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

A study was conducted to identify biophysical markers which change in respo nse to changing chlorophyll organization during plant development. When hea ted to around 70 degrees C in the dark, barley thylakoids emit red thermolu minescence (TL). This is a pure chemiluminescence signal and distinct from the lower-temperature TL bands of thylakoids that are seen only with preill umination. The development of the light-independent, 70 degrees C TL band w as investigated following transfer of dark-grown barley leaves to the light . Because of the rapidly increasing chlorophyll content of the plastid memb rane, the TL signal was normalized against either chlorophyll or tissue mas s of the starting material. In either case, the extent of the TL signal rea ched a maximum in the early hours of greening and then declined. The drop i n signal over 20 h was approximately 50% for TL per unit tissue mass, and w ell over 90% for TL per unit chlorophyll. Exposure of plastid membrane samp les to hydrogen peroxide for several minutes caused a large increase in lig ht-independent TL, while addition of ascorbate caused substantial quenching . The fluorescence profiles of dark-grown barley leaves were recorded followi ng transfer to the light. Basal fluorescence (F-o) reaches a substantial le ver after just seconds of illumination. Over the next few hours, F-o increa ses only slightly and then starts to decline. The decline in F-o is correla ted with an increase in variable fluorescence (F-v) which indicates the app earance of active photosystem II. It is concluded that the early peak in F- o reflects a state in which the leaves contain a maximum amount of disorgan ized chlorophyll. Considering the TL and fluorescence data together, we propose the following : When chlorophyll first appears in the system, it is not properly assemble d into the complexes that offer photochemical or non-photochemical quenchin g of the excited state. Thus, fluorescence and parallel chlorophyll triplet formation are prevalent. The triplets cause generation of active oxygen re sulting in lipid peroxidation and/or other radical-generating processes. Wh en the membranes are healed, increased interaction of the radicals with chl orophyll generates chemiluminescence. We thus conclude that light-independe nt thermoluminescence is a marker for actual damage arising from poor chlor ophyll organization and propose that this parameter might be usefully appli ed for assessing stress effects.