Persistent high temperature decreases the yield and quality of crops, including many important herbs. White clover (Trifolium repens) is a perennial herb with high feeding and medicinal value, but is sensitive to temperatures above 30 °C. The present study was conducted to elucidate the impact of changes in endogenous γ-aminobutyric acid (GABA) level by exogenous GABA pretreatment on heat tolerance of white clover, associated with alterations in endogenous hormones, antioxidant metabolism, and aquaporin-related gene expression in root and leaf of white clover plants under high-temperature stress. Our results reveal that improvement in endogenous GABA level in leaf and root by GABA pretreatment could significantly alleviate the damage to white clover during high-temperature stress, as demonstrated by enhancements in cell membrane stability, photosynthetic capacity, and osmotic adjustment ability, as well as lower oxidative damage and chlorophyll loss. The GABA significantly enhanced gene expression and enzyme activities involved in antioxidant defense, including superoxide dismutase, catalase, peroxidase, and key enzymes of the ascorbic acid–glutathione cycle, thus reducing the accumulation of reactive oxygen species and the oxidative injury to membrane lipids and proteins. The GABA also increased endogenous indole-3-acetic acid content in roots and leaves and cytokinin content in leaves, associated with growth maintenance and reduced leaf senescence under heat stress. The GABA significantly upregulated the expression of PIP1-1 and PIP2-7 in leaves and the TIP2-1 expression in leaves and roots under high temperature, and also alleviated the heat-induced inhibition of PIP1-1, PIP2-2, TIP2-2, and NIP1-2 expression in roots, which could help to improve the water transportation and homeostasis from roots to leaves. In addition, the GABA-induced aquaporins expression and decline in endogenous abscisic acid level could improve the heat dissipation capacity through maintaining higher stomatal opening and transpiration in white clovers under high-temperature stress.

Creeping bentgrass (Agrostis stolonifera), a widely used cool-season turfgrass, is highly susceptible to heat stress, which severely impairs its growth and physiological functions. In this study, two cultivars with contrasting heat tolerance, the heat-tolerant 13M and the heat-sensitive Seaside II (SII), were pretreated with diethyl aminoethyl hexanoate (DA-6) or distilled water and then exposed to either normal temperature or heat-stress conditions. Physiological traits and lipidomics were analyzed to investigate the regulatory role of DA-6 in lipid remodeling under high-temperature stress. Results showed that exogenous DA-6 application significantly mitigated physiological damage in both genotypes under heat stress. Under heat stress, compared with their corresponding untreated plants, DA-6 pretreatment increased the Fv/Fm by 15% in 13M and by 33% in SII; for the PI_ABS, DA-6 pretreatment increased it by 32% in 13M and by 55% in SII; for electrolyte leakage, DA-6 pretreatment reduced it by 24% in 13M and by 11% in SII. The analysis of lipidomics found that heat stress significantly reduced the accumulation of total lipids, phospholipids (PLs), glycolipids (GLs), and sphingolipids (SLs) in two genotypes, but under heat stress, 13M maintained significantly higher content of these lipids than SII. Exogenous DA-6 application significantly alleviated the heat-induced decline in photosynthesis-related glycolipids in SII. Specifically, MGDG, DGDG, and SQDG increased by 186%, 85%, and 32% in heat-stressed SII + DA-6, respectively, relative to heat-stressed SII without DA-6 pretreatment. In addition, DA-6 treatment also alleviated the heat-induced reduction in chloroplast- and mitochondria-associated lipids, including PG, LPG, and CL, in both genotypes. For heat-stressed 13M + DA-6, these lipids increased by 20%, 114%, and 22%, respectively, compared with heat-stressed 13M without DA-6 pretreatment; for heat-stressed SII + DA-6, they increased by 141%, 76%, and 184%, respectively, compared with heat-stressed SII without DA-6 pretreatment. These changes may contribute to improved stability of chloroplasts and mitochondria under heat stress. Furthermore, DA-6 application significantly promoted the accumulation of PC, PE, LPC, LPE, Cer, CerP, and Hex3Cer in both genotypes under heat stress. For 13M, the increases ranged from 18% to 120%; for SII, from 44% to 254%. In heat-stressed SII + DA-6 only, DA-6 also increased PA, PS, MLCL, DLCL, Hex1Cer, and Hex2Cer by 82%, 45%, 84%, 59%, 53%, and 41%, respectively, relative to heat-stressed SII without DA-6 pretreatment. These PLs and SLs are essential for maintaining plasma membrane integrity and mediating stress signal transduction. In addition, the application of DA-6 significantly reduced the heat-induced increase in unsaturation levels of total lipids in both genotypes, indicating that the DA-6 improved lipid saturation levels to better adapt to heat stress. Current findings demonstrated that the DA-6 application improved heat tolerance of creeping bentgrass associated with its regulation of lipid remodeling. Future investigations incorporating multi-omics approaches could comprehensively dissect the DA-6-induced signaling pathways and regulatory networks underlying heat-stress response in cool-season grass species.