In vivo 1H NMR methods to study dynamics of chloroplast water and thylakoid membrane lipids in leaves and in photosynthetic microorganisms

Dynamics of thylakoid membranes and mobility of pigment-protein complexes therein are essential for survival of photosynthetic organisms under changing environmental conditions. The published approaches to probe mobility of the thylakoid membrane lipids and protein complexes are either dependent on the use of external labels or are used only for <em>in vitro</em> studies. Here, we present non-invasive 1H NMR methods (DOSY and DRCOSY) to study dynamics of water in chloroplasts, lipids in oil bodies and in thylakoid membranes and pigment-protein complexes under complete <em>in vivo</em> conditions in leaf disks of <em>F. benjamina</em> and <em>A. platanoides</em> and in suspensions of the green alga <em>Chlamydomonas reinhardtii </em>and blue-green alga <em>Synechocystis</em>sp.PCC 6803. In leaf disks of <em>Ficus benjamina</em> and<em> Acer </em><em>platanoides</em>, water in chloroplasts could be clearly discriminated from other pools. Both water in chloroplasts, and water in vacuoles of palisade and spongy cells showed resonances in the high field part of the spectra (with respect to pure water), in contrast to what has been reported in literature. Subepidermal cells (present only in <em>F. benjamina</em> but not in <em>A. platanoides</em>) may act as a water storage, buffer pool during drought. This pool prevented the fast loss of water from the chloroplasts. Nutrient stress and excess salt stress resulted in accumulated lipid bodies and in striking differences in the dynamics and spectra/composition of the different components. T2 values of the different components are compared with those observed in suspensions of <em>Synechocystis</em>sp.PCC 6803. The differences in membrane composition (ratio of the different membrane lipids) were clearly observed in the DANS of the oil bodies and the (thylakoid) membranes, but the diffusion coefficients were quite comparable. Also the DANS of the component that is assigned to the pigment-protein complexes are quite different, reflecting the differed composition. The diffusion coefficients of this component in isolated spinach thylakoids and in C. reinhardtii are very comparable, but about a factor of 10 lower with respect to that of Synechocystis at short diffusion times. The dynamics of these complexes in these systems are thus quite different.