GhGLDH35A gene-mediated ROS homeostasis and stomatal movement via the ascorbic acid pathway confers alkaline stress tolerance

Yapeng Fan; Ning Wang; Shuai Wang; Zhining Yang; Xiao Chen; Xuke Lu; Hui Huang; Xiugui Chen; Lanjie Zhao; Menghao Zhang; Yuping Sun; Junjuan Wang; Lixue Guo; Lidong Wang; Ruize Song; Jing Wang; Xinrui Zhang; Xin Yu; Yi Liu; Xue-Rong Zhou; Junting Meng; Keyun Feng; Maohua Dai; Wuwei Ye

doi:10.1016/j.jare.2025.06.018

GhGLDH35A gene-mediated ROS homeostasis and stomatal movement via the ascorbic acid pathway confers alkaline stress tolerance

J Adv Res. 2025 Jun 15:S2090-1232(25)00428-X. doi: 10.1016/j.jare.2025.06.018. Online ahead of print.

Authors

Yapeng Fan¹, Ning Wang², Shuai Wang¹, Zhining Yang¹, Xiao Chen¹, Xuke Lu¹, Hui Huang¹, Xiugui Chen¹, Lanjie Zhao¹, Menghao Zhang¹, Yuping Sun¹, Junjuan Wang¹, Lixue Guo¹, Lidong Wang¹, Ruize Song¹, Jing Wang¹, Xinrui Zhang¹, Xin Yu¹, Yi Liu¹, Xue-Rong Zhou³, Junting Meng¹, Keyun Feng², Maohua Dai⁴, Wuwei Ye⁵

Affiliations

¹ Institute of Cotton Research of Chinese Academy of Agricultural Sciences / Zhengzhou Research Base, State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, School of Agricultural Sciences, Zhengzhou University / National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land, Anyang 455000, Henan, China.
² Institute of Crop Sciences, Gansu Academy of Agricultural Sciences, Lanzhou 730070 Gansu, China.
³ CSIRO Agriculture and Food, PO Box 1700, Canberra, ACT 2601, Australia.
⁴ Dryland Farming Institute, Hebei Academy of Agricultural and Forestry Sciences / Hebei Key Laboratory of Crops Drought Resistance, Hengshui 053000, Hebei, China. Electronic address: daimaohua@sina.com.
⁵ Institute of Cotton Research of Chinese Academy of Agricultural Sciences / Zhengzhou Research Base, State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, School of Agricultural Sciences, Zhengzhou University / National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land, Anyang 455000, Henan, China. Electronic address: yew158@163.com.

PMID: 40527445
DOI: 10.1016/j.jare.2025.06.018

Abstract

Introduction: Ascorbic acid (AsA) is involved in plant responses to various abiotic stresses. However, its specific function in alkaline stress tolerance remains poorly understood. The L-galactono-1,4-lactone dehydrogenase (GLDH) gene is crucial for AsA synthesis, yet the precise role of GLDH in modulating plant resistance to alkaline stress has not been comprehensively characterized.

Objectives: To investigate the role of GLDH genes in enhancing tolerance to alkaline stress.

Methods: Bioinformatics analysis of the GLDH gene family members was conducted, and an evolutionary tree was constructed using MEGA software. Cis-acting elements and gene structures were analyzed using TBtools. Gene expression levels were quantified by qRT-PCR, while the function of the GhGLDH35A gene was validated through VIGS (Virus-induced gene silencing) in cotton, heterologous overexpression in Arabidopsis thaliana, and complementation assays in yeast.

Results: Our study investigated the effects of salt-alkaline stress on cotton and found that alkaline stress caused significantly more severe damage than salt stress. The GLDH family genes were identified and analyzed, revealing a high degree of evolutionary conservation. Most GhGLDH genes exhibited a positive response to alkaline stress and were regulated by light. Among them, GhGLDH35A, which is highly expressed within the GLDH family, was found to play a key role in conferring tolerance to alkaline stress. Subcellular localization analysis indicated that GhGLDH35A is localized in the mitochondria. Silencing of GhGLDH35A in cotton resulted in reduced tolerance to alkaline stress, disruption of ROS homeostasis, and impairment of photosynthesis and stomatal function. Conversely, overexpression of GhGLDH35A in Arabidopsis enhanced alkaline stress resistance by elevating AsA levels, increasing antioxidant enzyme activities to enhance ROS scavenging, sustaining photosynthesis, and promoting stomatal closure. Furthermore, heterologous expression of GhGLDH35A in yeast also improved its tolerance to alkaline stress.

Conclusions: GhGLDH35A positively regulates alkaline stress tolerance by enhancing antioxidant defenses and regulating stomatal movement.

Keywords: 4-lactone dehydrogenase; Alkaline stress; Ascorbic acid; L-galactono-1; ROS homeostasis; Stomatal movement.