Heavy metal stress is a leading environmental issue reducing crop growth and productivity, particularly in arid and semi-arid agro-ecological zones. Cadmium (Cd), a non-redox heavy metal, can indirectly increase the production of reactive oxygen species (ROS), inducing cell death. A pot experiment was conducted to investigate the effects of different concentrations of Cd (0, 5, 25, 50, 100 µM) on physiological and biochemical parameters in two sorghum (Sorghum bicolor L.) cultivars: JS-2002 and Chakwal Sorghum. The results showed that various concentrations of Cd significantly increased the Cd uptake in both cultivars; however, the uptake was higher in JS-2002 compared to Chakwal Sorghum in leaf, stem and root. Regardless of the cultivars, there was a higher accumulation of the Cd in roots than in shoots. The Cd stress significantly reduced the growth and increased the electrolyte leakage (EL), hydrogen peroxide (H₂O₂) concentration and malondialdehyde (MDA) content in both cultivars, but the Chakwal Sorghum showed more pronounced oxidative damage than the JS-2002, as reflected by higher H₂O₂, MDA and EL. Moreover, Cd stress, particularly 50 µM and 100 µM, decreased the activity of different antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). However, the JS-2002 exhibited higher SOD, POD and CAT activities than the Chakwal Sorghum under different Cd-levels. These findings revealed that JS-2002 had a stronger Cd enrichment capacity and also exhibited a better tolerance to Cd stress due to its efficient antioxidant defense system than Chakwal Sorghum. The present study provides the available information about Cd enrichment and tolerance in S. bicolor, which is used as an important agricultural crop for livestock feed in arid and semi-arid regions.

The objective of this study was to determine the effect of soaking with γ-aminobutyric acid (GABA) on white clover (Trifolium repens cv. Haifa) seed germination under salt stress induced by 100 mM NaCl. Seeds soaking with GABA (1 μM) significantly alleviated salt-induced decreases in endogenous GABA content, germination percentage, germination vigor, germination index, shoot and root length, fresh and dry weight, and root activity of seedling during seven days of germination. Exogenous application of GABA accelerated starch catabolism via the activation of amylase and also significantly reduced water-soluble carbohydrate, free amino acid, and free proline content in seedlings under salt stress. In addition, improved antioxidant enzyme activities (SOD, GPOX, CAT, APX, DHAR, GR and MDHR) and gene transcript levels (Cu/ZnSOD, FeSOD, MnSOD, CAT, GPOX, APX, MDHR, GPX and GST) was induced by seeds soaking with GABA, followed by decreases in O₂^∙−, H₂O₂, and MDA accumulation during germination under salt stress. Seeds soaking with GABA could also significantly improve Na⁺/K⁺ content and transcript levels of genes encoding Na⁺/K⁺ transportation (HKT1, HKT8, HAL2, H⁺-ATPase and SOS1) in seedlings of white clover. Moreover, exogenous GABA significantly induced the accumulation of dehydrins and expression of genes encoding dehydrins (SK2, Y2K, Y2SK, and dehydrin b) in seedlings under salt stress. These results indicate that GABA mitigates the salt damage during seeds germination through enhancing starch catabolism and the utilization of sugar and amino acids for the maintenance of growth, improving the antioxidant defense for the alleviation of oxidative damage, increasing Na⁺/K⁺ transportation for the osmotic adjustment, and promoting dehydrins accumulation for antioxidant and osmotic adjustment under salt stress.

