OsASR6 Enhances Salt Stress Tolerance in Rice

Abstract

High salinity seriously affects crop growth and yield. Abscisic acid-, stress-, and ripening-induced (ASR) proteins play an important role in plant responses to multiple abiotic stresses. In this study, we identified a new salt-induced ASR gene in rice (OsASR6) and functionally characterized its role in mediating salt tolerance. Transcript levels of OsASR6 were upregulated under salinity stress, H₂O₂ and abscisic acid (ABA) treatments. Nuclear and cytoplasmic localization of the OsASR6 protein were confirmed. Meanwhile, a transactivation activity assay in yeast demonstrated no self-activation ability. Furthermore, transgenic rice plants overexpressing OsASR6 showed enhanced salt and oxidative stress tolerance as a result of reductions in H₂O₂, malondialdehyde (MDA), Na/K and relative electrolyte leakage. In contrast, OsASR6 RNAi transgenic lines showed opposite results. A higher ABA content was also measured in the OsASR6 overexpressing lines compared with the control. Moreover, OsNCED1, a key enzyme of ABA biosynthesis, was found to interact with OsASR6. Collectively, these results suggest that OsASR6 serves primarily as a functional protein, enhancing tolerance to salt stress, representing a candidate gene for genetic manipulation of new salinity-resistant lines in rice.

Keywords: Oryza sativa; OsASR6; OsNCED1; abscisic acid; salt tolerance.

Figure 1

Expression profiles of OsASR6. Inducible expression pattern of OsASR6 in (A) the roots and (B) the shoot under ABA, H₂O₂, and NaCl stresses treatment. Rice Actin1 was used as an internal control for qRT-PCR analysis. Asterisks indicate significant difference compared to the transcription level of 0 h groups. Three independent experiments were performed. Data represent means ± SD. * p < 0.05, ** p < 0.01.

Figure 2

Subcellular localization and transcriptional self−activating activity of OsASR6. Localization of OsASR6 protein in (A) rice protoplasts (scale bar: 10 μm) and (B) tobacco leaf epidermal cells (scale bar: 20 μm). (C) Transactivation assay of OsASR6. +: pGBKT7-53/pGADT7-T (positive control), -: pGBKT7-lam/pGADT7-T (negative control).

Figure 3

Analysis of salt tolerance in WT and OsASR6 transgenic seedlings. (A) Phenotypes of wild-type (WT) and transgenic lines following 100 mM NaCl treatment for 4 d. (B) Germination rates of WT and OsASR6 transgenic plants following 100 mM NaCl treatment for 4 d. Data represent means ± SD (n = 30). (C) Phenotypes of all tested rice plants before and after salt treatment, and after re-watering. Four-week-old seedlings were used for salt treatment (bar = 5 cm). (D) Survival rates and (E) relative ion leakage following NaCl treatment for 4 d. Asterisks indicate significant differences between means of WT and transgenic lines. Three independent experiments were carried out with similar results. Data represent means ± SD. * p < 0.05, ** p < 0.01.

Figure 4

Analysis of Na⁺ and K⁺ content. (A) Na⁺ content, (B) K⁺ content, and (C) Na/K ratio of wild-type (WT) and OsASR6 transgenic plants following 130 mM NaCl and control (CK) treatment, respectively. Three independent experiments were carried out with similar results. Asterisks indicate significant differences between means of WT and transgenic lines. Data represent means ± SD. * p < 0.05, ** p < 0.01.

Figure 5

Analysis of physiological indexes in WT and OsASR6 transgenic plants under salt treatment. (A) H₂O₂ content, (B) DAB staining, (bar = 1 cm), (C) the MDA content, and (D) SOD, (E) POD and (F) GPx enzyme activity. Asterisks indicate significant differences between means of WT and transgenic lines. Three independent experiments were carried out with similar results. Data represent means ± SD. ** p < 0.01.

Figure 6

Effects of MV on WT and OsASR6 transgenic rice. (A) Phenotypes of WT and transgenic seedling following treatment with 2 μM MV. Scale bar: 2 cm in the upper images, 1 cm in the lower images. (B) Shoot length and (C) chlorophyll contents following 2 μM MV and control (CK) treatment, respectively. Asterisks indicate significant differences between means of WT and transgenic lines. Three independent experiments were carried out with similar results. Data represent means ± SD (n = 30). * p < 0.05, ** p < 0.01.

Figure 7

ABA sensitivity of WT and OsASR6 transgenic rice. Phenotypes of wild-type (WT) and OsASR6 transgenic seedlings following treatment with (A) 5 and (B) 10 μM ABA (scale bar: 3 cm). (C) Shoot lengths following each treatment. Asterisks indicate significant differences between means of WT and transgenic lines. Data represent means ± SD (n = 30). * p < 0.05, ** p < 0.01.

Figure 8

OsASR6 interacts with OsNCED1. (A) OsASR6 was found to interact with OsNCED1 in a yeast two-hybrid system. (B) A BIFC assay further confirmed the interaction in rice leaf protoplasts (scale bar: 10 μm).

Figure 9

ABA contents in wild-type (WT) and OsASR6 transgenic plants following treatment with 130 mM NaCl and control treatment, respectively. Three independent experiments were carried out with similar results. Asterisks indicate significant differences between means of WT and transgenic lines. Data represent means ± SD. ** p < 0.01.