[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Fungal Genetics and Biology 10/2011; 48(12):1139-45. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: '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.
New Phytologist 11/2010; 188(3):655-73. · 6.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of plant physiology 03/2010; 167(10):805-11. · 2.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: The tropical tree Trichilia dregeana Sond. has recalcitrant seeds that cannot be stored by conventional seed banking methods that include drying and storage at low temperatures, or by using cryopreservation protocols that have been successfully applied to recalcitrant-seeded species such as Castanea sativa. We recently showed that in C. sativa both excision of the embryonic axes and subsequent dehydration cause transitory (5 min) bursts of extracellular superoxide (O2−) production. Here we show that both excision and partial dehydration of the embryonic axes from seeds of T. dregeana cause large, prolonged extracellular bursts of O2−. Furthermore, during rehydration after cryopreservation, another burst of O2− occurs with slightly different kinetics. Compared with C. sativa, rates of O2− production in T. dregeana are approximately twice as great and decline much more slowly, suggesting that excessive radical formation may be responsible for poor survival of the axes following cryopreservation. Fractionating the cell wall proteins of embryonic axes and cotyledons in conjunction with electrophoretic analyses of the fractions showed that most O2− was produced by two peroxidases with molecular masses of c. 50 and 80 kD that were loosely bound to the cell walls of the embryonic axes. Future successful cryopreservation of T. dregeana would appear to depend on manipulations of O2− production, and the discovery of peroxidases as the enzymes responsible described here may help in the development of more effective protocols.
[show abstract][hide abstract] ABSTRACT: Bidens pilosa L. is a weedy species in the Asteraceae producing dimorphic one-seeded fruits, with longer black seeds centrally situated in the capitulum, and shorter dormant brown peripheral seeds. While the outer seeds germinate readily after seed shedding, the shorter seeds possess various dormancy mechanisms, including requirements for after-ripening and red light. Here we show that applications of reactive oxygen species (ROS)-generating reagents can remove dormancy in the short seeds. In B. pilosa, reagents that generate hydroxyl radicals (OH) and superoxide (O2¯) partially replaced the requirement for after-ripening, while O2¯ generation replaced the requirement for red light. Hence, ROS appear to be implicated in the alleviation of dormancy in the seeds of B. pilosa.
[show abstract][hide abstract] ABSTRACT: 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 (O2·−) production upon desiccation in Castanea sativa seeds, mechanisms of O2·− production and the effect of exogenously supplied ROS. A transient increase in extracellular O2·− 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 (H2O2)-dependent O2·− production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of ∼50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O2·− production. H2O2, 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.
[show abstract][hide abstract] ABSTRACT: 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 126.96.36.199) and tyrosinase (o-diphenolase) (EC 188.8.131.52) 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.
Physiologia Plantarum 10/2009; 138(4):474-84. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Following our previous reports of extracellular laccase activity in lichenized ascomycetes, we investigated the diversity of laccase isoforms in lichens in 20 species from the suborder Peltigerineae. The molecular masses of the active forms of most laccases varied between 135 and 190 kD, although some lichens within the family Peltigeraceae had laccases with higher masses, typically varying from 200 to over 350 kD. Size exclusion chromatography (SEC) was used to confirm the accuracy of the electrophoretic estimates of molecular masses. SEC also clearly distinguished laccase from tyrosinase, another abundant cell wall oxidase in the Peltigerineae. Most species contained one oligomeric laccase isoform. Analysis of replicate collections of four species from different localities showed the isoform a given species contains does not vary with geographical location. The absorption spectra of lichen leachates suggested lichen laccases sometimes resemble the “yellow” laccases found in some free-living fungi. The thermostabilities of lichen laccases were only moderate; most of their activity remained after several hours at 40°C, but at 50°C activity was rapidly lost. Results indicate that considerable diversity in laccase isoforms exists in lichens.
[show abstract][hide abstract] ABSTRACT: Production of reactive oxygen species (ROS) is a widely reported response of plants to wounding. However, the nature of enzymes responsible for ROS production and metabolism in the apoplast is still an open question. We identified and characterized the proteins responsible for the wound-induced production and detoxification of ROS in the apoplast of wheat roots (Triticum aestivum L.). Compared to intact roots, excised roots and leachates derived from them produced twice the amount of superoxide (O2(*-)). Wounding also induced extracellular peroxidase (ECPOX) activity mainly caused by the release of soluble peroxidases with molecular masses of 37, 40 and 136 kD. Peptide mass analysis by electrospray ionization-quadrupole time-of-flight-tandem mass spectrometry (ESI-QTOF-MS/MS) following lectin affinity chromatography of leachates showed the presence of peroxidases in unbound (37 kD) and bound (40 kD) fractions. High sensitivity of O2(*-)-producing activity to peroxidase inhibitors and production of O2(*-) by purified peroxidases in vitro provided evidence for the involvement of ECPOXs in O2(*-) production in the apoplast. Our results present new insights into the rapid response of roots to wounding. An important component of this response is mediated by peroxidases that are released from the cell surface into the apoplast where they can display both oxidative and peroxidative activities.
Plant Cell and Environment 02/2009; 32(5):497-508. · 5.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Physiologia Plantarum 07/2008; 133(2):131-9. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: All organisms, even highly stress-tolerant lichens, produce a variety of reactive oxygen species (ROS) during and after stress. Furthermore, the cell walls of some lichens in Suborder Peltigerineae contain laccases, and therefore can produce quinone radicals that can break down to yield ROS. While the extracellular ROS produced by these enzymes probably play important roles in the biology of these lichens, they may also be potentially harmful and need to be rapidly broken down. To test this, rates of breakdown of exogenously supplied H2O2 were measured in a range of lichen species. Considerable diversity existed in rates of H2O2 breakdown but rates were on average almost double in members of Suborder Peltigerineae. While all lichens tested appeared to lack extracellular peroxidases and catalases, enzymes normally involved in breaking down H2O2, extracellular tyrosinase activity could be readily detected in the Peltigerineae. A role for tyrosinases in H2O2 breakdown was supported by the results from experiments involving inhibitors, and demonstration of the simultaneous release into an incubation solution of tyrosinase activity and the ability to breakdown H2O2. Rates of breakdown were very high, and tyrosinase appeared to break down H2O2 by a catalase-like mechanism. However, significant rates of breakdown of H2O2 also occurred in species that did not possess cell wall redox enzymes. These species probably took up the exogenously supplied H2O2 intracellularly and then broke it down by the usual catalases and peroxidases. The importance of H2O2 degradation is discussed in terms of its possible role in defence against the harmful effects of ROS.
Physiologia Plantarum 01/2007; 129(3):588 - 596. · 3.66 Impact Factor