Photosynthesis Research 53: 197–204, 1997. 197 c 1997 Kluwer Academic Publishers. Printed in the Netherlands. Regular paper Ascorbate in thylakoid lumen as an endogenous electron donor to Photosystem II: Protection of thylakoids from photoinhibition and regeneration of ascorbate in stroma by dehydroascorbate reductase Junichi Mano 1 , Takashi Ushimaru 2 & Kozi Asada 1 1 Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan (for corresponding and reprints); 2 Department of Biology, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422, Japan; Present address: Department of Biotechnology, Faculty of Engineering, Fukuyama University, 1 Gakuen-cho, Fukuyama-shi, Hiroshima 729-02, Japan Received 1 November 1996; accepted in revised form 12 June 1997 Key words: donor-side induced photoinhibition, electron spin resonance, monodehydroascorbate radical, monode- hyroascorbate reductase Abstract Photoinhibition of the electron transport activity from tyrosine Z (Y Z ) in PS II to NADP in Tris-treated thylakoids was suppressed by electron donation with either diphenylcarbazide or ascorbate (AsA) during the photoinhibition treatment. This suggests that AsA prevents donor side-induced photoinhibition in vivo as an endogenous donor. AsA in the lumen is photooxidized to monodehydroascorbate (MDA) in Tris-treated thylakoids, as detected by electron spin resonance spectrometry, but not in oxygenic thylakoids. Redox analysis of pyridine nucleotide in the presence of either MDA reductase or dehydroascorbate (DHA) reductase showed that the MDA photoproduced in the lumen is disproportionated to AsA and DHA, and the DHA leaking into the stroma is reduced to AsA by DHA reductase. No leakage of MDA through the thylakoid membrane was observed. Thus, the DHA-reducing enzyme system is indispensable in maintaining AsA concentrations in chloroplasts. Abbreviations: AsA – ascorbate; DHA – dehydroascorbate; DPC – 1,5-diphenylcarbazide; DTT – dithiothreitol; ESR – electron spin resonance; Fd – ferredoxin; GSH – reduced form of glutathione; GSSG – oxidized form of glu- tathione; MDA – monodehydroascorbate radical; Mes – 2-(N-morpholino)ethanesulfonic acid; P680 – reaction cen- ter chlorophyll of PS II; SOD – superoxide dismutase; Tris – tris(hydroxymethyl)aminomethane; UV – ultraviolet irradiation; VDE – violaxanthin de-epoxidase; Y D – Tyr-160 in D2 protein; Y Z – Tyr-161 in D1 protein Introduction Various environmental stresses on plants, such as drought, high- or low temperature, ultraviolet irradia- tion (UV), and pollutant gases, suppress CO 2 -utilizing capacity of chloroplasts, leading to an overflow of light energy which activates dioxygen. The resulting reactive species of oxygen ( 1 O 2 ,O 2 ,H 2 O 2 , OH), if not immediately scavenged, oxidize various target molecules to halt photosynthesis (photoinhibition) and promote destructive chain reactions to cell death. Ascorbate (AsA) is present at 10–50 mM levels in chloroplasts; it protects the organelle from photoin- hibition (Asada 1994). AsA suppresses the accumu- lation of photoproduced H 2 O 2 in the stroma by act- ing as the electron donor of AsA peroxidase to protect CO 2 -fixation enzymes from inactivation (Asada 1992). AsA peroxidase reduces H 2 O 2 to H 2 O, producing mon- odehydroascorbate (MDA), the univalently oxidized product of AsA (Hossain et al. 1984). AsA also non- enzymically reduces O 2 , OH, glutathione thiyl rad- ical, the chromanoxyl radical of -tocopherol, and