Nicholas Smirnoff

Hydroxyl radical scavenging activity of compatible solutes

Compatible solutes were assessed for their hydroxyl radical scavenging activity by their ability to compete in two different hydroxyl radical generating and detecting systems. Hydroxyl radicals were generated by ascorbate-hydrogen peroxide or by xanthine oxidase-hypoxanthine-hydrogen peroxide. They were detected by hydroxylation of salicylate or by denaturation of malate dehydrogenase. Of the compatible solutes tested, sorbitol, mannitol, myo-inositol and proline were effective hydroxyl radical scavengers. Glycinebetaine was ineffective. The role of compatible solutes as hydroxyl radical scavengers in vivo is discussed.

Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing

Humans are unable to synthesise L-ascorbic acid (L-AA, ascorbate, vitamin C), and are thus entirely dependent upon dietary sources to meet needs. In both plant and animal metabolism, the biological functions of L-ascorbic acid are centred around the antioxidant properties of this molecule. Considerable evidence has been accruing in the last two decades of the importance of L-AA in protecting not only the plant from oxidative stress, but also mammals from various chronic diseases that have their origins in oxidative stress. Evidence suggests that the plasma levels of L-AA in large sections of the population are sub-optimal for the health protective effects of this vitamin. Until quite recently, little focus has been given to improving the L-AA content of plant foods, either in terms of the amounts present in commercial crop varieties, or in minimising losses prior to ingestion. Further, while L-AA biosynthesis in animals was elucidated in the 1960s, 1 it is only very recently that a distinct biosynthetic route for plants has been proposed. 2 The characterisation of this new pathway will undoubtedly provide the necessary focus and impetus to enable fundamental questions on plant L-AA metabolism to be resolved. This review focuses on the role of L-AA in metabolism and the latest studies regarding its bio- synthesis, tissue compartmentalisation, turnover and catabolism. These inter-relationships are considered in relation to the potential to improve the L-AA content of crops. Methodology for the reliable analysis of L-AA in plant foods is briefly reviewed. The concentrations found in common food sources and the effects of processing, or storage prior to consumption are discussed. Finally the factors that determine the bioavailability of L-AA and how it may be improved are considered, as well as the most important future research needs. # 2000 Society of Chemical Industry

The biosynthetic pathway of vitamin C in higher plants

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown—one of the few primary plant metabolic pathways forwhich this is the case. L-ascorbate is abundant in plants (found at concentrations of 1–5 mM in leaves and 25 mM in chloroplasts,) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose anascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

Ascorbic acid in plants: biosynthesis and function.

ABSTRACT Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtcl mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifacetted molecule.

Plant resistance to environmental stress

A common effect of many environmental stresses is to cause oxidative damage; consequently, the antioxidant system is being intensively investigated. The use of transgenic plants to probe the role of the antioxidant system continues to be an important approach. The uncharted area of signal transduction in relation to oxidative stress is beginning to attract attention. Studies of drought response at the cellular level have focused on the role of compatible solutes (osmolytes) in acclimation to water stress. Information on signal transduction processes during drought is beginning to appear. As with the antioxidant system, there is increasing use of metabolic engineering in transgenic plants to introduce exotic compatible solutes. It is concluded that these potentially have a use in understanding, or even improving, drought resistance; however, there is a need for the assessment of stress tolerance of transgenics to be carried out at a more sophisticated level and for a critical analysis of the relevance for crop yield of the genes currently being manipulated.

Glycerol generates turgor in rice blast

Many plant pathogenic fungi are able to penetrate the cuticles of their host plants by elaborating specialized cells known as appressoria. The morphology and development of appressoria have been well studied, but little is known about how these cells are able to breach the tough plant surface. We have now found that the appressoria of rice blast fungus (Magnaporthe grisea) use glycerol to generate pressure which ruptures plant cuticles.

