The extremotolerant bacterium Deinococcus radiodurans is used as a model to explore the limits of life on Earth and beyond. In experiments performed in an ultra-high vacuum chamber with a vacuum ultraviolet (VUV) synchrotron beamline, this microorganism was exposed to conditions present on an extraterrestrial environment unprotected by an atmosphere, such as outside a spacecraft or on an asteroid, relevant in the context of planetary protection and panspermia hypothesis. Different methods were used to obtain the biologically relevant information from this investigation. Counting of colony forming units, the traditional approach for viability assessment, is limited to measuring the survival of the cells. For a more in-depth study of damage mechanisms at subcellular levels, specific molecular probes (propidium iodide and dihydrorhodamine 123) were applied and whole populations could be analysed, cell by cell, by flow cytometry. VUV radiation caused a substantial loss of viability, though only a fraction of the cells presented membrane damages even at the largest tested fluences. Additionally, intracellular oxidative stress was also detected upon exposure. These results point to significant VUV inactivating effects extending beyond the cells' outermost structures, in contrast to a more superficial role that could be expected due to the highly interacting nature of this radiation range. Nevertheless, it was observed that microscopic-level shading sufficed to allow the persistence of a small surviving subpopulation for the longer expositions. This study contributes to unveiling the response of biological systems under space conditions, assessing not just cell viability but also the mechanisms that lead to inactivation.

Interchangeable effects of temperature, moisture content and oxygen on seed longevity have been mostly examined to estimate seed viability during long-term dry storage, whereas few experiments have studied seed viability under near-natural conditions to evaluate seed persistence in the soil. To this end, we artificially aged seeds of Ranunculus baudotii, a hydrophyte widely distributed in temporary ponds constituting an abundant soil seed bank. Seeds were exposed to controlled ageing at three different relative humidities (RH) under both aerobic and anoxic conditions. Their viability, water content, membrane damage, oxidative stress and anti-oxidant enzymatic defence activity were evaluated. Seed survival was longer at higher relative humidity (97% RH), and lowest at a relative humidity (90% RH) simulating moist but not waterlogged soils. Anoxic conditions showed a protective role on viability at lower moisture contents (70% RH). Seed viability was negatively associated with hydrogen peroxide content and correlated with anti-oxidant enzyme activities, but not with membrane damage. Altogether, these results suggest negative roles for moist soils and anoxia in determining seed persistence in the field, but at higher moisture contents the negative effects of anaerobia diminished. The anti-oxidant system activation, even under unfavourable conditions, might recover seeds once all protective processes can operate, pointing out the plasticity of mechanisms involved in seed loss viability.

Removing lichen substances from dry lichen thalli using pure acetone is the least detrimental method. Measurements of properties strictly related to the photobiont, such as chlorophyll a fluorescence (ChlaF), are frequently used to verify acetone toxicity but they cannot reveal possible damage accumulated at the whole thallus level. Here, measurements of ChlaF have been integrated with others concerning the status of cell membranes and photobiont population (potassium leakage, malondialdehyde and photosynthetic pigment content). Dry thalli of Flavoparmelia caperata, Parmotrema perlatum and Xanthoria parietina were subjected to sequential acetone washings according to standard protocols. Membrane permeability was assessed before and after the washing treatment, and after a recovery period of 48 hours. Measurements of ChlaF were taken in a parallel experiment. Acetone washings increased potassium leakage in all the species from 3·9 to 6·6 times greater than the control level. After recovery, only P. perlatum returned to the control level. ChlaF was affected only in F. caperata, with a 20% decrease in Fv/Fm which had not fully recovered after 48 hours. There was neither an increase in lipid peroxidation of membranes nor a change in the photosynthetic pigment content. The sensitivity of F. caperata to this method and the impact of the results on its future application are discussed.

Solute leakage is used as an indicator of membrane damage during desiccation of recalcitrant seeds. We re-examined this phenomenon and its utility by comparing recalcitrant Spartina alterniflora and orthodox Spartina pectinata seeds, as well as drying isolated embryos or embryos isolated after whole-seed drying. During drying, intact seed leakage (electrical conductivity and absorbance at 280 nm) was independent of seed moisture content, dormant/non-dormant state or desiccation tolerance. Embryos from both Spartina species, isolated after drying within the intact seed, exhibited increased leakage, especially below 40% (dry weight basis, DWB) seed moisture, the critical water content for viability loss in Spartina alterniflora. When isolated embryos were dried, the pattern and extent of increased solute leakage were similar in both Spartina species, even though S. pectinata embryos were >95% viable and S. alterniflora embryos were < 20% viable. We conclude that increased solute leakage is an artefact of embryo excision and not an accurate indicator of desiccation damage to recalcitrant Spartina alterniflora seeds.

