Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses.

Molecular oxygen (O2) is the premier biological electron acceptor that serves vital roles in fundamental cellular functions. However, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species (ROS) such as superoxide (O2*-), hydrogen peroxide, and hydroxyl radical (OH*). If unabated, ROS pose a serious threat to or cause the death of aerobic cells. To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic and enzymatic antioxidant defenses. The latter include catalases, peroxidases, superoxide dismutases, and glutathione S-transferases (GST). Cellular ROS-sensing mechanisms are not well understood, but a number of transcription factors that regulate the expression of antioxidant genes are well characterized in prokaryotes and in yeast. In higher eukaryotes, oxidative stress responses are more complex and modulated by several regulators. In mammalian systems, two classes of transcription factors, nuclear factor kB and activator protein-1, are involved in the oxidative stress response. Antioxidant-specific gene induction, involved in xenobiotic metabolism, is mediated by the "antioxidant responsive element" (ARE) commonly found in the promoter region of such genes. ARE is present in mammalian GST, metallothioneine-I and MnSod genes, but has not been found in plant Gst genes. However, ARE is present in the promoter region of the three maize catalase (Cat) genes. In plants, ROS have been implicated in the damaging effects of various environmental stress conditions. Many plant defense genes are activated in response to these conditions, including the three maize Cat and some of the superoxide dismutase (Sod) genes.

Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses

Molecular oxygen (O2) is the premier biological electron acceptor that serves vital roles in fundamental cellular functions. However, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species (ROS) such as superoxide (O2À-), hydrogen peroxide, and hydroxyl radical (OHÀ). If unabated, ROS pose a serious threat to or cause the death of aerobic cells. To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic and enzymatic antioxidant defenses. The latter include catalases, peroxidases, superoxide dismutases, and glutathione S-transferases (GST). Cellular ROS-sensing mechanisms are not well understood, but a number of transcription factors that regulate the expression of antioxidant genes are well characterized in prokaryotes and in yeast. In higher eukaryotes, oxidative stress responses are more complex and modulated by several regulators. In mammalian systems, two classes of transcription factors, nuclear factor kB and activator protein-1, are involved in the oxidative stress response. Antioxidant-specific gene induction, involved in xenobiotic metabolism, is mediated by the "antioxidant responsive element" (ARE) commonly found in the promoter region of such genes. ARE is present in mammalian GST, metallothioneine-I and MnSod genes, but has not been found in plant Gst genes. However, ARE is present in the promoter region of the three maize catalase (Cat) genes. In plants, ROS have been implicated in the damaging effects of various environmental stress conditions. Many plant defense genes are activated in response to these conditions, including the three maize Cat and some of the superoxide dismutase (Sod) genes.

Structural organization, regulation, and expression of the chloroplastic superoxide dismutase Sod1 gene in maize.

A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined.

Differential antioxidant responses to norflurazon-induced oxidative stress in maize.

This study examined the contribution of catalase (CAT) and superoxide dismutase (SOD) in the overall antioxidant response to norflurazon (NF)-induced oxidative stress in leaves, mesocotyls and scutella of maize (Zea mays). Maize catalase null mutants were used to provide insights into the role(s) of these isozymes. A substantial increase in Cat1 and Cat2 transcript levels occurred in NF-treated leaves in all maize lines examined. However, these two transcripts did not show a particular pattern of change in NF-treated scutella from 5-day postimbibition (dpi) and 18-day postpollination (dpp) maize. The NF-induced increase in Cat1 appeared to be dependent on excessive light energy caused by a lack of photoprotectant carotenoids. especially in leaves. In NF-treated leaves, the chloroplastic Cu/Zn-SOD-1 isozyme responded strongly compared to the cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD-3 isozymes, suggesting the critical role of SOD-1 as a major component in chloroplastic antioxidant defenses. All SOD isozymes in the NF-treated scutella of various maize lines were consistent in their response to NF. The most significant increase was observed with Sod1 in NF-treated leaves; however, no significant Sod1 changes were observed in similarly treated scutella at 5 dpi and 18 dpp. These results suggest that the response of the Cat and Sod genes to NF is likely developmental and tissue-specific.

Transgenic tobacco plants expressing the maize Cat2 gene have altered catalase levels that affect plant-pathogen interactions and resistance to oxidative stress.

