Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition.

Factors influencing the rate of superoxide (O 2 (-) ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O 2 (-) )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O 2 (-) production by thylakoids, incubated at 25° C and 350 μmol photon · m(-2) · s(-1) (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 μmol · (mg chlorophyll)(-1) · h(-1). Incubation at 5° C and a moderate PFD, decreased the rate of O 2 (-) production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O 2 (-) -production at 25° C by 75-100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O 2 (-) production. In the absence of added electron acceptors, thylakoids produced O 2 (-) at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea reduced O 2 (-) production under all conditions tested. The results show that the rate of O 2 (-) production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O 2 (-) production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.