Light‐independent thermoluminescence from thylakoids of greening barley leaves. Evidence for involvement of oxygen radicals and free chlorophyll

Abstract

A study was conducted to identify biophysical markers which change in response to changing chlorophyll organization during plant development. When heated to around 70°C in the dark, barley thylakoids emit red thermoluminescence (TL). This is a pure chemiluminescence signal and distinct from the lower‐temperature TL bands of thylakoids that are seen only with preillumination. The development of the light‐independent, 70°C TL band was investigated following transfer of dark‐grown barley leaves to the light. Because of the rapidly increasing chlorophyll content of the plastid membrane, the TL signal was normalized against either chlorophyll or tissue mass of the starting material. In either case, the extent of the TL signal reached a maximum in the early hours of greening and then declined. The drop in signal over 20 h was approximately 50% for TL per unit tissue mass, and well over 90% for TL per unit chlorophyll. Exposure of plastid membrane samples to hydrogen peroxide for several minutes caused a large increase in light‐independent TL, while addition of ascorbate caused substantial quenching.The fluorescence profiles of dark‐grown barley leaves were recorded following transfer to the light. Basal fluorescence (F0) reaches a substantial level after just seconds of illumination. Over the next few hours, F0 increases only slightly and then starts to decline. The decline in F0 is correlated with an increase in variable fluorescence (Fv) which indicates the appearance of active photosystem II. It is concluded that the early peak in F0 reflects a state in which the leaves contain a maximum amount of disorganized chlorophyll.Considering the TL and fluorescence data together, we propose the following: When chlorophyll first appears in the system, it is not properly assembled into the complexes that offer photochemical or non‐photochemical quenching of the excited state. Thus, fluorescence and parallel chlorophyll triplet formation are prevalent. The triplets cause generation of active oxygen resulting in lipid peroxidation and/or other radical‐generating processes. When the membranes are heated, increased interaction of the radicals with chlorophyll generates chemiluminescence. We thus conclude that light‐independent thermoluminescence is a marker for actual damage arising from poor chlorophyll organization and propose that this parameter might be usefully applied for assessing stress effects.