The nature of live chloroplast gross morphology and thylakoid membrane architecture of higher plants in cells and in suspension was investigated. Chloroplasts were manipulated inside cells using laser optical tweezers. Moving a chloroplast without towing its neighbouring chloroplasts as well proved very difficult. This finding supports the notion of a cellular cytoskeleton structure holding chloroplasts in position with respect to each other. Routine electron microscopy of Pisum sativum leaves provided evidence for intimate physical contact between chloroplasts and mitochondria, particularly in samples harvested during the night period. A chloroplast stromule was also detected by electron microscopy Atomic force microscopy revealed connections between chloroplasts.
Manipulation of isolated chloroplasts with optical tweezers as well as the use of micro-turbulence in the surrounding buffer showed them to be cup-shaped rather than elliptical as seen in electron micrographs. In situ chloroplasts were concavo-convex to a lesser degree.
Thylakoid structure was studied principally with fluorescence based techniques including confocal and widefield de-convolution microscopy. PSII fluorescence from grana was easily detected by confocal microscopy, but stroma lamellae proved more cryptic. There was almost no fluorescence emitted from the intergranal regions.
According to most literature, based on biochemical and immunological evidence, the two photosystems are unevenly distributed in the thylakoid membrane. PSII quenchers or PSI enhancers mostly did not enhance PSI fluorescence in stroma lamellae, except for methyl viologen, which elicited some fluorescence from the intergranal region. An emission filter designed to exclude PSII chlorophyll fluorescence provided less bright but identical images to those supposedly of PSII predominantly.
Investigations of bundle sheath chloroplasts of C4 species, with negligible PSII activity showed confocal microscopy capable of detecting PSI fluorescence at room temperature. Therefore confocal microscopy must be able to detect PSI fluorescence in mesophyll chloroplasts. The confocal evidence suggests colocation of PSII and PSI in the grana of mesophyll chloroplasts. To visualise PSI directly we transformed Arabidopsis thaliana with a GFP-PsaE fusion construct. GFP-labelled PSI appeared as a membrane fretwork, quite different from the grana of chlorophyll fluorescence images. Comparison of GFP and chlorophyll images suggests considerable structural co-location of PSI and PSII.
Evidence for a coiled string type thylakoid membrane was provided by confocal microscope images of Pisum sativum, and Spathiphyllum sp. chloroplasts. However, the Arabidopsis thalia thylakoid membrane appeared as a three-dimensional fretwork, instead of a coiled string.
Experiments investigating the effects of diurnal light changes on Pisum sativum chloroplasts in situ revealed a significant decrease in their fluorescence after a few hours of darkness. The chloroplasts looked extremely dim and homogeneous. Pulse modulated fluorescence and calculated electron transport rates decreased correspondingly. Daylight induced a return to their bright granal appearance and high PAM fluorescence and electron transport rates.