Previously, we showed that ascorbate (Asc), by donating electrons to photosystem II (PSII), supports a sustained electron transport activity in leaves in which the oxygen-evolving complexes were inactivated with a heat pulse (49°C, 40 s). Here, by using wild-type, Asc-overproducing, and -deficient Arabidopsis (Arabidopsis thaliana) mutants (miox4 and vtc2-3, respectively), we investigated the physiological role of Asc as PSII electron donor in heat-stressed leaves (40°C, 15 min), lacking active oxygen-evolving complexes. Chlorophyll-a fluorescence transients show that in leaves excited with trains of saturating singleturnover flashes spaced 200 ms apart, allowing continual electron donation from Asc to PSII, the reaction centers remained functional even after thousands of turnovers. Higher flash frequencies or continuous illumination (300 mmol photons m 22 s 21 ) gradually inactivated them, a process that appeared to be initiated by a dramatic deceleration of the electron transfer from Tyr Z to P680 + , followed by the complete loss of charge separation activity. These processes occurred with half-times of 1.2 and 10 min, 2.8 and 23 min, and 4.1 and 51 min in vtc2-3, the wild type, and miox4, respectively, indicating that the rate of inactivation strongly depended on the Asc content of the leaves. The recovery of PSII activity, following the degradation of PSII proteins (D1, CP43, and PsbO), in moderate light (100 mmol photons m 22 s 21 , comparable to growth light), was also retarded in the Ascdeficient mutant. These data show that high Asc content of leaves contributes significantly to the ability of plants to withstand heat-stress conditions.

Previously, we showed that in barley (Hordeum vulgare) leaves with heat-inactivated oxygen-evolving complexes, photosystem II (PSII) has access to a large pool of alternative electron donors. Based on in vitro data, we proposed that this donor was ascorbate, yet this hypothesis has not been substantiated in vivo. In this paper, with the aid of chlorophyll a fluorescence induced by short (5-ms) light pulses and 820-nm absorbance transient measurements on wild-type and ascorbate-deficient (vtc2-1) mutant leaves of Arabidopsis (Arabidopsis thaliana), we show that in heat-treated leaves the rate of electron donation to PSII as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive electron transport toward photosystem I depend on the ascorbate content of the leaves: upon ascorbate treatment, the donation half-time in the wild type and the mutant decreased from 25 to 22 ms and from 55 to 32 ms, respectively. Thermoluminescence measurements show that Tyr Z + is involved in the electron transfer from ascorbate to PSII. These data and the similar ascorbate dependencies of the heat-treated and the tris(hydroxymethyl)aminomethane-treated thylakoid membranes, with maximal donation half-times of about 16 ms, show that ascorbate is capable of supporting a sustained electron transport activity in leaves containing inactivated oxygen-evolving complexes. This alternative electron transport appears to be ubiquitous in the plant kingdom and is present in the green alga Chlamydomonas reinhardtii, and its rate depends on the physiological state of the plants and on environmental conditions. Our data suggest that ascorbate, as an alternative PSII electron donor, plays a physiological role in heat-stressed plants.