Photoinhibitory printing on leaves, visualized by chlorophyll fluorescence imaging and confocal microscopy, is due to diminished fluorescence from grana

Abstract

By analogy with the starch printing technique, it was hypothesised that photoinhibition could be used to print images on leaves that would be invisible to the eye, but easily revealed by chlorophyll fluorescence imaging. We first illustrate the process of chlorophyll fluorescence printing on leaves of the shade plant, Cissus rhombifolia, using photographs of artefacts from starch printing experiments in the laboratory of Molisch. We then use portraits of current leaders in chlorophyll fluorescence research to demonstrate the stability of these images in living tissues. Text printing from microfilm of Ewart's pioneering studies in photoinhibition shows the resolution of the method with the fixed-focus, portable, imaging system used here. The stability of images, as well as quenching analysis of images and of leaves, suggests that localised photoinactivation, rather than sustained photoprotection, is responsible for the detail displayed by fluorescence printing. Electron micrograph positives of stained thylakoids can be printed to create an illusion of what is imagined to be the source of chlorophyll fluorescence at the membrane level. Individual chloroplasts in adjacent cells under the grid pattern of granal stacks printed on leaves were also examined using a confocal microscope. Compared with chloroplasts in the shaded parts of the grid, those in the photoinactivated parts of the grid show greatly reduced chlorophyll autofluorescence. Moreover, these chloroplasts have lost the localised bright fluorescence from grana. Comparisons of fluorescence yields show that relative chlorophyll autofluorescence from grana observed in the confocal microscope parallels that determined in leaves. Our experiments provide direct visual evidence that fluorescence from grana is lost following photoinactivation of photosystem II in vivo.