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. 1998 May;117(1):217–224. doi: 10.1104/pp.117.1.217

Two Structurally Similar Maize Cytosolic Superoxide Dismutase Genes, Sod4 and Sod4A, Respond Differentially to Abscisic Acid and High Osmoticum1

Lingqiang Guan 1, John G Scandalios 1,*
PMCID: PMC35006  PMID: 9576791

Abstract

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.

SOD (EC 1.15.1.1) is a metalloenzyme that is found in almost all organisms and catalyzes the dismutation of superoxide anion radical to hydrogen peroxide and molecular oxygen (Fridovich, 1978). This reaction is the first step for ROS scavenging. SODs are classified into three types: Mn-, Fe-, and Cu/Zn-SOD, depending on the metal found in the active site. In higher plants SODs are found in the cytosol, plastids, and mitochondria. In maize there are nine SOD isozymes: four Cu/Zn cytosolic isozymes (SOD-2, SOD-4, SOD-4A, and SOD-5), four mitochondrial-associated Mn SODs (SOD-3.1, SOD-3.2, SOD-3.3, and SOD-3.4), and one Cu/Zn chloroplast-associated isozyme (SOD-1; Baum and Scandalios, 1979; Scandalios, 1997, and refs. therein).

The cytosolic isozyme SOD-4A is biochemically indistinct from SOD-4. Deduced amino acid sequence analysis from cDNA clones showed that there are only two amino acid differences between the SOD-4 and SOD-4A proteins (Cannon and Scandalios, 1989), not enough to separate them by native gel electrophoresis or to produce a specific antibody for each protein. The cDNA sequence between the Sod4- and Sod4A-coding region is highly homologous (95% identity). Using gene-specific probes generated from the 3′ end of the cDNA, we isolated and characterized the Sod4 and Sod4A genomic clones (Kernodle and Scandalios, 1996). The sequence of these two genes showed that they share the same intron numbers and positions, and that their coding regions are highly homologous. However, the promoter region is different and shows almost no sequence homology between the two genes. This implies that Sod4 and Sod4A may be regulated differently and may respond differently to environmental signals. In fact, we have shown that Sod4 and Sod4A respond differently to light, hydrogen peroxide, cercosporin, and ethephon (Kernodle and Scandalios, 1996; Williamson and Scandalios, 1992b).

ABA has pleiotropic effects on plant growth and development. A number of ABA-responsive genes are normally expressed during late embryogenesis when seed tissues desiccate and the embryos become dormant (Finkelstein et al., 1985). ABA appears to mediate physiological processes in response to osmotic stress. Levels of endogenous ABA increase in tissues subjected to osmotic stress due to high osmoticum, salt, desiccation, and cold (Henson, 1984; Mohapatra et al., 1988). Under these conditions, specific genes are expressed that can also be induced in unstressed tissues by the application of exogenous ABA (Gomez et al., 1988; Mundy and Chua, 1988). It is thought that some of these ABA-responsive genes may encode proteins with osmoregulatory or other protective functions (Bartels et al., 1988). It was previously reported that Sod4 and Sod4A transcripts are highly expressed in immature embryos (Cannon and Scandalios, 1989); thus, we speculate that the Sod4 and Sod4A genes may be regulated in part by ABA during late embryogenesis. Herein we report the effect of ABA on Sod4 and Sod4A transcript accumulations in different tissues at different developmental stages.

MATERIALS AND METHODS

The inbred maize (Zea mays) lines W64A, M1A4 (Vp5/vp5), and Vp1 were used in these studies. W64A and M1A4 are maintained in this laboratory, and Vp1 was obtained from the Maize Stock Center at the University of Illinois (Urbana). The maize vp mutants contain either lowered amounts of ABA (M1A4, vp5) or are morphologically insensitive to normal endogenous levels of ABA (vp1), resulting in precocious germination in the ear. The viviparous kernels can be distinguished from wild-type kernels early in development based on endosperm and embryo color. The vp5 mutant in M1A4 interrupts ABA biosynthesis early in the biosynthetic pathway (Robichaud et al., 1980). Homozygous recessive kernels (vp5/vp5) lack carotenoids, resulting in white endosperm and embryos, easily distinguished from the yellow wild-type kernels (Vp5/−). In the vp1 mutant line, production of the Vp1 transcription factor is greatly reduced (McCarty et al., 1991). In addition, this mutant results in decreased anthocyanin biosynthesis in the kernel. The homozygous mutant (vp1/vp1) is characterized by yellow kernels, in contrast to the wild-type red/purple kernels (Vp1/−). Since both of these recessive mutations are lethal in the homozygous state, they are maintained as heterozygotes.