Protein composition of sunflower seeds (Helianthus annuus L.) was analysed to identify a possible marker for germination capacity and to evaluate seed viability precociously after harvest and after natural ageing during storage. Electrophoresis on 2-D gels was used to compare total soluble and in vivo synthesized proteins in two seed lots showing different germination capacities. Differences were observed to a greater extent in in vivo protein synthesis profiles than in total soluble proteins. Inhibition of [35S]methionine uptake and incorporation in seeds with low germinability was also observed. The precise relationship between protein composition and prediction of germinability could not be determined. Other aspects such as membrane damage, capacity for protein synthesis and seed lot homogeneity in relation to germination are discussed.

Desiccation tolerance is one of the most fundamental properties of seeds. It is acquired late in seed development and is considered necessary for the completion of the plant's life cycle, as an adaptive strategy to enable seed survival during storage or environmental stress, and to ensure better dissemination of the species. The role of water status in desiccated tissues and problems related to testing tolerance in seeds are reviewed. The molecular mechanisms of desiccation tolerance has received extensive consideration only during this past decade. There is a general consensus that desiccation tolerance involves the protection of cellular membranes from the deleterious effect of water removal and the resultant necessity to maintain the bilayer structure in the absence of an aqueous environment. Therefore, some aspects of desiccation-induced membrane injury are described. Several strategies for coping with cellular desiccation have been identified the presence of high amounts of non-reducing sugars, the efficiency of free radical-scavenging systems and the expression of desiccation- and/or ABA-regulated genes. These molecular mechanisms allowing cellular protection are reviewed together with their respective role in dessication tolerance. It is concluded that desiccation tolerance is not likely to be ascribed to a single mechanism but rather to a multifactorial property in which each component is equally critical.

Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85% glycerol. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis were incubated in 85% glycerol. The influence of duration of incubation and temperature on ultrastructure and viability were investigated. Unstressed cultures served as controls. Survival was studied after 24–36 h and 10 days incubation in 85% glycerol at 4°C and 36°C with transmission electron microscopy (TEM) and flow cytometry using viability stains indicating membrane damage (SYTO9, propidium iodide) or esterase activity (carboxyfluorescein diacetate). TEM clearly demonstrated variability in morphological changes of bacteria suggesting different mechanisms of damage. Viability stains supported these findings with faster declining viable cell populations in 85% glycerol at 36°C compared with 4°C. Both methods demonstrated that Gram-negative species were more susceptible than Gram-positive species. In conclusion, 85% glycerol may have some additional antimicrobial effect. Temperature is an important factor herein and Gram-negatives are most susceptible. The latter finding probably reflects the difference in cell wall composition between Gram-positive and Gram-negative bacteria.

Summary 185

I. INTRODUCTION 186

II. EFFECT OF ZINC ON PRODUCTION OF REACTIVE OXYGEN SPECIES 186

1. Superoxide-generating NADPH oxidase 186

2. Zinc deficiency potentiates iron-mediated free radical production 189

(a) Iron accumulation in zinc-deficient plants 189

(b) Iron-induced production of free radicals 189

3. Zinc deficiency-enhanced photooxidation 191

(a) Decrease in photosynthesis 191

(b) Light-induced leaf chlorosis 192

(c) Decrease in indole-3-acetic acid 192

III. MEMBRANE DAMAGE BY REACTIVE OXYGEN SPECIES 193

1. Impairments in membrane structure 193

2. Phospholipids and –SH groups 195

3. Alterations in ion absorption 195

(a) Membrane-bound ATPases 195

(b) Nutrient uptake 197

(c) Changes in activity of ion channels 197

IV. DETOXIFICATION OF REACTIVE OXYGEN SPECIES 198

1. Superoxide dismutases 198

2. H2O2-scavenging enzymes 198

V. INVOLVEMENT OF ZINC IN PLANT STRESS TOLERANCE 199

VI. CONCLUSIONS 199

Acknowledgements 200

References 200

Zinc deficiency is one of the most widespread micronutrient deficiencies in plants and causes severe reductions in crop production. There are a number of physiological impairments in Zn-deficient cells causing inhibition of the growth, differentiation and development of plants. Increasing evidence indicates that oxidative damage to critical cell compounds resulting from attack by reactive O2 species (ROS) is the basis of disturbances in plant growth caused by Zn deficiency. Zinc interferes with membrane-bound NADPH oxidase producing ROS. In Zn-deficient plants the iron concentration increases, which potentiates the production of free radicals. The Zn nutritional status of plants influences photooxidative damage to chloroplasts, catalysed by ROS. Zinc-deficient leaves are highly light-sensitive, rapidly becoming chlorotic and necrotic when exposed to high light intensity. Zinc plays critical roles in the defence system of cells against ROS, and thus represents an excellent protective agent against the oxidation of several vital cell components such as membrane lipids and proteins, chlorophyll, SH-containing enzymes and DNA. The cysteine, histidine and glutamate or aspartate residues represent the most critical Zn- binding sites in enzymes, DNA-binding proteins (Zn-finger proteins) and membrane proteins. In addition, animal studies have shown that Zn is involved in inhibition of apoptosis (programmed cell death) which is preceded by DNA and membrane damage through reactions with ROS.