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Overexpression of a Common Wheat Gene GALACTINOL SYNTHASE3 Enhances Tolerance to Zinc in Arabidopsis and Rice Through the Modulation of Reactive Oxygen Species Production

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Abstract

Galactinol synthase (GolS, EC 2.4.1.123), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), plays roles in plant growth and developmental processes. The in vitro roles of GolS in plant responses against heavy metal stress are not well clarified. In the present study, a suppression-subtractive hybridization (SSH) cDNA library has been constructed using RNA extracted from wheat cultivar Jinan 18 treated with ZnCl2 as the tester and RNA from untreated seedlings as the driver. Sixteen expressed sequence tags (ESTs) highly homologous with known proteins associated with stress tolerance have been obtained. Among these, a 1000-bp cDNA sequence encoding GolS protein has been isolated and designated as TaGolS3. Real-time quantitative PCR (qPCR) analysis revealed that TaGolS3 was mainly expressed in young roots and upregulated by exogenous ABA treatment and several abiotic stresses, such as ZnCl2, CuCl2, low temperature, and NaCl. Subcellular localization analysis showed that TaGolS3 protein is a nuclear-localized protein. A detailed analysis of Arabidopsis and rice transgenic plants overexpressing TaGolS3 gene displayed that transgenic plants exhibited increased lateral root number, primary root length, plant survival rate, and plant height. Moreover, in comparison with the wild-type (WT) plants, the TaGolS3-overexpressing lines showed a higher expression of ROS-scavenging genes, activities of antioxidative enzymes, proline contents, and a lower level of malondialdehyde (MDA) contents and electrolyte leakage under zinc stress. These results confirmed the positive roles of TaGolS3 in improving plant tolerance to heavy metal stress, indicating a potential resource in the transgenic breeding to enhance heavy metal stress tolerance in crop plants.

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Acknowledgments

This work was supported by the project of Modern Seed Industry Enterprise Science and Technology Development of Shandong (SDKJ2012QF003).

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    Authors and Affiliations

    1. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

      Yuange Wang

    2. Postdoctoral Research Center of Shandong Shengfeng Seeds Co., Ltd., Shandong Shengfeng Seeds Co., Ltd., Jiaxiang, 272400, Shandong, China

      Huaihua Liu, Shuping Wang & Hongjie Li

    3. Yantai Academy of Agricultural Sciences, Yantai, 265500, China

      Qingguo Xin

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    1. Yuange Wang
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    Correspondence to Qingguo Xin.

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    Yuange Wang and Huaihua Liu contributed equally to this work.

    Electronic supplementary material

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    Supplementary Table S1

    (DOCX 20 kb)

    Supplementary Table S2

    (DOCX 18 kb)

    Supplementary Table S3

    (DOCX 21 kb)

    Supplementary Fig. S1

    The nucleotide sequence and the deduced amino acid sequence of TaGolS3. (PPTX 76 kb)

    Supplementary Fig. S2

    Phylogenetic tree analysis between TaGolS3 and GolS protein sequences from other plant species. The multiple alignments were performed by clustalX 2.0 and the phylogenetic tree was constructed with Mega 4.0 using NJ method. The GenBank accession numbers or locus name of selected GolSs are listed as follows: Arabidopsis thaliana: AtGolS1, NP_182240.1; AtGolS2, NP_176053; AtGolS3, NP_172406.1; AtGolS4, NP_176250.1; AtGolS5, NP_197768.1; AtGolS6, NP_567741.2; AtGolS7, NP_176248.1; AtGolS10, NP_850902. Glycine max: GmGolS, AAM96867. Zea mays: ZmGolS1, NP_001105748; ZmGolS2, XP_008654184.1; ZmGolS3, ACG39512.1. Brachypodium distachyon: BdGolS, XP_003559767.1. Gossypium hirsutum: GhGolS, AFG26331.1. Sorghum bicolor: SbGolS, XP_002467954.1. Oryza sativa: OsGolS1, NP_001060697.1; OsGolS2, NP_001049939.1. Hordeum vulgare: HvGolS, AK248482. Triticum aestivum: TaGolS1, BAF51565.1; TaGolS2, BAF51566.1; TaGolS4, Traes_2AS_7339C00EB.1; TaGolS5, Traes_2BS_BC7099C57.1; TaGolS6, Traes_2BS_E69390845.1; TaGolS7, Traes_2BL_1F96D8DB3.1; TaGolS8, Traes_4AS_AF7143BFC1.1. Populus trichocarpa: PtGolS1, ACA04027.1; PtGolS2, ACA04030.1; PtGolS3, ACA04032.1; PtGolS4, XP_002301531. (PPTX 53 kb)

    Supplementary Fig. S3

    Histochemical assay of GUS gene expression under the control of the TaGolS3 promoter in transgenic Arabidopsis plants. a Hypocotyl, b 2-day-old seedling, c 5-day-old seedling, d rosette leaf, e and f flowers, i silique. Arrow indicates the root tip. Bar: 1 mm. (PPTX 315 kb)

    Supplementary Fig. S4

    Analysis of H2O2 accumulation in WT and transgenic plants under zinc stress. Seedlings in Fig. 6b were used to examine the H2O2 levels in leaves. Brown color indicates production of hygrogen peroxide. (PPTX 275 kb)

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    Wang, Y., Liu, H., Wang, S. et al. Overexpression of a Common Wheat Gene GALACTINOL SYNTHASE3 Enhances Tolerance to Zinc in Arabidopsis and Rice Through the Modulation of Reactive Oxygen Species Production. Plant Mol Biol Rep 34, 794–806 (2016). https://doi.org/10.1007/s11105-015-0964-9

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