Transgenic tobacco genotypes expressing the maize Cat2 gene were developed with altered catalase (CAT) levels that resulted in a moderate increase of CAT activity in two transgenic lines. Bacterial infection, with a pathogen that does not share homology with the transgene, caused local and systemic down-regulation of the steady state mRNA levels of the 35S-driven transgene in a manner resembling post-transcriptional gene silencing (PTGS). Phenotypic symptoms of hypersensitive response (HR) and systemic acquired resistance (SAR) were similar in control SR1 and the transgenic genotypes. Induction of hin1, used as a molecular marker of plant responses to invading bacteria, displayed a similar pattern between control and transgenic lines, but some variation in the levels of expression was observed. The major difference was recorded in the ability of the plants to restrict bacterial growth during HR. All transgenic lines were more sensitive than control SR1, with two lines exhibiting a significantly reduced capacity to inhibit bacterial growth. This is consistent with the putative enhanced capacity of transgenic lines containing the maize Cat2 gene to more effectively remove H2O2, which may act as a direct antimicrobial agent. Steady state mRNA levels of PR-1 and PR-5 varied among the genotypes, possibly indicating differences in strength of the SAR signal. Transgenic line 2, which was the most sensitive during HR, was most effective in restricting bacterial growth during SAR. This indicates that a reverse correlation might exist between the severity of infection during HR and the ability to inhibit bacterial growth during SAR. Growth under high light conditions affected plant-pathogen interactions in control SR1, as well as in transgenic line 8. Early induction and higher expression of PR-1 and PR-5 was detected in both SR1 and line 8 in high light-grown plants as compared with their low light-grown counterparts. Our data indicate that growth under high light conditions can predispose plants to better resist pathogen attack, and may amplify local and systemic defense signals. Finally, one transgenic line, which exhibited 1.3-fold higher average CAT activity in comparison with the untransformed SR1 control, suffered significantly less methyl viologen (MV) damage than untransformed control plants at moderate and high MV concentrations.

Hydrogen-peroxide-mediated catalase gene expression in response to wounding.

The effect of wounding on catalase expression was examined in embryos and leaves of maize. All three Cat genes are upregulated in response to wounding in immature embryos. Cat expression also increased in response to jasmonic acid (JA), raising the possibility that JA and wounding may share a common signal transduction pathway in upregulating Cat mRNA in immature embryos. In young leaves, only Cat1 and Cat3 transcripts increase in response to wounding, but JA does not play a role. Cat1 and Cat3 transcript accumulation also increases in response to wounding in both wild-type and mutant leaves deficient in abscisic acid (ABA), implying that Cat1 and Cat3 induction in response to wounding is not mediated by ABA in leaves. Transient assays using the Cat1 promoter fused with the reporter gene Gus, showed that the DNA sequence motif responsible for Cat1 upregulation by wounding overlaps with the ABA-responsive element (ABRE, G-box) in the Cat1 promoter. The exact nature of the signals triggering the Cat responses to wounding is not clear at this point, but some evidence indicates that reactive oxygen species (ROS) play a role in this response. In fact, we have found that endogenous H(2)O(2) levels increase in wounded leaves. Thus, wounding may indirectly induce the production of H(2)O(2) in leaves, triggering the antioxidant response.

Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response.

The mechanisms by which the maize antioxidant Cat1 gene responds to abscisic acid (ABA) and osmotic stress have been investigated. Results show that during late embryogenesis, Cat1 expression in vivo is independent of endogenous ABA levels. However, exogenously applied ABA significantly enhances Cat1 expression. Transient assays using particle bombardment show that the proximal ABRE2 element on the Cat1 promoter is responsible for the induction of Cat1 expression by ABA. We further show that ABA induces the expression of Cat1 via the interaction between ABRE2 and one of its binding proteins, CBF1 (Cat1 binding factor 1). Using ABA-deficient mutant embryos, we show that osmotic stress induces Cat1 expression through two alternate signal transduction pathways: an ABA signaling pathway leading to the interaction between the ABRE2 motif and CBF1, and a pathway via the interaction of ABRE2 and CBF2 (Cat1 binding factor 2) that is independent of ABA. The data presented clearly suggest that hydrogen peroxide (H2O2) plays an important intermediary role in the ABA signal transduction pathway leading to the induction of the Cat1 gene.

