Topography of UV-Melanized Thalli of Lobaria pulmonaria (L.) Hoffm.

Lichens are unique extremophilic organisms due to their phenomenal resistance to adverse environmental factors, including ultraviolet (UV) irradiation. Melanization plays a special role in the protection of lichens from UV-B stress. In the present study, we analyzed the binding of melanins with the components of cell walls of the mycobiont of the upper cortex in the melanized lichen thalli Lobaria pulmonaria. Using scanning electron and atomic force microscopy, the morphological and nanomechanical characteristics of the melanized layer of mycobiont cells were visualized. Melanization of lichen thalli led to the smoothing of the surface relief and thickening of mycobiont cell walls, as well as the reduction in adhesion properties of the lichen thallus. Treatment of thalli with hydrolytic enzymes, especially chitinase and lichenase, enhanced the yield of melanin from melanized thalli and promoted the release of carbohydrates, while treatment with pectinase increased the release of carbohydrates and phenols. Our results suggest that melanin can firmly bind with hyphal cell wall carbohydrates, particularly chitin and 1,4-ß-glucans, strengthening the melanized upper cortex of lichen thalli, and thereby it can contribute to lichen survival under UV stress.

Effect of Melanization on Thallus Microstructure in the Lichen Lobaria pulmonaria.

Lichens often grow in microhabitats where they experience severe abiotic stresses. Some species respond to high UV radiation by synthesizing dark brown melanic pigments in the upper cortex. However, unlike the melanized structures of non-lichenized fungi, the morphology of the melanic layer in lichens remains unstudied. Here, we analyzed the morphology, ultrastructure, and elemental composition of the melanized layer in UV-exposed thalli of the lichen Lobaria pulmonaria (L.) Hoffm. Using light microscopy, we detected a pigmented layer sensitive to staining with 3,4-L-dihydroxyphenylalanine, a precursor of eumelanin, in the upper cortex of melanized thalli. Analysis of cross-sections of melanized thalli using scanning electron microscopy revealed that melanin-like granules are deposited into the hyphal lumens. Melanized thalli also possessed thicker hyphal cell walls compared to pale thalli. Energy-dispersive X-ray spectroscopy analysis of the elemental composition of the hyphal walls and extracted melanin indicated that the type of melanin synthesized by L. pulmonaria is eumelanin. Transmission electron microscopy was used to show that during melanization melanosome-like dark vesicles are transported to the cell surface and secreted into the cell walls of the fungal hyphae. Results from this study provide new insights into the effects of melanin synthesis on the microstructure of lichen thalli.

Cathodic Water Enhances Seedling Emergence and Growth of Controlled Deteriorated Orthodox Seeds.

All orthodox seeds eventually deteriorate during storage, a well-known problem in seed banking. Here we used a greenhouse study to test if priming deteriorated seeds with cathodic water can improve the emergence and subsequent seedling growth of three South African tree species, Bolusanthus speciosus, Combretum erythrophyllum and Erythrina caffra. Other priming solutions investigated were calcium magnesium (CaMg) solution and deionized water. In the present study, seeds were subjected to an artificial deterioration by increasing their water content to 14% and keeping them at 40 °C and 100% RH until they had lost 50% of their germination under laboratory conditions. Fresh and deteriorated seeds were primed with cathodic water, CaMg solution and deionized water, with non-primed fresh and deteriorated seeds as controls. Controlled deterioration significantly reduced total emergence and the biomass and photosynthetic parameters of the resulting seedlings. In one species (Bolusanthus speciosus), priming the deteriorated seeds with cathodic water significantly improved emergence parameters. However, in all species cathodic water significantly improved the total biomasses and other growth parameters of the seedlings derived from deteriorated seeds. Priming with CaMg solution and deionized water had little effect on emergence and while improving the growth of seedlings derived from deteriorated seeds, they were less effective than cathodic water. In fresh seeds, priming with all solutions resulted in small improvements in some parameters. Controlled deterioration of fresh seeds reduced the membrane stability index (MSI) in two of the three species and in all species increased the levels of the lipid oxidation products MDA and 4-HNE. Priming deteriorated seeds with cathodic water increased the MSI and reduced the MDA contents in all species and the 4-HNE content in one species. Other priming solutions were generally less effective in ameliorating oxidative stress. Results suggest that the strong antioxidative properties of cathodic water can explain its ability to ameliorate deterioration. In conclusion, the present study shows that priming with cathodic water is an effective way of invigorating deteriorated orthodox seeds and that it may have considerable potential in orthodox seed conservation.