Background: Auxin may have a positive effect on plants under drought stress. White clover is widely cultivated and often prone to water shortages. In the present study, we investigated the effects of exogenous indole − 3-acetic acid (IAA) on growth and physiological changes of white clover under drought stress condition. The contents of endogenous IAA and other hormones including ABA, CTK, JA, GA, IAA, and SA were assayed. Moreover, expressions of auxin-responsive genes, drought-responsive genes and leaf senescence-associated genes were detected in response to exogenous IAA. Results: Compared to control, drought stress alone significantly diminished stem dry weigh, relative water content (RWC) and total chlorophyll content (Chl). Exogenous IAA treatment significantly increased RWC and Chl, whereas L-AOPP treatment drastically decreased stem dry weight, RWC and Chl under drought stress condition. Additionally, exogenous IAA treatment significantly increased ABA content and JA content, up-regulated expression of auxin responsive genes (GH3.1, GH3.9, IAA8), drought stress responsive genes (bZIP11, DREB2, MYB14, MYB48, WRKY2, WRKY56, WRKY108715 and RD22), and down-regulated expressions of auxin-responding genes (GH3.3, GH3.6, IAA27) and leaf senescence genes (SAG101 and SAG102) in the presence of PEG. Contrarily, L-AOPP treatment significantly reduced contents of ABA, GA3 and JA, down-regulated expressions of GH3.1, GH3.9, IAA8, bZIP11, DREB2, MYB14, MYB48, WRKY2, WRKY56, WRKY108715, ERD and RD22, and up-regulated SAG101 and SAG102. Conclusions: Exogenous IAA improved drought tolerance of white clover possibly due to endogenous plant hormone concentration changes and modulation of genes involving in drought stress response and leaf senescence. These results provided useful information to understand mechanisms of IAA improved drought tolerance in white clover.

In order to investigate the physiological effects of exogenous γ-aminobutyric acid (GABA) on drought tolerance in white clover (Trifolium repens), GABA shunt, polyamines (PAs), and proline (Pro) metabolism were examined after plants pretreated with or without GABA (8 mM) and then exposed to water or 15% PEG-induced drought stress in growth chamber. In this study, exogenous application of GABA effectively alleviated drought-induced damage in leaves, as reflected by significantly higher relative water content, lower electrolyte leakage, lipid peroxidation, and leaf wilt. Exogenous GABA further promoted drought-induced increases in GABA transaminase and alpha ketone glutarate dehydrogenase activities, but inhibited glutamate decarboxylase activity under both control and drought conditions, resulting in an increase in endogenous glutamate (Glu) and GABA content. Besides, exogenous GABA could well accelerated PAs synthesis and suppressed PAs catabolism, which lead to the extremely enhanced different types of PAs content (free Put and Spd, insoluble bound Spd and Spm, soluble conjugated Spd and Spm, and total Put, Spd and Spm) under drought stress. In addition, exogenous GABA application further activated drought-induced Δ¹-pyrroline-5-carboxylate synthetase and proline dehydrogenase activities, but suppressed drought-facilitated ornithine -δ-amino transferase activities, leading to a higher Pro accumulation and metabolism in GABA-pretreated plants in the middle and last period of drought. The results suggested that increased endogenous GABA by exogenous GABA treatment could improve drought tolerance of white clover associated with a positive regulation in the GABA-shunt, PAs and Pro metabolism.

This study was designed to determine the effect of exogenous spermidine (Spd) (30 μM) on white clover seed germination under water stress induced by polyethylene glycol 6000. Use of seed priming with Spd improved seed germination percentage, germination vigor, germination index, root viability and length, and shortened mean germination time under different water stress conditions. Seedling fresh weight and dry weight also increased significantly in Spd-treated seeds compared with control (seeds primed with distilled water). Improved starch metabolism was considered a possible reason for this seed invigoration, since seeds primed with Spd had significantly increased α-amylase/β-amylase activities, reducing sugar, fructose and glucose content and transcript level of β-amylase gene but not transcript level of α-amylase gene. In addition, the physiological effects of exogenous Spd on improving seeds’ tolerance to water deficit during germination were reflected by lower lipid peroxidation levels, better cell membrane stability and significant higher seed vigour index in seedlings. Enhanced antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), ascorbate-glutathione cycle (ASC-GSH cycle) and transcript level of genes encoding antioxidant enzymes induced by exogenous Spd may be one of the critical reasons behind acquired drought tolerance through scavenging of reactive oxygen species (ROS) in water-stressed white clover seeds. The results indicate that Spd plays an important function as a stress-protective compound or physiological activator.