Catalase transcript accumulation in response to dehydration and osmotic stress in leaves of maize viviparous mutants.

The effect of osmotic stress and dehydration on maize catalase transcript accumulation was examined in leaves of abscisic acid (ABA)-deficient and ABA-insensitive mutants. We have found that the response of Cat1 to osmotic stress and dehydration is not via an ABA-mediated pathway in young leaves, suggesting that there are two different mechanisms by which Cat1 responds to osmotic stress in embryos and in leaves. The Cat2 transcript increased in response to osmotic stress, but was repressed by dehydration. On the other hand, the Cat3 transcript is up-regulated by dehydration and osmotic stress only in ABA-deficient mutant leaves, implying that ABA may act as a repressor for Cat3 expression in response to dehydration and high osmoticum. We also found that the VP1 trans-acting factor is not required for the induction of Cat1 by ABA in leaves, but may play a role in stabilizing the Cat1 transcript after an initial induction. The exact nature of the signals triggering Cat responses to osmotic stress and dehydration is not clear. We speculate that oxygen free radicals may play a role in this response. Osmotic stress and dehydration may indirectly induce production of oxygen free radicals in leaves, thus triggering the antioxidant response.

Differential response of antioxidant genes in maize leaves exposed to ozone.

Antioxidant enzymes function to eliminate reactive oxygen species (ROS) produced as a consequence of normal metabolic functions as well as environmental stress. In these studies, the responses of catalase (Cat), superoxide dismutase (Sod) and glutathione S-transferase (Gst), as well as D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) genes were analyzed in 9- and 15-day postimbibition maize seedlings exposed to various ozone (O3) concentrations and time periods. After a single (acute) 6 h exposure, or 3, 6 and 10 consecutive days (chronic) exposure to O3, Cat1, Cat3, Gst1, Sod3, Sod4 and Sod4A transcript levels generally increased, while Cat2, RbcS and Sod1 levels decreased. Such changes in mRNA levels do not necessarily reflect parallel changes in the protein products of these genes. Changes in transcript levels seemed to be correlated with the spatial location of the isozymes encoded by the genes. The results are discussed with respect to gene regulation and expression, and the localization and function of these antioxidant enzymes during ozone-mediated oxidative stress.

Catalase activity and hydrogen peroxide levels are inversely correlated in maize scutella during seed germination.

Temporal patterns of hydrogen peroxide (H2O2) levels and total catalase activity are presented for post-imbibition scutella from six maize inbred lines expressing variable catalase activity. In all lines examined, H2O2 levels were highest during the initial days post-imbibition (1-2 dpi) and decreased thereafter, while total catalase activity was lowest during early dpi (1-2 dpi) and reached maximal activity at 4-6 dpi. In three of the six lines tested, a simple inverse correlation between catalase activity and H2O2 level was significant by Spearman's rank (P < 0.01). In addition to the general decline in H2O2 level throughout the dpi period, a reproducible increase in H2O2 level was observed at 4-5 dpi in five of six lines examined. Mutant lines lacking CAT-3 activity demonstrated a temporal shift in the occurrence of this increase. The role of total catalase (and individual isozymes) in controlling H2O2 levels during germination and the role of H2O2 as a potential regulator of catalase expression during germination are discussed.

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.

Two structurally similar maize cytosolic superoxide dismutase genes, Sod4 and Sod4A, respond differentially to abscisic acid and high osmoticum.

The maize (Zea mays) superoxide dismutase genes Sod4 and Sod4A are highly similar in structure but each responds differentially to environmental signals. We examined the effects of the hormone abscisic acid (ABA) on the developmental response of Sod4 and Sod4A. Although both Sod4 and Sod4A transcripts accumulate during late embryogenesis, only Sod4 is up-regulated by ABA and osmotic stress. Accumulation of Sod4 transcript in response to osmotic stress is a consequence of increased endogenous ABA levels in developing embryos. Sod4 mRNA is up-regulated by ABA in viviparous-1 mutant embryos. Sod4 transcript increases within 4 h with ABA not only in developing embryos but also in mature embryos and in young leaves. Sod4A transcript is up-regulated by ABA only in young leaves, but neither Sod4 nor Sod4A transcripts changed in response to osmotic stress. Our data suggest that in leaves Sod4 and Sod4A may respond to ABA and osmotic stress via alternate pathways. Since the Sod genes have a known function, we hypothesize that the increase in Sod mRNA in response to ABA is due in part to ABA-mediated metabolic changes leading to changes in oxygen free radical levels, which in turn lead to the induction of the antioxidant defense system.

