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

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.

Figure 1

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.

Figure 2

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.

Figure 3

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).

Figure 4

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).

Figure 5

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.

Figure 6

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.

Figure 7

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.

Figure 8

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.