In plants, the thylakoid is a network of membranes contained in the chloroplast, storing chlorophyll, and in which photosynthesis, a highly regulated process sensitive to environmental and light variations, is initiated. Thanks to numerous electron microscopy studies, the internal organisation of the thylakoid membrane (granum) is now established, and it is known that this membrane architecture plays a major role in regulating photosynthesis. However, the question of how is controlled the flow of energy during the early stages of photosynthesis remains unresolved and, ideally, live cell imaging would enable structures and processes to be visualised in vivo or in a state close to the native state. Fluorescence imaging is the method of choice for observing photosynthetic membranes, as there is no need for staining or labelling procedures, as the functional architecture of the thylakoid membrane in chloroplasts can be examined through the natural fluorescence of chlorophyll molecules.
In this work, the researchers used a special fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) which offers a gain of 1.4 in lateral and axial resolution compared with confocal fluorescence microscopy and also allows 3D volumetric reconstruction from 2D fluorescence images. The 3D architecture of the thylakoids, revealing the complete photosynthetic membrane network in intact, non-chemically fixed chloroplasts, was visualised using high-resolution volume reconstructions obtained on isolated chloroplasts from peas (Pisum sativum), spinach (Spinacia oleracea) and the model plant Arabidopsis thaliana. In agreement with previous studies, SPiRI images reveal larger diameters of thylakoid membrane stacks when derived from plants subjected to low light regimes. The stromal connections between each granule was also determined and the fluorescence intensity in the stromal lamellae was compared with that of neighbouring granules.
3D illustration of the structure of a chloroplast © GettyImages/Tumeggy
Using their original SPiRI approach, the researchers resolved the 3D structure of the thylakoid membranes of native chloroplasts with very good resolution. A step towards a better understanding of the role of thylakoids in the photosynthetic adaptation of plants to their environment.
Contacts : Bruno Robert (bruno.robert@cea.fr); Andrew Gall (andrew.gall@cea.fr)
Chloroplasts, the intracellular organelles responsible for photosynthesis, contain the photosynthetic membranes or thylakoids, which are themselves organised as flattened sacs running lengthways along the plastid. These sacs can be juxtaposed, forming a granum that appears as green granules under the light microscope and as stacks under the electron microscope.