Modulation of alternative oxidase to enhance tolerance against cold stress of chickpea by chemical treatments.

The alternative oxidase (AOX) is the enzyme responsible for the alternative respiratory pathway. This experiment was conducted to examine the influence on cold tolerance ability of chickpea (Cicer aurentium cv. Müfitbey) seedlings of AOX activator (pyruvate), AOX inhibitor (salicylhydroxamic acid (SHAM)) and an inhibitor of the cytochrome pathway of respiration (antimycin A) treatments. 5mM pyruvate, 2μM antimycin A and 4mM SHAM solutions were exogenously applied to thirteen-day-old chickpea leaves and then the seedlings were transferred to a different plant growth chamber arranged to 10/5°C (day/night) for 48h. Cold stress markedly increased the activities of antioxidant enzymes compared to controls. Pyruvate and antimycin A significantly increased the cold-induced increase in antioxidant activity but SHAM decreased it. Cold-induced increases in superoxide anion, hydrogen peroxide, and lipid peroxidation levels were significantly reduced by pyruvate and antimycin A, but increased by SHAM treatment. Pyruvate and antimycin A application increased both the activity and protein expression of AOX in comparison to cold stress alone. However, SHAM significantly decreased activity of AOX but did not affect its expression. Total cellular respiration values (TCRV) supported the changes in activity and expression of AOX. While TCRV were increased by cold and pyruvate, they were significantly reduced by SHAM and especially antimycin A. These results indicate that pyruvate and antimycin A applications were effective in reducing oxidative stress by activating the alternative respiratory pathway as well as antioxidant activity. Furthermore, direct activation of AOX, rather than inhibition of the cytochrome pathway, was the most effective way to mitigate cold stress.