Overexpression of TaMAPK20-2 in Brachypodium reveals freezing and drought tolerance via modulation of sugar synthesis pathway

Abstract

Mitogen-activated protein kinase (MAPK) cascades play critical roles in plant responses to abiotic stress, yet their functional characterization in wheat remains limited. In this study, we investigated the function of a cold-responsive wheat MAPK gene, TaMAPK20-2, and its associated cascade components. Expression analysis revealed that TaMAPK20-2, TaMKK5, and TaMPKKK1 were significantly upregulated under cold stress. Subcellular localization and BiFC assays confirmed physical interactions among MPKKK1–MKK5–MAPK20-2 and MPKKK5–MKK6–MAPK20-2 modules, suggesting distinct signaling pathways. To assess its physiological role, we generated transgenic Brachypodium distachyon lines overexpressing TaMAPK20-2. Compared to wild-type (WT) plants, overexpression lines exhibited enhanced tolerance to both freezing and drought stress, as evidenced by higher survival rates, lower water loss, and reduced malondialdehyde (MDA) accumulation. Notably, OE plants showed increased soluble sugar, starch, sucrose, and glucose contents under non-stress conditions, but not fructose. These changes were supported by elevated expression of key carbohydrate metabolism genes (Susy, GolS3, SPS, Invertase) in the absence of stress. Additionally, OE lines showed pre-activation of the ICE–CBF–COR cold tolerance pathway, suggesting a priming effect. These findings demonstrate that TaMAPK20-2 positively regulates abiotic stress tolerance by modulating both signaling and metabolic pathways. This study provides new insights into MAPK-mediated stress responses and highlights TaMAPK20-2 as a promising target for improving wheat resilience to environmental stress.