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Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt

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Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes.

Abstract

The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding choline monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

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Acknowledgments

This work was supported by the Cultural Affairs and Missions Sector, Higher education ministry, Egypt, and by the Japan Society for the Promotion of Science KAKENHI Grant numbers 25440158, 26117720, 15K07130 (H.T. and K.T.), and 22580203 (S.T.).

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Correspondence to Susumu Takio.

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Communicated by Y.-I. Park.

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299_2015_1854_MOESM1_ESM.pdf

Supplementary Fig. 1 Genomic structure of Mn-SOD genes from Japanese and Egyptian S. maritima. (a) Comparison of the exon/intron structures of plant Mn-SOD genes. Exon and intron are indicated by black and white boxes, respectively. The map covers only the ORF region. (b) Comparison of corresponding introns between two Mn-SOD genes from Japanese and Egyptian S. maritima. Nucleotide sequence identity between the corresponding introns is summarized in the table. The position of an insertion containing more than five nucleotides is indicated by a red box (PDF 46 kb)

299_2015_1854_MOESM2_ESM.pdf

Supplementary Fig. 2 Alignment of nucleotide sequences of Fe-SOD ORF from Japanese and Egyptian S. maritima. Nucleotides that differed between the two plants are indicated with red letters. Start and stop codons are indicated by boxes (PDF 44 kb)

299_2015_1854_MOESM3_ESM.pdf

Supplementary Fig. 3 Comparison of the transcript levels of SOD isozyme-encoding genes in leaves and stems. Total RNA was isolated from leaves and stems of Japanese S. maritima plants. RT-PCR was performed (PDF 96 kb)

299_2015_1854_MOESM4_ESM.pdf

Supplementary Fig. 4 Effect of Fe deficiency on SOD transcript levels. Japanese S. maritima plants grown in normal medium were transferred to Fe-depleted medium and were then incubated. Total RNA was extracted from leaves and RT-PCR was performed (PDF 69 kb)

299_2015_1854_MOESM5_ESM.pdf

Supplementary Fig. 5 Effect of light intensity on SOD transcript levels. Japanese S. maritima plants grown under normal conditions (in the absence of NaCl at a light intensity of 30 µmol m−2 s−1) were grown in the absence or presence of 400 mM NaCl, and/or at different light intensities (30 or 200 µmol m−2 s−1) for 10 days. Cell-free extracts were then prepared from the leaves of the plants. The extracts (20 µg protein) were electrophoresed on a native polyacrylamide gel, and in-gel SOD activity staining was performed (PDF 242 kb)

Supplementary Table 1 Primers used in this study (PDF 54 kb)

Supplementary Table 2 The accession numbers of SOD isozyme genes isolated in this study (PDF 57 kb)

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Mohamed, E., Matsuda, R., El-khatib, A.A. et al. Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt. Plant Cell Rep 34, 2099–2110 (2015). https://doi.org/10.1007/s00299-015-1854-1

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  • DOI: https://doi.org/10.1007/s00299-015-1854-1

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