBF4 promoter and positively regulates its expression.

Conclusion

MsCBF4 enhances frost tolerance in Arabidopsis by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes.