Key message This study first reveals the CtBBX4–CtnsLTP18–CtIPCS1 module mediates drought tolerance via the antioxidant system in Cynanchum thesioides, off ering genetic resources for plant abiotic stress improvement. Abstract Cynanchum thesioides, a wild plant with both medicinal and edible values widely distributed in Inner Mongolia of China, exhibits excellent adaptability to arid environments. However, the regulatory network of its nonspecific lipid transfer protein (nsLTP) gene family in drought response remains elusive. In this study, we preliminarily identified the CtnsLTP gene family in C. thesioides and conducted an in-depth analysis focusing on CtnsLTP18. Phylogenetic analysis revealed that the CtnsLTP gene family comprises 18 structurally divergent members, which are clustered into 9 subfamilies. CtnsLTP18 was significantly upregulated at the early stage of drought stress, and its encoded protein was localized to the plasma membrane. Functional validation assays showed that overexpression of CtnsLTP18 in Saccharomyces cerevisiae enhanced its osmotic stress tolerance, while heterologous overexpression of this gene in Arabidopsis thaliana improved the drought tolerance of transgenic plants. In contrast, suppression of CtnsLTP18 expression in C. thesioides via virus-induced gene silencing (VIGS) resulted in a significant reduction in plant drought resistance. At the physiological level, CtnsLTP18 enhanced plant drought resistance by elevating the activity of antioxidant enzymes, reducing reactive oxygen species (ROS) accumulation and alleviating membrane lipid peroxidation damage. At the molecular level, yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and luciferase complementation assay (LCA) experiments confirmed a physical interaction between CtnsLTP18 and CtIPCS1. Furthermore, yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays demonstrated that the transcription factor CtBBX4 could directly bind to the promoter of CtnsLTP18 and positively regulate its expression. Taken together, this study is the first to elucidate the molecular mechanism by which the CtBBX4–CtnsLTP18–CtIPCS1 regulatory module mediates drought tolerance in C. thesioides through the modulation of the antioxidant system. This finding not only enriches our understanding of the drought adaptation strategies of C. thesioides but also provides valuable genetic resources for the abiotic stress-resistant genetic improvement of plants.

A nonspecific lipid transfer protein, CtnsLTP18, mediates drought stress resistance in Cynanchum thesioides

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发表日期：

2026年4月2日

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Xiaoyao Chang,
Xiaoyan Zhang,
Xiumei Huang,
Zhongren Yang,
Guihua Chen,
Fenglan Zhang

摘要

Key message

This study first reveals the CtBBX4–CtnsLTP18–CtIPCS1 module mediates drought tolerance via the antioxidant system in Cynanchum thesioides, off ering genetic resources for plant abiotic stress improvement.

Abstract

Cynanchum thesioides, a wild plant with both medicinal and edible values widely distributed in Inner Mongolia of China, exhibits excellent adaptability to arid environments. However, the regulatory network of its nonspecific lipid transfer protein (nsLTP) gene family in drought response remains elusive. In this study, we preliminarily identified the CtnsLTP gene family in C. thesioides and conducted an in-depth analysis focusing on CtnsLTP18. Phylogenetic analysis revealed that the CtnsLTP gene family comprises 18 structurally divergent members, which are clustered into 9 subfamilies. CtnsLTP18 was significantly upregulated at the early stage of drought stress, and its encoded protein was localized to the plasma membrane. Functional validation assays showed that overexpression of CtnsLTP18 in Saccharomyces cerevisiae enhanced its osmotic stress tolerance, while heterologous overexpression of this gene in Arabidopsis thaliana improved the drought tolerance of transgenic plants. In contrast, suppression of CtnsLTP18 expression in C. thesioides via virus-induced gene silencing (VIGS) resulted in a significant reduction in plant drought resistance. At the physiological level, CtnsLTP18 enhanced plant drought resistance by elevating the activity of antioxidant enzymes, reducing reactive oxygen species (ROS) accumulation and alleviating membrane lipid peroxidation damage. At the molecular level, yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and luciferase complementation assay (LCA) experiments confirmed a physical interaction between CtnsLTP18 and CtIPCS1. Furthermore, yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays demonstrated that the transcription factor CtBBX4 could directly bind to the promoter of CtnsLTP18 and positively regulate its expression. Taken together, this study is the first to elucidate the molecular mechanism by which the CtBBX4–CtnsLTP18–CtIPCS1 regulatory module mediates drought tolerance in C. thesioides through the modulation of the antioxidant system. This finding not only enriches our understanding of the drought adaptation strategies of C. thesioides but also provides valuable genetic resources for the abiotic stress-resistant genetic improvement of plants.