Treatment Conditions

For immature embryos, maize ears were harvested in the morning from greenhouse plants 28 dpp, and whole embryos (scutella plus axes) were excised from kernels on the same day. For germinating embryos, seeds were surface sterilized with 1% sodium hypochlorite for 10 min and rinsed with deionized water. The seeds were then soaked with deionized water for 24 h and placed in germination trays at 25°C in the dark from 1 to several days, depending on the experimental design. Germinating embryos were then excised and treated with the appropriate doses of ABA. Embryos isolated from different developmental stages of immature or mature seeds were treated with ABA for 24 h at 25°C in the dark. At the end of each treatment, scutella were isolated from embryos and used for RNA isolation. Seven-day-old seedlings were also treated with different concentrations of ABA solution for 24 h in the light, and total RNA was isolated from leaves for RNA analysis. For experiments with high osmoticum, embryos were isolated from different lines and placed on Murashige-Skoog medium containing 11% (w/v) mannitol and/or 20% (w/v) Suc for 24 h in the dark. Seven-day-old leaves were also treated with 11% mannitol solution for 24 h under constant light conditions.

RNA Analyses

Total RNA was isolated from control and ABA-treated samples by a modification of the cold phenol extraction method (Beachy et al., 1985). For northern-blot analysis, total RNA (10 or 20 μg) from each sample was separated in denaturing 1.2% agarose gels and transferred onto nylon membranes. The blots were sequentially hybridized with 32P-labeled gene-specific probes for Sod4 and Sod4A (Cannon and Scandalios, 1989) in modified Church buffer (Church and Gilbert, 1984) containing 7% SDS, 0.5 m EDTA, 0.5 m sodium phosphate, and 1% BSA. After these analyses had been performed, probes were stripped from the filters and reprobed first with insert DNA from clone p1015 containing the coding sequence of the ABA-regulated Em polypeptide (Williamson et al., 1985) and subsequently with a DNA fragment from clone pHA2 containing an 18S ribosomal sequence (Jorgensen et al., 1987).

RESULTS

Sod4 and Sod4A Transcript Accumulation in Scutella during Late Embryogenesis

We previously found that Sod4 and Sod4A transcripts accumulate to high levels in scutella of mature embryos (Cannon and Scandalios, 1989); however, the expression pattern of cytosolic Sod at middle to late embryogenesis had not been determined. Sod4 and Sod4A transcript accumulation was examined during middle to late embryogenesis (17–30 dpp). Our results show that Sod4 and Sod4A transcripts start to accumulate at 17 dpp and reach maximum levels 30 dpp (Fig. 1). The typical ABA-responsive Em transcript of wheat was used as a control and showed a similar accumulation pattern. The late embryogenesis-abundant gene-like expression of Sod4 and Sod4A accumulation led us to speculate that ABA may play a role in Sod transcript accumulation during middle to late embryogenesis. Since these two cytosolic Sod genes are almost identical in their coding region, similar expression patterns led us to believe that they may share some common sequences in their promoter region.

Figure 1.

Figure 1

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Accumulation of the Sod4 and Sod4A transcripts during middle to late embryogenesis. W64A ears were harvested from greenhouse-grown plants at different developmental stages. Embryos were isolated from 17-, 21-, 24-, 27-, and 30-dpp kernels and used for RNA isolation. Total RNA (10 μg) was separated on denaturing agarose gels and transferred onto nylon membranes. The filter was probed with gene-specific probes of the maize Sod4 and Sod4A genes, the Em of wheat, and finally, with 18S rDNA as a loading and transfer control.

