Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b₆f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.Desiccation tolerance, the ability to survive absolute water contents down to approximately 0.1 g water g⁻¹ dry weight, is a trait found in some bacteria, algae, fungi, as well as animals and plants. In the plant kingdom, desiccation tolerance is common in ferns, mosses, and most seeds and pollen of flowering plants (angiosperms). Resurrection plants, a diverse group of approximately 300 angiosperm species, possess this trait also in their vegetative tissues. These plants are able to withstand prolonged periods of dehydration and to recover within hours to a few days once water is available. A major and interesting aspect in the study of desiccation tolerance in resurrection plants is how they protect themselves against oxidative damage during dehydration, which is often accompanied by conditions of high irradiance (for review, see Bartels and Hussain, 2011; Farrant and Moore, 2011; Morse et al., 2011).A decrease in water content quickly results in lowered leaf stomatal conductance and, consequently, decreased uptake of CO₂. This hinders and ultimately blocks the Calvin cycle. The light-driven reactions, however, typically continue well after the onset of water deficiency, with intact chlorophyll-protein complexes absorbing light energy. The imbalance between the light reactions and the downward biochemical pathways results in a lack of electron sinks and in the system becoming overenergized. This, in turn, leads to enhanced generation of reactive oxygen species (ROS), which inflict damage onto photosynthetic components as well as onto other chloroplast and cellular constituents. At times, the damage may be severe and lead to irreversible impairment and finally plant death (Dinakar et al., 2012).Resurrection plants minimize such potential ROS damage by shutting down photosynthesis during early stages of dehydration (Farrant, 2000; Farrant et al., 2007). There are two mechanisms whereby this is achieved. In poikilochlorophyllous resurrection plants, chlorophyll, along with photosynthetic protein complexes, are degraded, and thylakoids, the membranes that host the photosynthetic pigment-protein complexes, are dismantled. This straightforward mechanism prevents the formation of ROS, yet it comes at the cost of resynthesizing photosynthetic components de novo upon rehydration. On the other hand, homoiochlorophyllous species retain most of their photosynthetic complement and so must rely on other means to protect themselves from oxidative damage in the desiccated state. Some of these, such as leaf folding or curling, which minimize the exposure of inner leaves and/or of adaxial (upper) leaf surfaces to the light, and the accumulation of anthocyanins in leaf surfaces, which act as sunscreens, and the presence of reflective hairs and waxy cuticles, reduce the overall absorption of radiation and thus protect against photodamage (Sherwin and Farrant, 1998; Farrant, 2000; Bartels and Hussain, 2011; Morse et al., 2011). ROS that are generated are dealt with by antioxidants, ROS scavengers, and in some cases also by anthocyanins and other polyphenols (Moore et al., 2005; Kytridis and Manetas, 2006; Farrant et al., 2007). Nevertheless, all of these mechanisms are insufficient to completely prevent and/or detoxify all ROS that are formed, necessitating additional means to prevent or deal with possible damage that ROS may inflict during dehydration and while desiccated (Dinakar et al., 2012).The major photoprotective mechanism in plants and algae is nonphotochemical quenching (NPQ), in which excess light energy absorbed at the antennae of PSII is dissipated as heat. NPQ has been shown to be active in desiccation-tolerant bryophytes and pteridiophytes (Eickmeier et al., 1993; Oliver, 1996), in homoiochlorophyllous angiosperms (Alamillo and Bartels, 2001; Georgieva et al., 2009; Dinakar and Bartels, 2012; Huang et al., 2012), and during the initial stages of drying in poikilochlorophyllous angiosperms (Beckett et al., 2012). Photoinhibition, when damage to PSII (mainly to its D1 subunit) exceeds the repair capacity, typically under conditions of light stress, is also observed in homoiochlorophyllous resurrection plants (e.g. Georgieva and Maslenkova, 2006). Other ways to avoid ROS-induced damage include the rerouting of reducing equivalents to alternative electron sinks, such as the water-water cycle and/or photorespiration, as well as structural rearrangements of PSII and light-harvesting antenna (LHCII) complexes into energy-dissipating states (for review, see Dekker and Boekema, 2005; Yamamoto et al., 2014). These latter processes, in particular the ones pertaining to possible changes in PSII-LHCII macrostructure, have not yet been characterized in homoiochlorophyllous resurrection plants.To gain insight into the ways homoiochlorophyllous resurrection plants cope with dehydration while retaining most of their photosynthetic apparatus, we combined microscopic, spectroscopic, and biochemical approaches. Investigation of the supramolecular organization of photosynthetic complexes was carried out using cryoscanning electron microscopy (cryo-SEM) of high-pressure frozen, freeze-fractured leaf samples; to our knowledge, this combination of procedures has not been utilized previously to investigate thylakoid membranes within plant tissues.The studies reveal that during dehydration, the density of PSII in grana membranes gradually decreases. Notably, in the dehydrated state, in which photosynthetic activity is halted, PSII complexes are also observed to be arranged into rows and two-dimensional arrays. These arrangements are proposed to represent quenched PSII complexes that likely minimize the generation of ROS during desiccation. Furthermore, we observe inverted hexagonal (H_II) phases in this dry state, and these two structural rearrangements are correlated with the massive accumulation of Suc. Biochemical studies of thylakoid membrane fractions support the finding that the relative level of PSII proteins decreases during dehydration. These analyses also reveal that the level of the cytochrome f subunit of the cytochrome b₆f complex decreases quite dramatically and early during dehydration. This provides evidence for an additional level of regulation that inhibits/shuts down the photosynthetic light reactions during desiccation.