Catalase gene expression in response to auxin-mediated developmental signals.

The effect of auxin on maize catalase gene expression was examined at several different developmental stages during embryo and seedling development. All three catalase genes and their respective proteins were induced by both natural and synthetic auxin in immature embryos. Total catalase (CAT) activity increased dramatically in response to high concentrations of auxin, with CAT-2, which is not normally expressed at this stage, being the isozyme most responsible for the increase. Cat1 transcript accumulated to high levels at 2-8 h after auxin treatment, while Cat2 and Cat3 transcripts increased dramatically, but only after 12 h. In CAT-2 null mutant lines, the CAT-1 isozyme compensated for the missing CAT-2 activity and was the major isozyme responsible for the observed increase in total CAT activity. Auxin treatment mimics the germination process (i.e. induces germination) in immature embryos. Thus, the observed early induction of CAT-1 and the later increase of CAT-2 during the germination process may be due, in part, to changes in auxin content. In germinating embryos, auxin also induces total CAT activity and Cat transcript accumulation, although to a lesser extent. Auxin also induces Cat1 transcript accumulation in young leaves. The involvement of ROS in the auxin response is discussed.

Salt-induced antioxidant metabolism defenses in maize (Zea mays L.) seedlings.

Salinity alters general metabolic processes and enzymatic activities, causing increased production of reactive oxygen species (ROS). Expression of antioxidant defense genes would, in turn, be triggered to defend the cell against oxidative damage. We report that salt disturbed antioxidant metabolism in maize seedlings, causing detrimental effects on the growth and development of maize plantlets, increased hydrogen peroxide production and altered antioxidant activities and transcripts profiles. Excessive ROS levels were accompanied by increased catalase (CAT) activity in photosynthesizing shoots, along with induction of mRNA accumulation. Increased accumulation of superoxide dismutase (SOD) transcripts was also observed although no significant changes in total SOD enzymatic activity and isozyme profiles were detected. Higher salt concentrations (above 0.25 M NaCl) were highly detrimental to the plants, causing arrested growth and severe wilting, among other effects. Histochemical detection of H(2)O(2) by 3,3-diaminobenzidine (DAB) staining indicated a collapse of the leaf veins, with hydrogen peroxide leaking to neighboring cells. In agreement to these observations, Sod1, Sod2, Sod4, Sod4A, as well as all Cat transcripts were severely inhibited in plants exposed to high salt concentrations.

Antioxidant gene responses to ROS-generating xenobiotics in developing and germinated scutella of maize.

There is circumstantial evidence implicating reactive oxygen species (ROS) in the highly ordered temporal and spatial regulation of expression of the Cat and Sod antioxidant genes during seed development and germination in maize. In order to understand and provide experimental data for the regulatory role of ROS, the expression patterns of the Cat1, Cat2, Cat3, GstI, Sod3, Sod4, and Sod4A genes, as well as catalase (CAT) and superoxide dismutase (SOD) activity responses, were examined after treatments with ROS-generating xenobiotics in developing and germinated maize scutella. CAT and SOD activities increased at both stages in response to each xenobiotic examined in a dose-dependent and stage-specific manner. Individual Cat gene expression patterns were co-ordinated with isozyme patterns of enzymatic activity in scutella of developing seeds. This was not observed in germinated seeds where, although Cat1 expression was highly induced by ROS, there was not a similar increase of enzymatic CAT1 activity, suggesting the involvement of post-transcriptional regulation. Enhanced enzyme activities were synchronous with increases in steady-state transcript levels of specific Sod genes. The steady-state transcript level of GstI was elevated in all samples examined. Gene expression responses derived from this study along with similar results documented in previous reports were subjected to cluster analysis, revealing that ROS-generating compounds provoke similar effects in the expression patterns of the tested antioxidant genes. This could be attributable to common stress-related motifs present in the promoters of these genes.

The maize alternative oxidase 1a (Aox1a) gene is regulated by signals related to oxidative stress.

We isolated and characterized the expression of Aox1a, a member of the maize alternative oxidase (Aox) small multigene family. Aox1a consists of four exons interrupted by three introns and its promoter harbors diverse stress-specific putative regulatory motifs pointing to complex regulation and response to multiple signals. Responses of Aox1a to such signals were examined and compared with those of maize glutathione S-transferase I (GstI), a typical oxidative stress inducible gene. Potassium cyanide (KCN) and hydrogen peroxide (H2O2) induced a rapid increase of the Aox1a and GstI transcripts, which was persisted in prolonged treatment at high H2O2 concentration only for Aox1a. High concentration of salicylic acid (SA) and salicyl hydroxamic acid (SHAM) induced Aox1a mRNA only after prolonged exposure, while GstI displayed an early strong induction, which declined thereafter. Nitric oxide (NO) induced a high increase of Aox1a after prolonged exposure at high concentration, while GstI displayed a weak response. Our results show that multiple signaling pathways, involved in stress responses, also participate and differentially regulate Aox1a and GstI in maize. A ROS-depended signaling event may be involved, suggesting an essential role of Aox1a under oxidative stress in maize.

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.

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases.

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.

Altered Cu metabolism and differential transcription of Cu/ZnSod genes in a Cu/ZnSOD-deficient mutant of maize: evidence for a Cu-responsive transcription factor.

Maize inbred line A351 exhibits extremely low levels of Cu/Zn superoxide dismutase (SOD) isozymes, three cytosolic and one chloroplastic, which are increased by supplying copper to near-toxic concentrations. Activities of the copper enzymes cytochrome c oxidase and ascorbate oxidase are also reduced. The level of expression of the maize copper chaperone for SOD is normal to elevated. The gene transcript encoding chloroplastic SOD-1 is present at normal levels, whereas RNA levels of the cytosolic SODs are low and increase with added copper, suggesting a promoter element and copper-dependent transcription factor common to the three genes. Although a reduced level of high-affinity copper transport in A351 cannot be ruled out, high transcript levels of a constitutively expressed metallothionein, suggesting increased copper chelation capacity and creating a general copper-deprivation effect, seem to be a likely cause of the reduced levels of copper enzyme activity and Cu/ZnSod gene transcripts. While exogenous copper does not affect the wild-type SOD activity or protein, it increases wild-type Cu/ZnSod transcript levels in a response similar to that of several yeast genes involved in copper sequestration and antioxidant defense. A sequence that is highly homologous to those of the copper-responsive transcription factors ACE1 (Saccharomyces cerevisiae) and AMT1 (Candida glabrata) is present in the promoters of three maize Cu/ZnSod genes.