Increased transcriptional levels of genes encoding antioxidant enzymes play important protective roles in coping with excessive accumulation of reactive oxygen species (ROS) in plants exposed to various abiotic stresses. To fully elucidate different evolutions and functions of ROS-scavenging enzymatic genes, we isolated iron superoxide dismutase (FeSOD), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) from white clover for the first time and subsequently tested dynamic expression profiles of these genes together with previously identified other antioxidant enzyme genes including copper zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), glutathione reductase (GR), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in response to cold, drought, salinity, cadmium stress and exogenous abscisic acid (ABA) or spermidine (Spd) treatment. The cloned fragments of FeSOD, DHAR and MDHAR genes were 630, 471 and 669 bp nucleotide sequences encoding 210, 157 and 223 amino acids, respectively. Phylogenetic analysis indicated that both amino acid and nucleotide sequences of these three genes are highly conservative. In addition, the analysis of genes expression showed the transcription of GR, POD, MDHAR, DHAR and Cu/ZnSOD were rapidly activated with relatively high abundance during cold stress. Differently, CAT, APX, FeSOD, Cu/ZnSOD and MnSOD exhibited more abundant transcripts compared to others under drought stress. Under salt stress, CAT was induced preferentially (3–12 h) compared to GR which was induced later (12–72 h). Cadmium stress mainly up-regulated Cu/ZnSOD, DHAR and MDHAR. Interestingly, most of genes expression induced by ABA or Spd happened prior to various abiotic stresses. The particular expression patterns and different response time of these genes indicated that white clover differentially activates genes encoding antioxidant enzymes to mitigate the damage of ROS during various environmental stresses.

Water stress decreases seed-germination characteristics and also hinders subsequent seedling establishment. Seed priming with bioactive compounds has been proven as an effective way to improve seed germination under normal and stressful conditions. However, effect and mechanism of seed priming with chitosan (CTS) on improving seed germination and seedling establishment were not well-understood under water-deficit conditions. White clover (Trifolium repens) seeds were pretreated with or without 5 mg/L CTS before being subjected to water stress induced by 18% (w/v) polyethylene glycol 6000 for 7 days of germination in a controlled growth chamber. Results showed that water stress significantly decreased germination percentage, germination vigor, germination index, seed vigor index, and seedling dry weight and also increased mean germination time and accumulation of reactive oxygen species, leading to membrane lipid peroxidation during seed germination. These symptoms could be significantly alleviated by the CTS priming through activating superoxide dismutase, catalase, and peroxidase activities. In addition, seeds pretreated with CTS exhibited significantly higher expression levels of genes encoding dehydration-responsive transcription factors (DREB2, DREB4, and DREB5) and dehydrins (Y2K, Y2SK, and SK2) than those seeds without the CTS priming. Current findings indicated that the CTS-induced tolerance to water stress could be associated with the enhancement in dehydration-responsive pathway during seed germination.

Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover (Trifolium repens) plants were pretreated with 70 μM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic Arabidopsisthaliana overexpressing a TrSAMS encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the TrSAMS overexpression in Arabidopsisthaliana could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the TrSAMS-transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type Arabidopsisthaliana in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.

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.

Salinity stress threatens global agriculture through three main synergistic effects: osmotic stress, ionic toxicity, and oxidative stress. γ-Aminobutyric acid (GABA) is recognized as a critical regulator of plant tolerance to multiple abiotic stresses, but its positive effects on mitigating salt damage to plants have not been fully elucidated. This study aimed to investigate beneficial effects of exogenous GABA pretreatment on physiological homeostasis in salt-stressed white clover (Trifolium repens) and further elucidate potential role of the GABA shunt in regulating lipid reprogramming for maintaining the stability and functionality of cell membrane systems under salt stress. Results demonstrated that salt stress exerted negative impacts on white clover, including oxidative damage, disruption to water balance, and inhibition of photosynthesis. However, exogenous GABA pretreatment significantly reduced reactive oxygen species level and membrane lipid peroxidation, while enhancing net photosynthetic rate, photochemical efficiency, and water balance via improved osmotic adjustment and water use efficiency in leaves of white clover when exposed to salt stress. Furthermore, exogenous GABA significantly improved endogenous GABA content and GABA transaminase (GABA-T) activity, thereby enhancing GABA branched metabolism to support tricarboxylic acid (TCA) cycle during salt stress. Additionally, lipidomic analysis found that salt stress altered the accumulation and composition of lipids, and both GABA-pretreated and untreated plants increased DGDG/MGDG and PC/PE ratios to adapt to high-salt conditions. More importantly, GABA-pretreated plants maintained significantly higher contents of phospholipids, glycolipids, and sphingolipids as well as lower unsaturation level of lipids contributing to the stability and functionality of membrane systems. In addition, the GABA could enhance the conversion of lipids and pyruvate into acetyl-CoA to feed the TCA cycle. These findings highlight protective role of GABA against salt stress by improving physiological homeostasis, optimizing energy metabolism, and remodeling membrane lipids.