Circadian expression of the maize catalase Cat3 gene is highly conserved among diverse maize genotypes with structurally different promoters.

The Cat3 gene of maize exhibits a transcriptionally regulated circadian rhythm. In the present study we examined the following: (1) the extent of the circadian Cat3 expression between maize genotypes of diverse origin; (2) the functional significance of a Tourist transposable element located in the Cat3 promoter of the inbred line W64A, which harbors putative regulatory elements (GATA repeat, CCAAT boxes) shown to be involved in the light induction and circadian regulation of the Arabidopsis CAB2, as well as other plant genes; and (3) aspects of the physiological role of CAT-3 in maize metabolism. Results confirm that the circadian Cat3 expression is a general phenomenon in maize. Regulation of Cat3 gene expression is not dependent on the presence of the Tourist element in the promoter of the gene nor on the presence of motifs similar to those found significant in the circadian expression of the Arabidopsis CAB2 gene. Structural diversity was revealed in the Cat3 promoters of maize genotypes of diverse origins. However, highly conserved regions with putative regulatory motifs were identified. Relevance of the conserved regions to the circadian regulation of the gene is discussed. Possible physiological roles of CAT-3 are suggested.

Response of the maize catalases to light.

The three maize catalase genes respond differentially to light signals. Expression of Cat1 is light independent while expression of Cat2 and Cat3 is light responsive. Upon exposure to light there is rapid accumulation of CAT-2 protein in leaves, due to both increased transcript accumulation and increased translation of the Cat2 message. Short UV light pulses also cause a strong transient induction of Cat2 gene expression, while long term exposure to UV does not affect the rate of Cat2 transcription. The Cat3 gene of maize exhibits a transcriptionally regulated circadian rhythm. The induction of the Cat3 circadian expression in etiolated leaves is probably regulated by a very low fluence phytochrome response; the involvement of a blue light/UV-A and a UV-B photoreceptor is also possible. Regulatory elements located on the Cat3 promoter have recently been identified and their significance in the complex light response of the gene is being investigated. Possible physiological role(s) of the light responding maize catalases Cat2 and Cat3 are discussed.

Influence of UV-light on the expression of the Cat2 and Cat3 catalase genes in maize.

The effects of UV (ultraviolet) -irradiation on the expression of the antioxidant genes Cat2 and Cat3, encoding the CAT-2 and CAT-3 catalases in maize were examined. Cat2 and Cat3 transcript accumulation was analyzed in leaves of maize seedlings grown under different light conditions, and subsequently exposed to UV-light. Under DD-(constant darkness) and LL- (continuous light) conditions, as well as under a 12h D/L- (dark/light) photoperiod, the Cat2 mRNA was expressed at low and constant levels. In contrast, Cat3 transcript accumulation was constant and about 10 times higher than that of Cat2 under DD or LL, while the expression of Cat3 exhibits a typical circadian rhythm under a 12h D/L photoperiod. UV- light pulses in the range of 290 to 400 nm strongly induce the expression of Cat2. Upon removing the UV-B portion (290-310 nm) of the UV-spectrum the maximal Cat2 transcript level was reduced by about 60%. On applying UV-light of the same quality in addition to visible light, the expression of Cat2 was induced. DNA damage caused by UV-light and induction mediated by a UV-light photosensory system are suggested. Further, it is suggested that the Cat3 circadian rhythm may be regulated by a blue light/UV-A and a UV-B photoreceptor. If DD and LL grown plants that exhibit no circadian oscillation, were exposed to constant UV-light in the range of 290 to 400 nm a circadian rhythm was induced; indicating that UV-light may function as an additional environmental cue to entrain the Cat3 circadian rhythm. Since, Cat2 and Cat3 showed distinct responses to UV light, it is suggested that both genes may act to scavenge reactive oxygen species (ROS) generated by UV-light to protect the plant from oxidative damage.

A comparison of the structure and function of the highly homologous maize antioxidant Cu/Zn superoxide dismutase genes, Sod4 and Sod4A.

