Protection of the photosynthetic apparatus during desiccation in resurrection plants

In this project, we studied the photosynthetic apparatus during dehydration and rehydration of the homoiochlorophyllous resurrection plant Craterostigmapumilum (retains most of the photosynthetic components during desiccation). Resurrection plants have the remarkable capability to withstand desiccation, being able to revive after prolonged severe water deficit in a few days upon rehydration. Homoiochlorophyllous resurrection plants are very efficient in protecting the photosynthetic machinery against damage by reactive oxygen production under drought. The main purpose of this BARD project was to unravel these largely unknown protection strategies for C. pumilum. In detail, the specific objectives were: (1) To determine the distribution and local organization of photosynthetic protein complexes and formation of inverted hexagonal phases within the thylakoid membranes at different dehydration/rehydration states. (2) To determine the 3D structure and characterize the geometry, topology, and mechanics of the thylakoid network at the different states. (3) Generation of molecular models for thylakoids at the different states and study the implications for diffusion within the thylakoid lumen. (4) Characterization of inter-system electron transport, quantum efficiencies, photosystem antenna sizes and distribution, NPQ, and photoinhibition at different hydration states. (5) Measuring the partition of photosynthetic reducing equivalents between the Calvin cycle, photorespiration, and the water-water cycle.   At the beginning of the project, we decided to use C. pumilum instead of C. wilmsii because the former species was available from our collaborator Dr. Farrant. In addition to the original two dehydration states (40 relative water content=RWC and 5% RWC), we characterized a third state (15-20%) because some interesting changes occurs at this RWC. Furthermore, it was not possible to detect D1 protein levels by Western blot analysis because antibodies against other higher plants failed to detect D1 in C. pumilum.   We developed growth conditions that allow reproducible generation of different dehydration and rehydration states for C. pumilum. Furthermore, advanced spectroscopy and microscopy for C. pumilum were established to obtain a detailed picture of structural and functional changes of the photosynthetic apparatus in different hydrated states. Main findings of our study are: 1. Anthocyan accumulation during desiccation alleviates the light pressure within the leaves (Fig. 1). 2. During desiccation, stomatal closure leads to drastic reductions in CO2 fixation and photorespiration. We could not identify alternative electron sinks as a solution to reduce ROS production. 3. On the supramolecular level, semicrystalline protein arrays were identified in thylakoid membranes in the desiccated state (see Fig. 3). On the electron transport level, a specific series of shut downs occur (summarized in Fig. 2). The main events include: Early shutdown of the ATPase activity, cessation of electron transport between cyt. bf complex and PSI (can reduce ROS formation at PSI); at higher dehydration levels uncoupling of LHCII from PSII and cessation of electron flow from PSII accompanied by crystal formation. The later could severe as a swift PSII reservoir during rehydration.   The specific order of events in the course of dehydration and rehydration discovered in this project is indicative for regulated structural transitions specifically realized in resurrection plants. This detailed knowledge can serve as an interesting starting point for rationale genetic engineering of drought-tolerant crops.