Multilayered omics reveals PEG6000 stimulated drought tolerance mechanisms in white clover (Trifolium repens L.)

Drought stress is a major environmental factor that severely impacts plant growth and productivity, especially in the context of global climate change. This study aims to systematically compare the drought tolerance and underlying mechanisms of three white clover genotypes — ‘Haifa’ (YS), ‘Purple’ (PL), and ‘Red-flowered’ (RF) —by integrating physiological measurements, transcriptomic, and metabolomic analyses. The results showed that the RF genotype exhibited superior drought tolerance, maintaining higher leaf relative water content (LWC), lower electrolyte leakage (EL) and malondialdehyde (MDA) levels, stable fast chlorophyll fluorescence induction curve (OJIP) transient curves, and high PI ABS values under drought stress. The PL genotype displayed the most extensive transcriptomic response, with significant enrichment in pathways related to metal ion homeostasis, light perception, and mitochondrial function, but its physiological performance was poorer, indicating higher sensitivity to drought. The YS genotype demonstrated moderate drought tolerance, primarily relying on antioxidant mechanisms, as evidenced by the upregulation of flavonoid biosynthesis to counteract oxidative damage caused by drought. Transcriptomic and metabolomic analyses revealed both common and specific mechanisms among the three genotypes in response to drought stress. The RF genotype showed significant enrichment in pathways related to ribosome biogenesis, chlorophyll metabolism, and zeatin biosynthesis, suggesting its ability to maintain photosynthetic efficiency and growth under stress conditions. This study provides valuable insights into the complex responses of white clover to drought stress through multi-layered omics analyses, revealing the molecular basis of drought tolerance. These findings highlight potential targets for enhancing drought tolerance in white clover through breeding or biotechnological approaches, contributing to sustainable agricultural practices. • Difference of drought tolerance exists among different white clover genotypes. • Multi-layered omics analyses were applied. • Different white clover genotypes showed specific drought tolerance mechanisms. • The red-flowered genotype exhibits superior drought tolerance. • Providing valuable insights into the complex responses to drought stress.