SUPEROXIDE PRODUCTION BY THYLAKOIDS DURING CHILLING AND ITS IMPLICATION IN THE SUSCEPTIBILITY OF PLANTS TO CHILLING-INDUCED PHOTOINHIBITION

Factors influencing the rate of superoxide (O2-) 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 20-degrees-C or 45-degrees-C. Superoxide production was determined by electron-spin-resonance spectroscopy of the O2--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 O2- production by thylakoids, incubated at 25-degrees-C and 350-mu-mol photon.m-2. s-1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5-mu-mol . (mg chlorophyll)-1. h-1. Incubation at 5-degrees-C and a moderate PFD, decreased the rate of O2BAR production 40% and 15% by thylakoids from S. oleracea and 20-degrees-C-grown N. oleander, chilling-insensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45-degrees-C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O2--production at 25-degrees-C by 75-100%. With these electron acceptors, lowering the temperature to 5-degrees-C caused only a slight decrease in O2- production. In the absence of added electron acceptors, thylakoids produced O2- 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 O2- production under all conditions tested. The results show that the rate of O2- 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 O2- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.