Antioxidants and reactive oxygen species in plants

1. Glutathione.Christine H. Foyer, Leonardo Gomez and Philippus D. R. van Heerden, Rothamsted Research, Harpenden, UK.2. Plant thiol enzymes and thiol homeostasis in relation to thiol-dependent redox regulation and oxidative stress.Karl-Josef Dietz, Lehrstuhl fur Biochemie und Physiologie der Pflanzen, Fakultat fur Biologie, Universitat Bielefeld, Germany.3. Ascorbate, tocopherol and carotenoids: metabolism, pathway engineering and functions.Nicholas Smirnoff, School of Biological and Chemical Sciences, University of Exeter, UK.4. Ascorbate peroxidase.Ron Mittler, Department of Biochemistry, University of Nevada, Reno, USA and Thomas L. Poulos, Department of Molecular Biology and Biochemistry, University of California, Irvine, USA.5. Catalases in plants: molecular and functional properties and role in stress defence.Jurgen Feierabend, Institute of Botany, J. W. Goethe Universitat, Frankfurt, Germany.6. Phenolics as antioxidants.Stepehen C. Grace, Biology Department, University of Arkansas at Little Rock, Arkansas, USA.7. Reactive oxygen species as signalling molecules.Radhika Desikan, John Hancock and Steven Neill, Centre for Research in Plant Science, University of the West of England, Bristol, UK.8. Reactive oxygen species in plant development and pathogen defence.Mark A. Jones and Nicholas Smirnoff, School of Biological and Chemical Sciences, University of Exeter, UK.9. Reactive oxygen species in cell walls.Robert A. M. Vreeburg and Stephen C. Fry, School of Biological Sciences, University of Edinburgh, UK.10. Reactive oxygen species and photosynthesis.Barry Logan, Biology Department, Bowdoin College, Brunswick, Maine, USA.11. Plant responses to ozone.Pinja Jaspers, Hannes Kollist, Christian Langebartels, and Jaakko Kangasjarvi, Department of Biological and Environmental Sciences, University of Helsinki, Finland.References.Index

Independent Signaling Pathways Regulate Cellular Turgor during Hyperosmotic Stress and Appressorium-Mediated Plant Infection by Magnaporthe grisea

The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase–encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea Δosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Δosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol.

Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease

One of the first responses of plants to microbial attack is the production of extracellular superoxide surrounding infection sites. Here, we report that Magnaporthe grisea, the causal agent of rice blast disease, undergoes an oxidative burst of its own during plant infection, which is associated with its development of specialized infection structures called appressoria. Scavenging of these oxygen radicals significantly delayed the development of appressoria and altered their morphology. We targeted two superoxide-generating NADPH oxidase-encoding genes, Nox1 and Nox2, and demonstrated genetically, that each is independently required for pathogenicity of M. grisea. Δnox1 and Δnox2 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. The initiation of rice blast disease therefore requires production of superoxide by the invading pathogen.

L-Ascorbic Acid Metabolism in the Ascorbate-Deficient Arabidopsis Mutant vtc1

The biosynthesis of L-ascorbic acid (vitamin C) is not well understood in plants. The ozone-sensitive Arabidopsis thaliana mutant vitamin c-1 (vtc1; formerly known as soz1) is deficient in ascorbic acid, accumulating approximately 30% of wild-type levels. This deficiency could result from elevated catabolism or decreased biosynthesis. No differences that could account for the deficiency were found in the activities of enzymes that catalyze the oxidation or reduction of ascorbic acid. The absolute rate of ascorbic acid turnover is actually less in vtc1 than in wild type; however, the turnover rate relative to the pool of ascorbic acid is not significantly different. The results from [U-14C]Glc labeling experiments suggest that the deficiency is the result of a biosynthetic defect: less L-[14C]ascorbic acid as a percentage of total soluble 14C accumulates in vtc1 than in wild type. The feeding of two putative biosynthetic intermediates, D-glucosone and L-sorbosone, had no positive effect on ascorbic acid levels in either genotype. The vtc1 defect does not appear to be the result of a deficiency in L-galactono-1,4-lactone dehydrogenase, an enzyme able to convert L-galactono-1,4-lactone to ascorbic acid.