Influence of Cathodic Water Invigoration on the Emergence and Subsequent Growth of Controlled Deteriorated Pea and Pumpkin Seeds.

The quality of seeds in gene banks gradually deteriorates during long-term storage, which is probably, at least in part, a result of the progressive development of oxidative stress. Here, we report a greenhouse study that was carried out to test whether a novel approach of seed invigoration using priming with cathodic water (cathodic portion of an electrolysed calcium magnesium solution) could improve seedling emergence and growth in two deteriorated crop seeds. Fresh seeds of Pisum sativum and Cucurbita pepo were subjected to controlled deterioration to 50% viability at 14% seed moisture content (fresh weight basis), 40 °C and 100% relative humidity. The deteriorated seeds were thereafter primed with cathodic water, calcium magnesium solution and deionized water. In addition, to study the mechanism of the impacts of invigoration, the effects of such priming on the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and on the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase and catalase were also determined in the fresh and deteriorated seeds. All priming treatments improved seed emergence parameters, subsequent seedling photosynthesis and growth relative to the unprimed seeds. In general, cathodic water was most effective at invigorating deteriorated seeds. Analysis of the lipid peroxidation products and antioxidant enzyme activities in invigorated seeds provided support for the hypothesis that the effectiveness of cathodic water in invigoration of debilitated orthodox seeds in general and of pea and pumpkin seeds in particular derive from its ability to act as an antioxidant.

Membrane sterols and genes of sterol biosynthesis are involved in the response of Triticum aestivum seedlings to cold stress.

Cold stress can significantly alter the composition and functioning of the major membrane lipids in plants. However, the roles of the sterol component of plant membranes in stress tolerance remain unclear. In the work presented here we investigated the role of sterols in the response of wheat to cold stress. Initial experiments demonstrated that the roots and leaves of wheat seedlings are differentially sensitive to low positive temperatures. In the roots, cold stress induced disturbance of membrane integrity and accumulation of ROS followed by the induction of autophagy. The absence of such changes in leaves suggests that in wheat, the roots are more sensitive to cold than the leaves. The roots display a time-dependent parabolic pattern of cold stress response, characterized by raised levels of sterols and markers of oxidative stress during short-term treatment, and a decline of these parameters after prolonged treatment. MßCD-induced sterol depletion aggravated the negative effects of cold on the roots. In the leaves the changes also displayed parabolic patterns, with significant changes occurring in 24-ethyl sterols and major PLs. Constitutively high levels of sterols, glycolipids and PLs, and up-regulation of TaSMTs in the leaves may provide membrane stability and cold tolerance. Taken together, results suggest that sterols play important roles in the response of wheat seedlings to cold stress.

Extracellular redox cycling and hydroxyl radical production occurs widely in lichenized Ascomycetes.

Some free-living Ascomycetes and white and brown rot Basidiomycetes can generate hydroxyl radicals using extracellular redox cycling. However, the mechanisms of hydroxyl radical production differ between white and brown rot Basidiomycetes, and are unknown for Ascomycetes. Here, we present a survey of extracellular hydroxyl radical production by a range of lichenized Ascomycetes. Results show that given a quinone and chelated ferric ions, many lichens can readily produce hydroxyl radicals, and this is accompanied by the reduction of Fe3+ to Fe2+. In white rot fungi, extracellular redox enzymes have been proposed to be involved in hydroxyl radical generation. However, a survey of a wide range of lichens suggests that in these fungi hydroxyl radical production does not directly correlate with the activity of laccases and peroxidases. Rather, radicals are probably produced by a mechanism like that proposed for brown rot fungi. Potential roles of hydroxyl radicals produced by lichens include the breakdown of lignocellulosic residues in the soil which may allow lichens to live a partially saprotrophic existence, the breakdown of toxic soil chemicals and the formation of an 'oxidative burst' to deter potential pathogens.

A proposed interplay between peroxidase, amine oxidase and lipoxygenase in the wounding-induced oxidative burst in Pisum sativum seedlings.

