John G. Scandalios

Catalases in Plants: Gene Structure, Properties, Regulation, and Expression

Catalase action in plant and animal tissues was first observed in 1818 by Thenard, who noted that such tissues readily degraded hydrogen peroxide, a substance he had also discovered some years earlier (Aebi and Sutter 1971). Loew (1901) first established that the degradation of H 2 O 2 in tissues was due to the effect of an individual, separable enzyme, which he named “catalase.” Warburg (1923) suggested that catalase is an iron-containing enzyme, because it is inhibited by cyanide. Evidence for its hematin prosthetic group was presented by Zeile and Hellstrom (1930). Catalase was first purified and crystallized from beef liver, and its identity was made clear by Sumner and Dounce (1937). The earliest genetic studies on catalase were reported by the Russian biologist Koltzoff (1927), who demonstrated that blood catalase levels in several animal species are inherited and segregate according to Mendelian rules. Catalase has been found in all plants examined, and has been most thoroughly studied biochemically, genetically, and molecularly in the agronomically important species Zea mays L. (Scandalios 1990). That catalases can exist in multiple molecular forms or isozymes encoded by multiple genes, in any organism, was first demonstrated by Scandalios (1965 Scandalios (1968) with the maize catalases and has since been found to be the rule rather than the exception, as originally perceived. OXYGEN AND REACTIVE OXYGEN SPECIES During respiration, molecular oxygen accepts four electrons to produce two molecules of H 2 O. However, because of spin restrictions, O 2 cannot accept four electrons at once but accepts them one at a time...

Response of plant antioxidant defense genes to environmental stress.

Publisher Summary This chapter discusses the genomic responses of plants to the oxidative stress—particularly in maize . The chapter also discusses several mechanisms that the plants and other aerobes have evolved for protection against the toxic effects of reduced oxygen species. Particular emphasis is placed on the discussion of the molecular dissection of two antioxidant gene families—catalase and superoxide dismutase—in an effort to unravel the mechanisms by which the genome perceives and responds to oxidative stress signals. Among higher eukaryotes, plants have evolved diverse ways of responding to their environment. Plants have incorporated a variety of environmental signals into their developmental pathways that have provided for their wide range of adaptive capacities over time. An example of such an environmental signal is light that—in addition to driving photosynthesis—serves as a trigger and modulator of complex regulatory and developmental mechanisms. The chapter presents an example of photosynthesis to illustrate the catalase gene expression in maize.

The rise of ROS

I gratefully acknowledge the generous, and continuous, support of my work over many years by the US Department of Energy, National Institute of Health, National Science Foundation, Department of Agriculture, and Environmental Protection Agency.

A PROCEDURE FOR THE SMALL-SCALE ISOLATION OF PLANT RNA SUITABLE FOR RNA BLOT ANALYSIS

A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity.

Analysis of variants affecting the catalase developmental program in maize scutellum

SummaryThe catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6–67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6–67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.

Modulation of antioxidant responses by arsenic in maize.

The effects of arsenic on the expression of the antioxidant genes encoding superoxide dismutase, catalase, and glutathione S-transferase, as well as the activity of SOD and CAT enzymes, were examined at different developmental stages and in different tissues. Both CAT and SOD activities increased in response to low concentrations (0.01-0.1 mM) of arsenic in developing maize embryos. In germinating embryos both CAT and SOD activities increased in response to a wide range of arsenic concentrations (0.01-10 mM). Cat1 transcript increased in response to arsenic in developing and germinating embryos and in young leaves. Conversely, Cat2 increased at low concentrations of arsenic only in germinating embryos. Cat3 transcript levels increased in response to low concentrations of arsenic only in developing embryos. Sod3 transcript increased at low concentrations of arsenic in developing, germinating embryos and in leaves. The cytosolic Sod4 and Sod4A increased in response to arsenic in germinating embryos, while only Sod4 transcript increased in response to arsenic in leaves. Expression of Gst1 was similar to that of Cat1 in all tissues examined. These results indicate that arsenic triggers tissue and developmental stage specific defense responses of antioxidant and detoxification related genes in maize.

Isolation and characterization of the cytosolic and mitochondrial superoxide dismutases of maize

Abstract The cytosolic and mitochondrial forms of Superoxide dismutase have been purified to homogeneity from an inbred line of maize. The cytosolic isozymes SOD-2 and SOD-4 are dimers with a molecular weight of 31,000–33,000, composed of apparently equal subunits, and are remarkably similar with respect to their ultraviolet absorption spectra, antigenic specificity, and sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate. These and other data suggest that both isozymes belong to the family of copper and zinc-containing Superoxide dismutases. The mitochondrial isozyme, SOD-3, is unlike the cytosolic isozymes in every parameter studied and appears to be similar to the mitochondrial manganese-containing Superoxide dismutases purified from other eukaryotic organisms. It is a tetramer with a molecular weight of approximately 90,000, composed of apparently equal subunits, and is insensitive to both 1 m m cyanide and hydrogen peroxide.

Characterization of catalase transcripts and their differential expression in maize

In maize, the three unlinked catalase (EC 1.11.1.6) structural genes (Cat1, Cat2 and Cat3) are differentially expressed temporally, spatially and in response to environmental signals in the developing seedling. In order to understand more fully the molecular mechanisms involved in catalase gene expression, full-length cDNA clones representing the maize Cat1, Cat2 and Cat3 transcripts were isolated and characterized. DNA sequence analysis confirmed that each cDNA encodes a unique catalase protein. Gene-specific probes for the three maize catalase cDNAs were isolated and used to probe blots of poly(A)+ RNA isolated from various maize tissues. Cat1 mRNA was found in scutella, milky endosperm of immature kernels, leaves and epicotyls. The Cat2 mRNA was present primarily in post-germinative scutella, with lower levels in leaves and epicotyls. Cat3 mRNA was detected primarily in epicotyls and, to a lesser extent, in leaves and scutella. The gene-specific probes hybridized with maize genomic DNA blots in simple, but unique patterns, indicating that there is one, or a very few copies of each catalase gene. The coding region of the Cat3 cDNA comprised 66% G + C, which led to a strong codon usage bias in this gene. This codon bias was also seen with the Cat2 transcripts, but not with those for Cat1. A high degree of similarity was found between the maize catalase nucleic acid and deduced amino-acid sequences and those of sweet potato and rat liver catalase.

Cat3, a third gene locus coding for a tissue-specific catalase in maize: Genetics, intracellular location, and some biochemical properties

SummaryA new and unique catalase isozyme, CAT-3, has been found in Zea mays. It is encoded in the Cat3 nuclear structural gene which is distinct from the two previously described catalase structural genes, Cat1 and Cat2. The Cat3 gene is both tissue- and time-dependent in its expression, being expressed primarily in young leaves and in the pericarp of nearly mature kernels. Cell fractionation experiments, utilizing epicotyl (coleoptile+primary leaf) and mesocotyl cells, suggest that CAT-3 is associated with the mitochrondria where it may play a role in the alternate oxidase pathway. CAT-3 was purified and characterized with respect to some of its biochemical properties. While CAT-3 differs in some of its properties from CAT-1 and CAT-2, it is similar to these and to other catalases in most respects.

Oxidative stress responses - what have genome-scale studies taught us?

Oxidative stress arises from an imbalance between generation and elimination of reactive oxygen species, often leading to cell death. Genomic tools are expanding our understanding of the antioxidant defenses aerobes have evolved and the recently discovered role(s) of reactive oxygen species in signaling.