A amount of environmental strains can result in enhanced creation of superoxide within seed tissues, and plant life are thought to depend on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. regarded as important in switching O2? to H2O2 through the pathogen-induced oxidative burst in pet phagocytic immune system cells and in seed cells (Desikan et al., 1996; Babior et al., 1997). In plant life contact with photoinhibitory light, ozone, or various other environmental circumstances that trigger oxidative tension can boost O2? amounts (Yruela et al., 1996; Vaartnou and Runeckles, 1997); however, it isn’t very clear whether SOD has an essential function in attenuating seed oxidative tension in these circumstances. To time the protective function of SOD in plant life continues to be explored by transgenic techniques, mainly through overexpression or by relationship of SOD appearance to the amount of oxidative tension resistance (Bowler et al., 1994; Alscher et al., 1997; Scandalios, 1997). Both approaches have yielded inconclusive and sometimes contradictory results about the role of SOD in plant oxidative stress responses (Bowler et al., 1994; Alscher et al., 1997; Scandalios, 1997). The existence of three classes of SOD enzymes, each typically encoded by a small gene family, complicates the elucidation of the roles of SOD in plants. This situation is exacerbated by the fact that past work has generally focused on one member of a gene family. To circumvent this problem, we have initiated a thorough analysis of the 827022-33-3 manufacture Arabidopsis SOD genes. The availability of large numbers of cDNA and genomic DNA sequences in Arabidopsis (Newman et al., 1994; Rounsley et al., 1996; Delseny et al., 1997) has made it possible to identify the complete SOD arsenal of this plant. We describe progress toward the detailed understanding of SOD genes and their functions in Arabidopsis, and report on the sequencing of three SOD cDNAs and the map locations of seven SOD structural genes. Information is also provided on the characterization of antisera against five of the seven SOD proteins. Finally, we describe the regulation of SOD activity and mRNA and protein levels in response to ozone, UV-B light, and variations in incident light fluences. MATERIALS AND METHODS Strains and Materials Unless otherwise noted, Arabidopsis ecotype Columbia was planted in a soil mixture (Cornell University, Ithaca, NY) and grown at 70% RH, 21C, 70 mol m?2 s?1 PAR from 400 W lamps (Multi-Vapor, General Electric) under a 16-h photoperiod (Landry et al., 1995). Whole rosette tissue was collected for RNA and protein analyses unless otherwise indicated. All treatments were replicated twice with duplicate RNA samples and triplicate protein analysis per replication. strain XL1 Blue (Stratagene) (F::Tn10 lacI[and Columbia was amplified using the standard microsatellite reaction mixture and program, but with an annealing temperature of 58C (Bell and Ecker, 1994). The products were separated on a 4% agarose gel, revealing a 4-bp polymorphism between the two ecotypes. This polymorphism was used to map TAMUBAC30D05, 827022-33-3 manufacture and therefore the CSD2 structural gene, using the recombinant inbred lines generated by Lister and Dean (1993). Table I Primers used in this study CICYAC Pool PCR Yeast strains containing a multiplexed CICYAC library were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). Yeast was grown and CICYAC DNA was isolated as described in the provider’s handout. Three microliters of the pooled CICYAC DNA was amplified in 25 L of 2.5 mm MgCl2, 1 mm dNTP, 0.6 m of each primer, and 3 units of TAQ (Promega) in the follow manner: 2 min at 94C, followed by 30 cycles of 30 s at 94C, 30 s at 52C, 90 s at 72C, and 10 min at 72C. CICYACs that produced an amplification product were obtained Rabbit Polyclonal to ADCK5 from the Arabidopsis Biological Resource Center and individually tested for amplification. MSD1 CAPS The MnSOD structural gene was amplified using the MnSOD 1F and 1R primers (Table ?(TableI)I) and the standard CAPS PCR protocol, except that an annealing temperature of 60C and 1.5 mm MgCl2 were employed (Jarvis et al., 1994). The purified 827022-33-3 manufacture PCR product was sequenced using the MnSOD 1F, 1R, and 3A primers. A Tai1 site was identified in the Columbia sequence that was not in the Landsberg sequence. Digestion of the Landsberg PCR product amplified by MnSOD 1F and 1R with Tai1 yielded DNA fragments of 1 1 kb and 230 bp, whereas the corresponding Col fragments were 700, 300, and 230 bp. This polymorphism was used to map the MnSOD structural gene using the recombinant inbred lines generated by Lister and Dean (1993). RNA and Immunoblot Techniques All RNA.