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Oryza sativa protein phosphatase 1a (OsPP1a) involved in salt stress tolerance in transgenic rice

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Abstract

Serine/threonine protein phosphatases (PPs) can be grouped into PP1 and PP2 based on enzymological criteria using specific inhibitors and activators. PP2 receives abscisic acid as signal and negative regulation of tolerance of high salt, drought, and cold stresses, yet the role of PP1 in stress tolerance remains unknown. The objective of this study was to transfer OsPP1a cDNA to rice via Agrobacterium tumefaciens strain EHA105 to assess the mechanisms by which it can confer tolerance to salt treatment. All OsPP1a transcript levels except for OsPP1a-4 detected in transgenic lines were significantly higher than in non-transgenic (NT) plants. Transgenic plants overexpressing OsPP1a showed enhanced tolerance to high salt treatment, SnRK1A, and two stress-responsive genes, OsNAC5 and OsNAC6, which were up-regulated in transgenic OsPP1a-2, OsPP1a-3, and OsPP1a-6 lines. The gene expression profiles of lines OsPP1a-2, -3, and -6 were well-matched with the data for ascorbate peroxidase (APX) and superoxide dismutase (SOD) activity, scavenging the DPPH radical, and malondialdehyde content in NT and transgenic plants under salt stress treatment. In addition, transgenic rice plants also exhibited higher survival rates and plant heights, fewer oxidative injuries, and grew faster than NT plants exposed to salt treatment. Thus, the overexpression of OsPP1a in rice may be useful for enhancing tolerance in high-salt areas.

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    Authors and Affiliations

    1. Department of Biotechnology, Ming Chuan University, Taoyuan, 333, Taiwan

      Yu-Duan Liao, Chiu-Chen Chen & Chih-Ming Chiang

    2. Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, 700000, Vietnam

      Kuan-Hung Lin

    3. Department of Horticulture and Biotechnology, Chinese Culture University, Taipei, 111, Taiwan

      Kuan-Hung Lin

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    Correspondence to Chih-Ming Chiang.

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    Yu-Duan Laio and Kuan-Hung Lin contributed equally to this work.

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    Supplemental Fig. S1 Vector maps of pENTR/OsPP1a (A) and pPZP200/OsPP1a (B) (DOCX 272 kb)

    11032_2016_446_MOESM2_ESM.docx

    Supplemental Fig. S2 Analysis of transgenic rice by genomic PCR and Southern blot analysis for HPT genes. (A) The expected size of the HPT gene fragment (indicated by arrowhead) was 689 bp. M, 100 bp marker; NT, non-transgenic rice; P, positive control (using pPZP200/OsPP1a plasmid as the template); lanes 4–9, OsPP1a-1, OsPP1a-2, OsPP1a-3, OsPP1a-4, OsPP1a-5, and OsPP1a-6, respectively. (B) Genomic DNA (15 μg/lane) from NT and OsPP1a-1, -2, -3, -4, -5, and -6 transgenic plants were digested with restriction enzyme HindIII and electrophoresed through 1 % agarose gel. 32P-labeled hyg DNA was used as a probe (DOCX 124 kb)

    11032_2016_446_MOESM3_ESM.docx

    Supplemental Fig. S3 Phylogenetic tree inferred from the Protein Phosphatase amino acid sequences by the Neighbor Joining method. Numbers indicate bootstrap support for individual nodes. Similarities of amino acids (%) between OsPP1a and others are also shown using OsPP1a as 100 % (DOCX 212 kb)

    11032_2016_446_MOESM4_ESM.docx

    Supplemental Fig. S4 RNA expression in the OsNAC gene family and SnRK1 genes under salt stress by RT-PCR (A) in NT plant and T1 transgenic lines (OsPP1a-2, OsPP1a-3, and OsPP1a-6). The transcript levels of OsNAC5 and OsNAC6 were also assessed in NT and transgenic plants receiving sodium butyrate (NaB) treatments and without NaB as control (B). Total RNA in all tested plants was extracted from 5-day-old plants (DOCX 302 kb)

    11032_2016_446_MOESM5_ESM.docx

    Supplemental Fig. S5 Measurement of the seed germination rate in salt-containing solutions with 0, 100, 150, 200, 250, and 300 mM of NaCl. All tested plants were germinated for 5 days and for high-salt stress treatment. Transgenic plants were grown in nutrient soil under normal conditions for 14 days and transferred to 150 mM NaCl for 3 days (DOCX 73 kb)

    11032_2016_446_MOESM6_ESM.docx

    Supplemental Fig. S6 Endogenous OsPP1a was expressed in different tissues of germinating rice seedling by RT-PCR. Paired primers OsPP1a 5F and OsPP1a 3R (Table 1) were used for confirmation of OsPP1a expression in endosperm, embryo, root, and leaf tissues of NT seedlings. Tubulin (TUB) gene was used as internal control (DOCX 59 kb)

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    Liao, YD., Lin, KH., Chen, CC. et al. Oryza sativa protein phosphatase 1a (OsPP1a) involved in salt stress tolerance in transgenic rice. Mol Breeding 36, 22 (2016). https://doi.org/10.1007/s11032-016-0446-2

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