Two highly similar cytosolic Cu/Zn Sod (Sod4 and Sod4A) genes have been isolated from maize. Sod4A contains eight exons and seven introns. The Sod4 partial sequence contains five introns. The introns in both genes are located in the same position and have highly homologous sequences in several regions. The largest intron (> 1200 bp) interrupts the 5' leader sequence. The presence of different regulatory motifs in the promoter region of each gene may indicate distinct responses to various conditions. Zymogram and RNA blot analyses show that Sod4 and Sod4A are expressed in all tissues of the maize plant. The developmental profiles of Sod4 and Sod4A mRNA accumulation differ in scutella during sporophytic development. RNA blot analysis of the respective Sod mRNAs indicates a differential, tissue-specific response of each gene to certain stressors. RNA isolated from stem tissue of ethephon-treated seedlings shows an increase in the Sod4 but not the Sod4A transcript while there is no change in transcripts of either gene in leaves or roots. There is differential mRNA accumulation between the two genes in leaf and stem tissue of paraquat-treated seedlings. Other agents that can cause oxidative stress were also tested for differential expression of the genes.

Molecular evolution of maize catalases and their relationship to other eukaryotic and prokaryotic catalases.

We have compared the nucleotide and protein sequences of the three maize catalase genes with other plant catalases to reconstruct the evolutionary relationship among these catalases. These sequences were also compared with other eukaryotic and prokaryotic catalases. Phylogenies based on distances and parsimony analysis show that all plant catalases derive from a common ancestral catalase gene and can be divided into three distinct groups. The first, and major, group includes maize Cat1, barley Cat1, rice CatB, and most of the dicot catalases. The second group is an apparent dicot-specific catalase group encompassing the tobacco Cat2 and tomato Cat. The third is a monocot-specific catalase class including the maize Cat3, barley Cat2, and rice CatA. The maize Cat2 gene is loosely related to the first group. The distinctive features of monocot-specific catalases are their extreme high codon bias at the third position and low degree of sequence similarity to other plant catalases. Similarities in the intron positions for several plant catalase genes support the conclusion of derivation from a common ancestral gene. The similar intron position between bean catalases and human catalase implies that the animal and plant catalases might have derived from a common progenitor gene sequence.

Isolation, characterization and expression of the maize Cat2 catalase gene.

The maize Cat2 gene was isolated by direct cloning and PCR. The clones were mapped and sequenced. The start site of transcription was determined by primer extension. Computer analysis of the 1.6 kb Cat2 promoter sequence has revealed an obvious TATA box, tow GC boxes, a putative GA response element, and several ACGT core sequences known to have diverse regulatory functions in plants. Several other protein binding motifs were also identified within 800 bp upstream from the transcriptional start site. Five introns were identified in the Cat2 coding region. All five Cat2 introns are located in exactly the same position as five of the six introns in Cat1. Two of the Cat2 introns are located in the same position as the two Cat3 introns. The identical positioning of these introns suggests an evolutionary link between all three maize catalase genes. The response of the CAt2 gene to plant growth regulators was examined. Results clearly showed that the response of CAt2 to several environmental factors are developmental stage-dependent. Thus, complex regulatory mechanisms appear to be involved in the regulation of Cat2 expression in maize.

Developmentally related responses of maize catalase genes to salicylic acid.

The response of the maize catalase genes (Cat1, Cat2, and Cat3) to salicylic acid (SA) was examined at two distinct developmental stages: embryogenesis and germination. A unique, germination-related differential response of each maize catalase gene to various doses of SA was observed. During late embryogenesis, total catalase activity in scutella increased dramatically with 1 mM SA treatment. The accumulation of Cat2 transcript and CAT-2 isozyme protein provided the major contribution to the observed increase in total catalase activity. This increase was paralleled by the enhanced growth of germinated embryos at that stage. In a CAT-2 null mutant line, a full compensation of total catalase activity by the CAT-1 isozyme was observed in the presence of SA. This suggests that catalase is important for maintenance of normal cellular processes under stress conditions. SA at 1 mM, which enhances growth of precociously germinated embryos, appeared to inhibit seed germination at 1 day after inhibition. Furthermore, Cat2 transcript accumulation was inhibited at this stage. SA is probably not a direct signal for the induction of the catalase genes. Other signals, possibly germination-related regulator(s), might be responsible for the induction of the catalase genes. The effect of SA on the activity of purified catalase protein was also examined.