Molecular Cloning and Functional Characterization of Heat Stress-Responsive Superoxide Dismutases in Garlic (Allium sativum L.)

Abstract

Superoxide dismutases (SODs) are key antioxidant enzymes that can detoxify the superoxide radicals generated by various stresses. Although various plant SODs have been suggested to improve stress tolerance, SODs in garlic, an economically important vegetable grown worldwide, remain relatively unknown. In this study, we found that heat stress strongly induced the activities of Cu/ZnSODs, FeSODs, and MnSODs in garlic leaves. In addition, we cloned four garlic SODs (AsSODs) and suggest that heat stress-increased SOD activity was reflected at least by the induction of these AsSODs. The results of the agro-infiltration assay suggested that the cloned AsSODs encoded functional SOD enzymes belonging to the Cu/ZnSOD and MnSOD families. As a first step toward understanding the enzymatic antioxidant system in garlic plants, our results provide a solid foundation for an in-depth analysis of the physiological functions of the AsSOD family.

Keywords: antioxidant; garlic; heat stress; superoxide dismutase.

Figure 1

Physiological response of garlic plants under heat stress. Changes in phenotypes (A), H₂O₂ level (B), malondialdehyde level (C), total SOD activity level (D), and the activity pattern of SOD isozymes (E) were determined. In-gel SOD activity assay was performed using total protein obtained from heat-treated samples (45 °C for 8 h). The bars represent the mean ± SE across five independent experiments. Different letters indicate statistically significant differences (p < 0.05).

Figure 2

Phylogenetic tree, domain architectures (A), and expression patterns of putative garlic SODs (AsSODs) in different organs (B) and in response to heat stress (C). Scale bar in phylogenetic tree represents distance scale. For organ-specific expression analysis, the expression level of each gene was normalized relative to actin, whereas the normalized expression levels of AsSODs were expressed relative to their value at 0 h for analysis of the heat-induced expression pattern. The bars represent the mean ± SE across five independent experiments. Different letters indicate statistically significant differences (p < 0.05).

Figure 3

Functional characterization of putative garlic SODs (AsSODs) using the agro-infiltration assay. Total SOD activity (A) and activity pattern of SOD isozymes (B) were determined in N. benthamiana leaves infiltrated with Agrobacterium containing either each AsSOD overexpression construct or a GUS-overexpression construct (treated control). Arrows indicate a distinctive SOD activity band. The bars represent the mean ± SE across three independent experiments. Different letters indicate significant differences (p < 0.05).