Plant surfaces form the barrier between a plant and its environment. Upon damage, the wound healing process begins immediately and is accompanied by a rapid production of extracellular reactive oxygen species (ROS), essential in deterring pathogens, signalling responses and cell wall restructuring. Although many enzymes produce extracellular ROS, it is unclear if ROS-producing enzymes act synergistically. We characterised the oxidative burst of superoxide (O2(·-)) and hydrogen peroxide (H2O2) that follows wounding in pea (Pisum sativum L.) seedlings. Rates of ROS production were manipulated by exogenous application of enzyme substrates and inhibitors. The results indicate significant roles for di-amine oxidases (DAO) and peroxidases (Prx) rather than NADPH oxidase. The burst of O2(·-) was strongly dependent on the presence of H2O2 produced by DAO. Potential substrates released from wounded seedlings included linoleic acid that, upon exogenous application, strongly stimulated catalase-sensitive O2(·-) production. Moreover, a 65kD plasma membrane (PM) guaiacol Prx was found in the secretome of wounded seedlings and showed dependence on linoleic acid for O2(·-) production. Lipoxygenases are suggested to modulate O2(·-) production by consuming polyunsaturated fatty acids in the apoplast. Overall, a O2(·-)-producing mechanism involving H2O2-derived from DAO, linoleic acid and a PM-associated Prx is proposed.

Oxidoreductases and cellulases in lichens: possible roles in lichen biology and soil organic matter turnover.

Lichens are symbiotic associations of a fungus (usually an Ascomycete) with green algae and/or a cyanobacterium. They dominate on 8 % of the world's land surface, mainly in Arctic and Antarctic regions, tundra, high mountain elevations and as components of dryland crusts. In many ecosystems, lichens are the pioneers on the bare rock or soil following disturbance, presumably because of their tolerance to desiccation and high temperature. Lichens have long been recognized as agents of mineral weathering and fine-earth stabilization. Being dominant biomass producers in extreme environments they contribute to primary accumulation of soil organic matter. However, biochemical role of lichens in soil processes is unknown. Our recent research has demonstrated that Peltigeralean lichens contain redox enzymes which in free-living fungi participate in lignocellulose degradation and humification. Thus lichen enzymes may catalyse formation and degradation of soil organic matter, particularly in high-stress communities dominated by lower plants. In the present review we synthesize recently published data on lichen phenol oxidases, peroxidases, and cellulases and discuss their possible roles in lichen physiology and soil organic matter transformations.

A heme peroxidase of the ascomyceteous lichen Leptogium saturninum oxidizes high-redox potential substrates.

Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosinase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum (LsaPOX) by fast protein liquid chromatography and then partially characterized. The oligomeric protein occurs as both 79 kDa dimeric and 42 kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6-dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auricula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fischeri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant peroxidases. LsaPOX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow.

What is stress? Concepts, definitions and applications in seed science.

'Stresses' that impact upon seeds can affect plant reproduction and productivity, and, hence, agriculture and biodiversity. In the absence of a clear definition of plant stress, we relate concepts from physics, medicine and psychology to stresses that are specific to seeds. Potential 'eustresses' that enhance function and 'distresses' that have harmful effects are considered in relation to the seed life cycle. Taking a triphasic biomedical stress concept published in 1936, the 'General Adaptation Syndrome', to the molecular level, the 'alarm' response is defined by post-translational modifications and stress signalling through cross-talk between reactive oxygen and nitrogen species, and seed hormones, that result in modifications to the transcriptome. Protection, repair, acclimation and adaptation are viewed as the 'building blocks' of the 'resistance' response, which, in seeds, are the basis for their longevity over centuries. When protection and repair mechanisms eventually fail, depending on dose and time of exposure to stress, cell death and, ultimately, seed death are the result, corresponding to 'exhaustion'. This proposed seed stress concept may have wider applicability to plants in general.

Extracellular production of reactive oxygen species during seed germination and early seedling growth in Pisum sativum.

Extracellularly produced reactive oxygen species (ROS) play key roles in plant development, but their significance for seed germination and seedling establishment is poorly understood. Here we report on the characteristics of extracellular ROS production during seed germination and early seedling development in Pisum sativum. Extracellular superoxide (O2(.-)) and hydrogen peroxide (H2O2) production and the activity of extracellular peroxidases (ECPOX) were determined spectrophotometrically, and O2(.-) was identified by electron paramagnetic resonance. Cell wall fractionation of cotyledons, seed coats and radicles was used in conjunction with polyacrylamide gel electrophoresis to investigate substrate specificity and molecular masses of O2(.-)-producing enzymes, and the forces that bind them to the cell wall. Seed imbibition was accompanied by an immediate, transient burst of redox activity that involved O2(.-) and other substances capable of oxidizing epinephrine, and also H2O2. At the final stages of germination, coinciding with radicle elongation, a second increase in O2(.-) but not H2O2 production occurred and was correlated with an increase in extracellular ECPOX activity. Electrophoretic analyses of cell wall fractions demonstrated the presence of enzymes capable of O2(.-) production. The significance of extracellular ROS production during seed germination and early seedling development, and also during seed aging, is discussed.