Sequence Comparison of the Promoter of Sod4 and Sod4A

We compared the promoter sequence of Sod4 and Sod4A up to the start of transcription. Under the best alignment conditions, Sod4 and Sod4A share only 40% of sequence identity. Sequence alignment also revealed that the ABA-responsive element is found in the promoter of the Sod4 gene (212, CACGTGGT; 322, GACGTACC) but cannot be located in the promoter of Sod4A (Fig. 2). Two Y-box elements are located only in the promoter region of the Sod4A. The Y-box motif mediates redox-dependent transcriptional activation (Duh et al., 1995). These data imply that Sod4 transcript accumulation during late embryogenesis may be due to changes in endogenous ABA levels, whereas the accumulation of Sod4A transcript at the same developmental stage may be due to signals other than ABA. To prove this hypothesis, we examined the accumulation of the Sod4 and Sod4A transcripts in response to exogenously applied ABA in 28-dpp embryos.

Figure 2.

Figure 2

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Sequence comparison between the promoter of the maize Sod4 and Sod4A genes. The Sod4 and Sod4A promoter sequences were aligned using MegAlign of the DNAStar program. Identical nucleotides in the Sod4 and Sod4A promoter are shaded. Two ABA-responsive elements (ABRE), TATA box in Sod4, and two Y-box motifs (mediates redox-dependent transcriptional activation) in Sod4A are boxed.

Sod4 and Sod4A Transcript Accumulation in Response to ABA in Late-Developing Embryos

The effects of ABA on accumulation of the Sod4 and Sod4A transcripts in scutella of developing maize embryos were investigated. The steady-state levels of the Sod4 transcript increased with increasing concentrations of ABA. The Sod4A transcript increased in control embryos (−ABA for 24 h) in comparison with untreated embryos (in planta) but did not increase further in response to ABA (Fig. 3, top). The Em transcript increased in response to all ABA concentrations applied. In a time-course experiment the Sod4 transcript started to accumulate after 4 h of ABA (10−4 m) treatment and reached highest levels at 24 h. An interesting finding was that the Sod4 transcript in control embryos (−ABA) was also increased between 4 and 24 h. The Sod4A transcript in control embryos increased at 8 to 24 h, whereas transcript accumulation was repressed in ABA-treated embryos (Fig. 3, bottom). The Em transcript increased in response to ABA within 4 h and reached its maximum 12 h after ABA treatment.

Figure 3.

Figure 3

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Changes in Sod4 and Sod4A transcript accumulation in 28-dpp developing embryos in the presence of ABA. Embryos were isolated from 28-dpp kernels of greenhouse-grown W64A plants and incubated on Murashige-Skoog medium supplemented with either increasing concentrations of ABA for 24 h (top) or with (+)/without (−) 10−4 m ABA in the dark for 0, 2, 4, 8, 12, and 24 h (bottom). Scutella were isolated for RNA isolation. Total RNA (10 μg) was used for northern-blot analysis. Blot was probed with the Sod4, Sod4A gene-specific probes, Em, and 18S rDNA. ip, In planta control (0 h of treatment).

Effects of ABA on Sod4 and Sod4A Transcript Accumulation in Germinating Embryos

The effects of ABA on Sod4 and Sod4A expression were also examined in mature germinating maize embryos. We used 5-dpi germinating embryos to conduct the same ABA treatments as described above for developing embryos. Maize embryos (scutella plus axes) were isolated from 5-dpi W64A seedlings and incubated on Murashige-Skoog medium supplemented with increasing doses of ABA (0, 10−5, 10−4, and 10−3 m) for 24 h. The Sod4 transcript is low in 5-dpi scutella (in planta control) and increased dramatically after 24 h of ABA treatment. The maximum increase in Sod4 transcript was observed at 10−3 m ABA (Fig. 4, top). The Sod4A transcript is relatively high in 5-dpi scutella, but it did not change in response to ABA. A time-course experiment was conducted with 5-dpi embryos. Northern-blot analysis showed that the Sod4 transcript started to accumulate after 2 h of ABA treatment and continued to increase to high levels at 24 h (Fig. 4, bottom). On the other hand, the Sod4A transcript showed a slight decrease in response to ABA throughout the 24-h treatment period.

Figure 4.

Figure 4

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Changes in Sod4 and Sod4A transcript accumulation the presence of ABA in postgermination embryos. Embryos were excised from 5-dpi germinating W64A seeds and treated with either increasing concentrations of ABA for 24 h (top) or with 0 (−) and 10−3 m ABA (+) for 2, 4, 12, and 24 h (bottom). Scutella were isolated from treated embryos and used for RNA isolation and northern-blot analysis. Total RNA (10 μg) was used for northern blot and probed with Sod4 and Sod4A gene-specific probes. The 18S rRNA was used as a loading control. ip, In planta control (0 h of treatment).

