The apoplastic oxidative burst as a key factor of hyperhydricity in garlic plantlet in vitro

The phenomenon of hyperhydricity, a physiological disorder occurring frequently in tissue culture, causes ultrastructural modification and metabolic alteration of shoots. Reactive oxygen species (ROS) accumulation and oxidative stress induction are common features during the development of hyperhydricity, but the relationship between organelle redox homeostasis and hyperhydricity with ultrastructural abnormalities is unclear. To investigate the origin of oxidative stress-induced hyperhydricity, changes in oxygen metabolism in different subcellular compartments of garlic plantlets in vitro were studied. Under exogenous hydrogen peroxide (H2O2) stress, the chloroplastic and mitochondrial ultrastructure was disrupted, which was concomitant with aggravated frequency and severity of hyperhydricity. The addition of H2O2 to the growth medium enhanced superoxide anion generation and H2O2 content in the subcellular compartments. Accumulation of ROS was the highest in apoplasts. Compared with control shoots, in apoplasts exogenous H2O2 stimulated a sharp increase in superoxide dismutase activity within 4 days and a sharp increase in ascorbate peroxidase and glutathione reductase activities and in ascorbic acid and glutathione contents after 8 days of H2O2 treatment. In the other subcellular compartments, dramatic improvement of the antioxidant system occurred after 12 days. Thus, the apoplast was the most sensitive compartment among those investigated. Apoplastic ROS might play a signaling role to participate in the coordination of stress adaptation. The apoplastic oxidative burst in garlic plantlets in vitro is an early response to the development of hyperhydricity.