3-D STRUCTURE OF GRANUM-STROMA THYLAKOID ASSEMBLIES IN HIGHER

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The helical arrangement of the granal thylakoidal system is evident mostly from serial section EM studies on chemically fixed specimens on single granum or on a pair of grana. These do not reveal the 3D arrangement (network) of grana that interconnects with stroma thylakoids. The ontogeny of grana, the variation of its size, its flexibility under various environmental conditions (high light  low light, short term adaptation, long-term acclimation), structural consequences of the phosophorylation of membranes, and the role of the physico-chemical environment (osmolality, ionic strength) are also poorly undrestood. Futher, there are some uncertainties because of possible artifacts due to the chemical fixation. Granal chloroplasts have not been studied by electron tomographic techniques  perhaps because of the large size and dense structure of whole, mature chloroplasts. Thus we plan, in addition, to attempt the imaging of intact chloroplasts (and thylakoid membranes, i.e. plastids without their envelope membranes and soluble components), by the use of plastids with small-sized grana, and granum stroma assemblies isolated by sonication and/or cleaved by detergents. We have detailed knowledge concerning the organization of complexes and lamellae in the granum-stroma assemblies by a wealth of other techniques, such as circular dichroism spectroscopy that reveals the macrodomain organization (300-400 nm in diameter) and small angle X-ray and neutron scattering which provide information on the variations in the periodic ultrastructure, as well as their photosynthetic functions. It has also been established that these structures are capable of undergoing light-induced reversible structural organization by a previously unknown physical mechanism in biology, the thermo-optic effect. In the previous reporting period, several tomographic reconstructions were made. Drs. Must¿rdy and Garab visited the RVBC for one week. They observed and participated in data collection and tomographic reconstruction. A tomogram was made showing how the grana are linked within the thylakoid, a point they had been hoping to clarify. They presented a lecture showing a variety of techniques brought to bear on understanding structure/function of the chloroplast.