Effects of Osmotic Stress on the Expression of Sod4 and Sod4A in Developing and Germinating Embryos

We also examined the effect of high osmoticum (11% mannitol) on Sod4 and Sod4A transcript accumulation. In 28-dpp developing embryos the Sod4 transcript showed a similar accumulation pattern (in comparison with ABA response) in response to osmotic stress (11% mannitol; Fig. 5, top). The Sod4 transcript started to increase in 4 h and reached maximum levels by 24 h in response to mannitol (11%, w/v). Similar to the ABA response, the Sod4A transcript accumulation decreased in response to mannitol. In 5-dpi embryos the Sod4 transcript increased in response to mannitol (11%, w/v), whereas Sod4A did not change (Fig. 5, bottom). The Em transcript normally cannot be detected at that stage. After 24 h of ABA treatment, a different Em transcript was induced with higher Mr than the transcript normally found in developing embryos. Litts et al. (1987) reported that Em comprises a multigene family, but variable Em transcript accumulation in response to ABA has not been previously documented.

Figure 5.

Figure 5

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Accumulation of the Sod4 and Sod4A transcripts in response to high osmoticum in developing and germinating embryos. Top, Embryos were dissected from developing kernels of W64A 28 dpp and incubated on Murashige-Skoog medium in the dark for 0, 2, 4, 8, 12, and 24 h with (+) or without (−) mannitol (11%, w/v). Bottom, Embryos were excised from 5-dpi germinating W64A seeds and treated with 20% (w/v) Suc, 11% (w/v) mannitol, or 10−4 m ABA for 24 h in the dark. Scutella were isolated and used for RNA isolation and northern-blot analysis. Total RNA (10 μg) was separated on 1.2% denaturing gel and transferred to a nylon membrane. The blot was sequentially hybridized with Sod4, Sod4A, Em, and 18S probes.

Accumulation of the Sod4 and Sod4A Transcripts in Scutella of an ABA-Deficient Mutant in Response to ABA and High Osmoticum

To understand the possible role of ABA in the accumulation of Sod transcripts in developing embryos treated with high osmoticum, the accumulation of Sod4 and Sod4A transcripts was examined in an ABA-deficient mutant (vp5/vp5) and its wild-type sibling (Vp5/−). Ears were harvested from 25-dpp heterozygous plants. Embryos were isolated and placed on Murashige-Skoog medium supplemented with 10−4 m ABA, 20% (w/v) Suc, or 11% (w/v) mannitol for 24 h in the dark. Scutella were collected for RNA isolation. Exogenous application of ABA resulted in increased accumulation of the Sod4 transcript in both wild-type (Vp5/−) and mutant embryos (vp5/vp5). However, increased accumulation of the Sod4 transcript in response to high osmoticum was detected only in wild-type embryos and not in the ABA-deficient mutant (Fig. 6), suggesting that the accumulation of the Sod4 transcript in response to high osmoticum is due to an increase in the endogenous ABA levels. In contrast, the Sod4A transcript did not change in either the wild-type or the vp5 mutant in response to ABA or high osmoticum.

Figure 6.

Figure 6

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Changes in Sod4 and Sod4A transcript accumulation in response to ABA and high osmoticum in an ABA-deficient mutant (vp5/vp5) and its wild-type sibling (Vp5/−). Embryos were excised from 25-dpp kernels of vp5 and its wild-type sibling and treated with 20% Suc, 11% mannitol, or 10−4 m ABA. After the treatment scutella were collected and used to examine the transcript accumulation for Sod4, Sod4A, and the Em of wheat. The 18S rRNA served as a loading control.

