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Reactive oxygen species (ROS), namely superoxide radical (O2(-)) and hydrogen peroxide (H(2)O(2)) are generated when plant tissues endure a variety of environmental stresses, including light stress. The extremely short life times of ROS makes the study of their production in planta very difficult. The use of ROS-specific tracer dyes, 3-3' diaminobenzidine and nitroblue tetrazolium, together with high-resolution imaging provides the opportunity to identify sites of photooxidative stress response by ROS accumulation. This technique was applied to grapevine during the first 7 days after transfer from in vitro to ex vitro under an irradiance 4-fold higher than in vitro. ROS accumulation was detected in the first days of analysis, which gradually decreased to levels comparable to greenhouse leaves. (O2(-)) was uniformly distributed while H(2)O(2) accumulated preferentially in veins, wounds and stomatal guard and surrounding cells. To evaluate the role of H(2)O(2 )in stomatal functioning and its crosstalk with abscisic acid (ABA) we focused on the percentage of coloured structures, stomatal aperture and ABA concentration. We propose that the high H(2)O(2) level triggered by increased light is responsible for the activation of a signalling pathway over stomatal cells, in a process apparently irrespective of ABA regulation prior to root protrusion. This could explain the gain of function of a low yet consistent percentage of stomatal cells, essential for plant survival during the ontogenic period in analysis.

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