Chitosan and spermine enhance drought resistance in white clover, associated with changes in endogenous phytohormones and polyamines, and antioxidant metabolism.

The interaction of chitosan and polyamines (PAs) could be involved mitigating drought stress in white clover (Trifolium repens L.). This research aimed to determine the effect of chitosan and PAs, and co-application of chitosan and PAs on improving drought tolerance associated with growth, phytohormones, polyamines and antioxidant metabolism. Plants were pretreated with or without 1gL-1 chitosan, 0.5mM spermine, or 1gL-1 chitosan+0.5mM spermine, then subjected to drought induced by polyethylene glycol (PEG) 6000 (-0.5MPa) in growth chambers for 14 days. Exogenous chitosan and spermine improved the level of PAs by regulating arginine decarboxylases, S-adenosyl methionine decarboxylase, copper-containing amine oxidase and polyamine oxidase activity, and expression of the genes encoding these enzymes under drought. Application of exogenous chitosan improved ABA content under normal and drought conditions. In addition, chitosan and spermine significantly enhanced the levels of cytokinin and GA, but reduced IAA levels during drought stress. Exogenous chitosan and spermine improved antioxidant defence, including enzyme activity, gene expression and the content of ascorbate and glutathione compounds, leading to a decline in superoxide anion radicals, H2O2 and malondialdehyde, effectively mitigating drought-induced oxidative damage. Other protective metabolites, such as total phenols and flavonoids, increased considerably under application of chitosan and spermine. These results suggest that chitosan-induced drought tolerance could be involved in PA metabolism, changes in endogenous phytohormones and antioxidant defence in white clover. Co-application of chitosan and spermine was more effective than either chitosan or spermine alone in mitigating drought stress.