The alterations of endogenous polyamines and phytohormones induced by exogenous application of spermidine regulate antioxidant metabolism, metallothionein and relevant genes conferring drought tolerance in white clover

• There is a cross-talk between polyamines (PAs) and phytohormones under drought and well-watered conditions.
• The changes of endogenous PAs and phytohormones resulted in accelerated proline metabolism and elevated AsA–GSH cycle and relevant genes expression.
• Other metabolites such as total phenols, flavonoid, cysteine and metallothionein (MT) were also elevated with significantly increased endogenous PAs content.
• On the contrary, the deficiency in PAs weakened antioxidant defense and proline metabolism, inhibited MT genes expression leading to aggravation of drought-induced stress damage.

Cross-talk between polyamines (PAs) and phytohormones may be involved in the regulation of drought tolerance in plants. The objective of this study was to determine the effects of exogenous spermidine (Spd) and a Spd inhibitor, dimethylthiourea (DCHA), on drought responses of white clover (Trifolium repens). Metabolic, protein, and gene transcript levels were evaluated at leaf relative water content levels of 87% for non-stressed plants and 50% for drought-stressed plants. The exogenous application of Spd (0.05 mM) significantly increased endogenous PAs content whereas DCHA generally reduced PAs content. Phytohormone responses to Spd application included an elevated gibberellic acid (GA) and indole-3-acetic acid (IAA) content under non-stressed condition, and an increased GA and cytokinin (CTK) content as well as decreased IAA content under water deficit condition. The increase in endogenous PAs had no significant effect on abscisic acid (ABA) accumulation under either water treatment. Spd treatment resulted in enhanced antioxidant defense via maintenance of greater antioxidant enzyme activities and transcript levels, especially ascorbic acid (AsA). Other metabolites such as total phenols, flavonoids, proline, metallothionein (MT), and cysteine (Cyt) were also elevated with Spd treatment. On the contrary, inhibition of Spd biosynthesis weakened antioxidant defense, decreased proline metabolism, and inhibited MT genes expression leading to aggravation of drought-induced stress damages. These results suggest that changes of PAs and phytohormones play critical roles in the improvement of drought tolerance in white clover, which are attributed to the alleviation of oxidative stress, inhibition of leaf senescence, and maintenance of growth under drought stress.