Effects of Abscisic Acid and Nitric Oxide on Chilling Resistance and Activation of the Antioxidant System in Walnut Shoots In Vitro

Abstract

The roles of abscisic acid (ABA) and nitric oxide (NO) and the relationship between NO and ABA on chilling resistance and activation of antioxidant activities in walnut (Juglans regia) shoots in vitro under chilling stress were investigated. Walnut shoots were treated with ABA, the NO donor sodium nitroprusside (SNP), ABA in combination with the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), PTIO, SNP in combination with the ABA biosynthesis inhibitor fluridone (Flu), and Flu. Their effects on chilling tolerance, reactive oxygen species (ROS) levels, and the antioxidant defense system were analyzed. The results showed that ABA treatment markedly alleviated the decreases in the maximal photochemical efficiency and survival and the increases in electrolyte leakage and lipid peroxidation induced by chilling stress, suggesting that application of ABA could improve the chilling tolerance. Further analyses showed that ABA enhanced antioxidant defense and slowed down the accumulation of ROS caused by chilling. Similar results were observed when exogenous SNP was applied. ABA in combination with PTIO or PTIO alone differentially abolished these protective effects of ABA. However, treatment with NO in combination with Flu or Flu alone did not affect the SNP-induced protective effect against CI or the activation of antioxidant activities under conditions of chilling stress. In addition, ABA treatment increased the NO content under chilling conditions, which was suppressed by the ABA biosynthesis inhibitor Flu or NO scavenger PTIO. Conversely, SNP application induced the same ABA rise observed in control plants in response to chilling. Taken together, these results suggested that ABA may confer chilling tolerance in walnut shoots in vitro by enhancing the antioxidant defense system, which is partially mediated by NO, preventing the overproduction of ROS to alleviate the oxidative injury induced by chilling.