Cell wall peroxidases in the liverwort Dumortiera hirsuta are responsible for extracellular superoxide production, and can display tyrosinase activity.

In our earlier work, we showed that the liverwort Dumortiera hirsuta produces an extracellular oxidative burst of superoxide radicals during rehydration following desiccation stress. The oxidative burst is a common early response of organisms to biotic and abiotic stresses, with suggested roles in signal transduction, formation of protective substances such as suberin, melanin and lignin and defense against pathogens. To discover which enzymes are responsible for the extracellular superoxide production, we isolated apoplastic fractions from D. hirsuta, surveyed for the presence of potential redox enzymes, and performed non-denaturing polyacrylamide gel electrophoresis activity stains. Various isoforms of peroxidase (EC 1.11.1.7) and tyrosinase (o-diphenolase) (EC 1.10.3.1) were present at significant levels in the apoplast. In-gel activity staining revealed that some peroxidases isoforms could produce superoxide, while tryosinases could readily metabolize 3,4-dihydroxy phenyl l-alanine (l-dopa) into melanins. Interestingly, some peroxidase isoforms could oxidize the native tyrosinase substrate l-dopa at significant levels, even in the absence of hydrogen peroxide, while others could do so only in the presence of hydrogen peroxide. In D. hirsuta, peroxidases may play an important role in melanin formation. Possible functions for these diverse oxidases in liverwort biology are discussed.

Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds.

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O(2)(.-)) production upon desiccation in Castanea sativa seeds, mechanisms of O(2)(.-) production and the effect of exogenously supplied ROS. A transient increase in extracellular O(2)(.-) production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H(2)O(2))-dependent O(2)(.-) production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of approximately 50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O(2)(.-) production. H(2)O(2), applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.

An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation.

Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut (Castanea sativa). Axis excision was accompanied by a burst of superoxide (O(2)(*-)), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial 'burst' in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O(2)(*-) production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.

Effect of water content components on desiccation and recovery in Sphagnum mosses.

BACKGROUND AND AIMS: The basic parameters of water relations were measured in Sphagnum mosses. The relationships of these parameters to the photosynthetic response to desiccation and the ecology of these mosses were then tested. METHODS: The water relations parameters of six Sphagnum species (mosses typical of wet habitats) and Atrichum androgynum (a moss more typical of mesophytic conditions) were calculated from pressure-volume isotherms. Photosynthetic properties during and after moderate desiccation were monitored by chlorophyll fluorescence. KEY RESULTS: When desiccated, the hummock-forming species S. fuscum and S. magellanicum lost more water before turgor started dropping than other sphagna inhabiting less exposed habitats (73 % compared with 56 % on average). Osmotic potentials at full turgor were similar in all species, with an average value of -1.1 MPa. Hummock sphagna had clearly more rigid cell walls than species of wet habitats (epsilon = 3 x 55 compared with 1 x 93 MPa). As a result, their chlorophyllous cells lost turgor at higher relative water contents (RWCs) than species of wet habitats (0 x 61 compared with 0 x 46) and at less negative osmotic potentials (-2 x 28 compared with -3 x 00 MPa). During drying, Phi(PSII) started declining earlier in hummock species (at an RWC of 0 x 65 compared with 0 x 44), and F(v)/F(m) behaved similarly. Compared with other species, hummock sphagna desiccated to -20 or -40 MPa recovered more completely after rehydration. Atrichum androgynum responded to desiccation similarly to hummock sphagna, suggesting that their desiccation tolerance may have a similar physiological basis. CONCLUSIONS: Assuming a fixed rate of desiccation, the higher water-holding capacities of hummock sphagna will allow them to continue metabolism for longer than other species. While this could be viewed as a form of 'desiccation avoidance', hummock species also recover faster than other species during rehydration, suggesting that they have higher inherent tolerance. This may help them to persist in drought-exposed hummocks. In contrast, species growing in wet habitats lack such strong avoidance and tolerance mechanisms. However, their turgor maintenance mechanisms, for example more elastic cell walls, enable them to continue metabolizing longer as their water contents fall to the turgor-loss point.

Co-occurrence of the multicopper oxidases tyrosinase and laccase in lichens in sub-order peltigerineae.