Accumulation of the Sod4 and Sod4A Transcripts in Excised Embryos of vp1 Mutants and Their Wild-Type Siblings in Response to ABA

The change in Sod4 and Sod4A transcript accumulation was also examined in the ABA-insensitive mutant vp1. Ears containing both mutant (vp1/vp1) and wild-type (Vp1/−) kernels were harvested from heterozygous plants at 18 dpp. Embryos were isolated and placed on Murashige-Skoog medium supplemented with 10−4 m ABA for 24 h in the dark. Scutella were collected for RNA isolation after treatments. Results showed that the Sod4 transcript increased in response to ABA in both the wild type and the vp1 mutant, but the absolute levels of the Sod4 transcript are much higher in the wild type than in the vp1 mutant (Fig. 7). The Sod4A transcript was detectable but showed no change in response to ABA in both wild-type and vp1 mutant embryos. In contrast to Sod4, the Em transcript exhibits a significant increase in wild-type ABA-treated embryos but had very low detectable transcript levels in 18-dpp vp1 mutant embryos in response to ABA.

Figure 7.

Figure 7

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Changes in Sod4 and Sod4A transcript accumulation in response to 10−4 m ABA in 18-dpp vp1 embryos and their wild-type sibling. Embryos were collected from mutant (vp1/vp1) and wild type (Vp1/−) at 18 dpp and treated with or without 10−4 m ABA for 24 h. Total RNA (10 μg) was used for northern-blot analysis and probed with Sod4 and Sod4A gene-specific probe, as well as the Em of wheat. The 18S rRNA was used as a loading and transfer control.

Accumulation of Sod Transcripts in Response to ABA and High Osmoticum in Young Leaves

To understand the mechanisms of cytosolic Sod gene responses to ABA, we also examined the effect of ABA on Sod4 and Sod4A expression in young maize leaves. Seven-day-old light-grown W64A seedlings were harvested and roots were soaked in a solution containing 10−4 m ABA or 11% mannitol for 2, 4, 8, 12, and 24 h in the light. After treatment, leaves were collected and total RNA was isolated for northern-blot analysis. The Sod4 transcript was almost undetectable in untreated leaves but increased dramatically in response to ABA within 4 h and reached highest levels at 12 to 24 h. The Sod4 transcript increased in response to mannitol at 8 and 12 h. The Sod4A transcript increased in response to ABA after 12 to 24 h of treatment. The Em transcript could not be detected in young leaves. Thus, we used an ABA-responsive maize Cat1 transcript as a control (Williamson and Scandalios, 1992a). The Cat1 transcript showed a pattern similar to Sod4 in response to ABA; however, the Cat1 transcript was also increased dramatically in response to mannitol at 12 to 24 h of treatment, whereas Sod4 and Sod4A showed no transcript increase in response to mannitol after 24 h of treatment (Fig. 8). These data clearly indicate that the Sod4 transcript can be induced by ABA not only in immature embryos but also in mature embryos and in young leaves. These data also suggest that the Sod4 mRNA accumulation in response to ABA is not developmental stage dependent and might represent a general stress-response mechanism in which the Sod4 gene product increases to protect plants from ABA-mediated stress. The change of Sod4A transcript levels in response to ABA is dependent on tissue and developmental stages. These data also suggest that there are different pathways for the response of Sod genes to ABA and mannitol in young leaves.

Figure 8.

Figure 8

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Kinetics of accumulation of the Sod4 and the Sod4A transcripts in response to ABA and osmotic stress in 7-d-old leaves. Seven-day-old young W64A seedlings were harvested and roots were soaked with 10−4 m ABA solution or 11% mannitol solution for 2, 4, 8, 12, and 24 h with constant light. Total RNA was isolated from leaves and 20 μg of RNA from each sample was used for northern-blot analysis and probed with Sod4, Sod4A, and Cat1 gene-specific probes. The 18S rDNA was used as a loading control.

DISCUSSION

We have examined the Sod4 and Sod4A transcript accumulation during embryo development and in response to ABA at late embryogenesis, postgermination, and in young leaves. Our results indicate that the two closely related cytosolic Sod4 and Sod4A genes responded differently to ABA at each developmental stage examined. Both Sod4 and Sod4A transcripts increased to high levels during late embryogenesis; however, only the Sod4 transcript was up-regulated in response to ABA in 28-dpp scutella, in 5-dpi scutella, and in young leaves. The Sod4A transcript, on the other hand, showed no increase in response to ABA in developing and germinating embryos but increased in response to ABA after 12 h in young leaves. The Sod4 transcript increased only after a few hours following ABA treatment at all three stages. This implies that Sod4 might be directly responding to ABA, whereas the response of Sod4A to ABA in leaves is likely to be indirect, responding to ABA-mediated metabolic changes or stress.

