NO is involved in spermidine-induced drought tolerance in white clover via activation of antioxidant enzymes and genes.

Nitric oxide (NO), a key signaling molecule, can be induced by polyamines (PAs), which play an important role in improving drought tolerance in plants. This study was to further investigate the role of NO in spermidine (Spd)-induced drought tolerance associated with antioxidant defense in leaves of white clover (Trifolium repens) under drought stress induced by -0.3 MPa polyethylene glycol (PEG-6000) solution. A hydroponic growth method was used for cultivating plants in a controlled growth chamber for 30-33 days until the second leaves were fully expanded. Two relative independent experiments were carried out in our study. One is that exogenous application of Spd or an NO donor (sodium nitroprusside (SNP)) significantly improved drought tolerance in whole plants, as demonstrated by better phenotypic appearance, increased relative water content (RWC), and decreased electrolyte leakage (EL) and malondialdehyde (MDA) content in leaves as compared to untreated plants. For another detached leaf experiment, PEG induced an increase in the generation of NO in cells and significantly improved activities of nitrate reductase (NR) and nitric oxide synthase (NOS). These responses could be blocked by pre-treatment with a Spd biosynthetic inhibitor, dicyclohexyl amine (DCHA), and then reversed by application of exogenous Spd. Meanwhile, PEG induced up-regulation of activities and gene transcript levels of corresponding antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) to varying degrees, while these effects were partially blocked by pre-treatment with DCHA, the scavenger of NO, the inhibitors of NR or NOS. In addition, Spd-induced antioxidant enzyme activities and gene expression also could be effectively inhibited by an NO scavenger as well as inhibitors of NR and NOS. These findings suggest that both Spd and NO can enhance drought tolerance. Spd was involved in drought stress-activated NR and NOS pathways associated with NO release, which mediated antioxidant defense and thus contributed to drought tolerance in white clover.