BACKGROUND AND AIMS: Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied. METHODS: Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis. KEY RESULTS: Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. CONCLUSIONS: Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents.

Occurrence of laccases in lichenized ascomycetes of the Peltigerineae.

Following our previous findings of high extracellular redox activity in lichens, the results of the work presented here identify the enzymes involved as laccases. Despite numerous data on laccases in fungi and flowering plants, this is the first report of the occurrence of laccases in lichenized ascomycetes. Extracellular laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from suborder Peltigerineae, 18 displayed laccase activity, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases was confirmed by the ability of lichen leachates to readily metabolize substrates such as 2,2'-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in the absence of hydrogen peroxide, sensitivity of the enzymes to cyanide and azide, the enzymes having typical laccase pH and temperature optima, and an absorption spectrum with a peak at 614nm. Desiccation and wounding stimulated laccase activity. Laccase activity was not increased after treatment with normal inducers of laccase synthesis, suggesting that they are constitutively expressed. Electrophoresis showed that the active form of laccase from Peltigera malacea was a tetramer with an unusually high molecular mass of 340kDa and an isoelectric point (pI) of 4.7. The finding of abundant extracellular redox enzymes known to actively produce reactive oxygen species suggest that their roles may include increasing nutrient supply to lichens by delignification, and deterring pathogens by contributing to the oxidative burst. Furthermore, once released into the environment, they may participate in the carbon cycle by facilitating the breakdown or formation of humic substances.

Hardening by partial dehydration and ABA increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon.

BACKGROUND AND AIMS: The ability of partial dehydration and abscisic acid pretreatments to increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon was tested. * METHODS: Net photosynthesis and respiration were measured using infrared gas analysis during a drying and rehydration cycle. At the same time, the efficiency of photosystem two was measured using chlorophyll fluorescence, and the concentrations of chlorophyll a were spectrophotometrically assayed. Heat production was also measured during a shorter drying and rehydration cycle using differential dark microcalorimetry. * KEY RESULTS: Pretreating lichens by dehydrating them to a relative water content of approx. 0.65 for 3 d, followed by storing thalli hydrated for 1 d in the light, significantly improved their ability to recover net photosynthesis during rehydration after desiccation for 15 but not 30 d. Abscisic acid pretreatment could substitute for partial dehydration. The improved rates of photosynthesis during the rehydration of pretreated material were not accompanied by preservation of photosystem two activity or chlorophyll a concentrations compared with untreated lichens. Partial dehydration and ABA pretreatments appeared to have little direct effect on the desiccation tolerance of the mycobiont, because the bursts of respiration and heat production that occurred during rehydration were similar in control and pretreated lichens. * CONCLUSIONS: Results indicate that the photobiont of P. polydactylon possesses inducible tolerance mechanisms that reduce desiccation-induced damage to carbon fixation, and will therefore improve the supply of carbohydrates to the whole thallus following stress. In this lichen, ABA is involved in signal transduction pathways that increase tolerance of the photobiont.

Biochemical traits of lichens differing in relative desiccation tolerance.

• Oxidative stress arises when desiccation restricts photosynthesis and light energy is transferred from photo-excited pigments onto ground state oxygen. We tested whether a highly desiccation tolerant lichen, Pseudevernia furfuracea, displays better protection against oxidative stress than more sensitive species, Lobaria pulmonaria and Peltigera polydactyla. • We rehydrated lichens after desiccation periods of 2, 7 and 9 weeks and assessed their viability by measuring CO2 exchange using IRGA. During desiccation and rehydration, photosynthetic pigments and the antioxidant α-tocopherol were analysed by HPLC, and peroxidases by spectrophotometry. • Pseudevernia furfuracea contained considerably lower chlorophyll, α-tocopherol and ß-carotene concentrations and peroxidase activity than the two other lichens. However, it recovered photosynthesis rapidly, even after remaining in the desiccated state for 2 months while there was a significant delay in the onset of photosynthesis in L. pulmonaria and P. polydactyla. • We conclude that high antioxidant concentrations do not necessarily indicate better adaptation to desiccation. Rather, the ability to rapidly re-establish the species-specific normal antioxidant concentrations during rehydration, even after longer desiccation times, is a characteristic of well-adapted species.

Revival of a resurrection plant correlates with its antioxidant status.

Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, beta-carotene, ascorbate, alpha-tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, gamma-tocopherol, lutein, neoxanthin and glucose-6-phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re-watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of alpha-tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, beta-carotene and alpha-tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.