The Maize Sod4 Gene Is Unique and Different in Its Response to ABA in Comparison with a Typical ABA-Regulated Gene, Em

We found that Sod4 and the typical ABA-regulated gene Em have similar expression patterns in response to ABA in W64A developing embryos; however, Sod4 is also unique and different from Em. An interesting finding is that the Sod4 transcript in control embryos (−ABA) also increased at 4 h and reached high levels in 24 h, whereas the Em transcript in controls increased slightly only after 12 h. It is likely that Sod4 responds to developmental signals other than ABA in the control embryos (−ABA), and the effects of ABA are superimposed on the developmental signal in ABA-treated embryos. In germinating embryos and in leaves the developmental signal no longer affects Sod4 gene expression. Thus, the effect of ABA on Sod4 expression is clearly observed within a few hours after ABA treatment. Previous studies showed that the Sod4 transcript accumulates to high levels in 3- to 6-dpi scutella, whereas the Sod4A transcript accumulates to high levels 5 to 10 dpi (Kernodle and Scandalios, 1996). This implies that the Sod4 transcript accumulates earlier than Sod4A after germination. They may respond to different signals during that period. The ABA-responsive Em transcript decreases to undetectable levels after 1 dpi and this is also coincident with the decrease in endogenous ABA levels after 1 dpi (Williamson and Scandalios, 1994). Both Sod4 and Sod4A transcripts increased in control scutella in 28-dpp embryos. The explanation for this is that when immature embryos are isolated and cultured in Murashige-Skoog medium without the presence of ABA, they will start to germinate. Given the fact that the Sod4 and the Sod4A transcripts accumulate to high levels after seed germination, the increase in Sod4 and Sod4A transcripts in control developing embryos (−ABA, 24 h) may be caused by the same developmental signals that induce the Sod4 and Sod4A transcripts after seed germination.

There Are at Least Two Pathways Involved in the Response of the Maize Sod4 Gene to ABA

Utilizing the ABA-insensitive mutant line Vp1, we found that Sod4 is induced by ABA in both wild-type (Vp1/−) and mutant (vp1/vp1) 18-dpp embryos with the highest Sod4 mRNA level found in ABA-treated wild-type embryos. The Em transcript is induced only by ABA in wild-type embryos and there is almost no detectable Em mRNA in 18-dpp vp1 mutant embryos. In immature embryos the Vp1 trans-acting factor is present (McCarty et al., 1989) and is required for maximizing the ABA-mediated induction of Sod4 in wild-type embryos where the highest Sod4 mRNA was detected in response to ABA. However, in germinating embryos and in young leaves, Vp1 factor is not present (McCarty et al., 1989) and is not required for the ABA-mediated induction of Sod4 in vitro. We previously reported that the Sod4 transcript accumulates to high levels in germinating embryos when ABA and Vp1 are not present (Kernodle and Scandalios, 1996). This suggests that the expression of Sod4 during postgermination is regulated by signals other than ABA in vivo. From the above observations we concluded that at least two independent pathways are involved in ABA-mediated Sod4 expression. During embryogenesis, the Vp1-dependent pathway is most likely to be involved in the ABA-induced expression of Sod4 because the Sod4 transcript accumulates to highest levels in ABA-treated wild-type (Vp1/−) embryos. In germinating embryos and in leaves the induction of Sod4 mRNA by ABA is not dependent on Vp1 because Vp1 transcript is not present during this period.

Sod4 Transcript Accumulation in Response to Osmotic Stress in Maize Embryos Is Due to Increased Endogenous ABA Levels

