Leaf Extracellular Ascorbate Metabolism in Relation to Ozone Tolerance in Two Soybean Cultivars

Abstract

Antioxidants in the leaf apoplast have the potential to detoxify ozone (O3) and active oxygen species (AOS), and thus may play a role in preventing plant injury. To investigate this possibility, two experiments were conducted to compare antioxidant metabolism in the leaf extracellular spaces of O3-tolerant (Essex) and O3-sensitive (Forrest) cultivars of soybean [Glycine max (L.) Merr.]. In the first experiment, a canopy profile consisting of the 2nd, 4th and 6th main stem trifoliates was sampled from plants grown for four weeks in a greenhouse supplied with charcoal-filtered air (CF) to assess genetic and leaf age effects under low stress conditions. In general, apoplastic ascorbate (AA) levels were low (< 30 nmol AA g-1 FW) in both cultivars. Apoplastic dehydroascorbate (DHA) levels were higher (> 50 nmol DHA g-1 FW) relative to AA, particularly in younger leaves (250-450 nmol DHA g-1 FW), resulting in a low ascorbate redox state. Ascorbate oxidase (AO) and ascorbate peroxidase (APX) activity were found in leaf apoplast samples, suggesting that the enzymes played a role in maintaining ascorbate primarily in the oxidized state. In the second experiment, the second main stem trifoliates of four week-old plants were compared in the two genotypes following exposure to CF or elevated O3 conditions. Following a six-day treatment period with 77 ppb O3 for 7 h d-1, foliar injury was greater in O3-treated Forrest than in Essex, and total leaf guaiacol peroxidase activity was correlated with the greater O3 sensitivity of Forrest. Under both CF and elevated O3 conditions, there was a significantly higher apoplastic total antioxidant capacity in Essex than in Forrest correlating with greater O3 tolerance. Although reduced AA concentration was greater in apoplasts sample from O3-treated Essex than Forrest, the difference (6.5 nmol g-1 FW) was unlikely to play a major role in the differential O3 sensitivity of these two cultivars. Apoplast AA was generally less than 30% of the total antioxidant capacity found in apoplast samples, suggesting that other antioxidants might be involved in the detoxification of O3 in the extracellular space in these soybean cultivars.