In numerous systems osmotic stress has been shown to increase endogenous ABA levels (Skriver and Mundy, 1990). Thus, it has been suggested that osmotic stress is mediated via an ABA-driven transduction pathway. Other reports indicate distinctions between the response to ABA and osmotic stress. A desiccation responsive gene (Rd 29) in Arabidopsis is rapidly induced by water and salt stress in an ABA-independent manner (Yamaguchi-Shinozaki and Shinozaki, 1993). The MMK4 (MAP kinase; mitogen-activated protein) gene in alfalfa accumulates after drought and cold treatment but not in response to ABA, indicating that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA (Jonak et al., 1996). Utilizing the ABA-deficient mutant vp5, we have demonstrated that the Sod4 transcript is increased only in wild-type embryos in response to osmotic stress; however, Sod4 mRNA increased to the same levels in wild-type and vp5 embryos in response to ABA. These data suggest that the increase of Sod4 transcript in response to high osmoticum is mediated by an increase in endogenous ABA levels in developing embryos. The Em transcript showed a similar expression in response to ABA and high osmoticum. It has been reported that the transcript of the wheat Em gene can also accumulate in seedlings in response to ABA and desiccation (Morris et al., 1988); however, we cannot detect Em transcript in young maize leaves under ABA treatment or under osmotic stress. In young leaves both Sod4 and Sod4A transcripts increase in response to ABA; however, Sod4 and Sod4A transcripts do not change in response to mannitol in leaves. These data imply that the Sod4 and Sod4A response to mannitol is dependent on the developmental stage. Their transcripts do not change in response to mannitol and may be due to different sensitivity to mannitol-mediated ABA accumulation in young leaves or simply due to different pathways between the mannitol response in leaves and in embryos. Another antioxidant gene, Cat1, responds positively to both ABA and mannitol in young leaves under the same treatment conditions.

Many genes regulated by ABA and/or high osmoticum have been isolated (Skriver and Mundy, 1990). Although many of these genes are stress induced, only a few are of known function. The SODs are among a few enzymes of known function with expression influenced by ABA and osmotic stress. We suggest that the observed changes in Sod transcripts in response to ABA may be caused in part by altered metabolic activity of cells leading to changes in ROS levels. Although there are data that suggest that elevated levels of ABA may lead to oxidative stress, no direct evidence has as yet been provided. However, ABA-induced stomatal closure can cause excess energy input that may result in increased oxidative stress. In addition, several antioxidant genes are induced by ABA, including the mitochondria-associated Mn Sod3 (Zhu and Scandalios, 1994) and the Cat1 genes of maize (Williamson and Scandalios, 1992a). These genes are also induced by certain xenobiotics that are known to cause oxidative stress (Scandalios, 1997; Scandalios et al., 1997), implying that there might be a link between ABA and oxidative stress. This matter is presently under further investigation.

Overall, the data obtained in this study suggest that the Sod genes in maize are regulated in a multilayered fashion. The response of Sod4 to ABA may be indirect because of the antioxidant nature of this gene product. In immature embryos the Vp1 trans-acting factor is used for the maximum induction of the Sod4 transcript by ABA. In mature embryos the induction of Sod4 by ABA is not dependent on the presence of the Vp1 protein. In fact, Sod4 RNA also increases in germinating embryos without the presence of ABA (Kernodle and Scandalios, 1996). The presence of ABA may cause an increase in endogenous ROS levels, leading to the activation of the Sod4 gene. The Sod4A gene responds to ABA only in young leaves. The effect of ABA on Sod4A expression is likely to be indirect. Developmental studies indicate that both Sod4 and Sod4A show similar expression patterns during embryogenesis and germination. The Sod4A transcript is always higher than Sod4 in immature embryos, mature embryos, and in leaves. The promoter sequence comparison also shows that the Sod4 and Sod4A promoters are distinct. The Sod4 promoter reveals substantial deletions or replacements. Because of the high sequence similarity in the coding region, the split of the Sod4 and Sod4A genes may be a recent evolutionary event. We hypothesize that Sod4A may represent the original form of cytosolic Sod, the transcript of which is constantly present at high levels, to deal with the constant production of ROS during normal metabolic processes. The expression of Sod4 transcript, on the other hand, is relatively low during normal growth conditions but can be highly induced under stress to deal with sudden elevated levels of ROS. Thus, the Sod gene system of maize may provide us with an excellent opportunity to study the mechanisms involved in the regulation of these important defense genes and the relationship between hormone-mediated metabolic changes and oxidative stress.

ACKNOWLEDGMENTS

We thank Stephanie Ruzsa and Sheri Kernodle for expert technical assistance.

Abbreviations:

Cat1

catalase-1

dpi

days postimbibition

dpp

days postpollination

ROS

reactive oxygen species

SOD

superoxide dismutase

Vp1

viviparous-1

Footnotes

1

This research was supported in part by grant no. R819360 from the U.S. Environmental Protection Agency.

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