OsLEA3-2, an abiotic stress induced gene of rice plays a key role in salt and drought tolerance

Abstract

Late embryogenesis abundant (LEA) proteins are involved in tolerance to drought, cold and high salinity in many different organisms. In this report, a LEA protein producing full-length gene OsLEA3-2 was identified in rice (Oryza sativa) using the Rapid Amplification of cDNA Ends (RACE) method. OsLEA3-2 was found to be only expressed in the embryo and can be induced by abiotic stresses. The coding protein localizes to the nucleus and overexpression of OsLEA3-2 in yeast improved growth performance compared with control under salt- and osmotic-stress conditions. OsLEA3-2 was also inserted into pHB vector and overexpressed in Arabidopsis and rice. The transgenic Arabidopsis seedlings showed better growth on MS media supplemented with 150 mM mannitol or 100 mM NaCl as compared with wild type plants. The transgenic rice also showed significantly stronger growth performance than control under salinity or osmotic stress conditions and were able to recover after 20 days of drought stress. In vitro analysis showed that OsLEA3-2 was able to protect LDH from aggregation on freezing and inactivation on desiccation. These results indicated that OsLEA3-2 plays an important role in tolerance to abiotic stresses.

Figure 1. In silico analysis of the OsLEA3-2 protein.

(A) Comparison of deduced amino acid sequence of OsLEA3-2 with OsLEA3 (AAD02421.1) and LEA3 proteins from A. thaliana (BAA11017.1) and B. napus (ACJ39155.1), underlines show the 11-mer repeats in the OsLEA3-2; (B) phylogenetic analysis of these group 3 LEA proteins; (C) hydropathy analysis of OsLEA3-2 protein.

Figure 2. OsLEA3-2 gene expression.

(A) Expression profile of OsLEA3-2 in different tissues of Oryza sativa. Lane 1, embryo; lane 2, root; lane 3, shoot base; lane 4, leaf. (B) RT-PCR analysis of OsLEA3-2 gene expression in Oryza sativa under different stresses. Irrigation with half-strength Hoagland solution (A, control), 400 mM mannitol (B), sprayed with 100 µM ABA in 0.02% Tween-20 on seedlings (C), 4°C incubation (D), 200 mM NaCl (E), 25% PEG-6000 (F). 1, roots; 2, shoot base; 3, leaves.

Figure 3. Subcellular localization of OsLEA3-2.

(A) Diagram of the T-DNA region of the binary vector pHB::GFP::OsLEA3-2 used for transformation; (B) Subcellular localization of OsLEA3-2. The green fluorescent signal of GFP:OsLEA3-2 was detected exclusively within the nucleus of onion epidermal cells, while GFP by itself was detected both in the cytoplasm and nucleus. DIC (Differential Interference Contrast), referring to bright field images of the cells.

Figure 4. Growth of S. cerevisiae cells expressing OsLEA3-2 and the control cell in the medium containing 1.2 M NaCl (A), 1.2 M KCl (B), and 2.0 M sorbitol (C).

Yeast cells were grown on YNBG medium for two days. Twenty microliters of the culture was inoculated into YNBG supplemented with either 1.2 M NaCl, 1.2 M KCl, or 2.0 M sorbitol. At each time point, 60 microliters of culture was removed, and OD₆₀₀ was measured with a spectrophotometer.

Figure 5. Effect of osmotic or salinity stress on Arabidopsis seedlings from wild type and OsLEA3-2-overexpressing transgenic lines (L35, L48, and L69).

(A) Diagram of the T-DNA region of the binary vector pHB::OsLEA3-2; (B) OsLEA3-2 expression level in transgenic Arabidopsis, wild type plants as control; (C, D) Sorbitol or NaCl sensitivity of wild type or transgenic seedlings. Photographs were taken following 9 days of growth on media containing 0 (control), 200 mM sorbitol, or 100 mM NaCl. (E) Fresh/dry weights of 9-day-old seedlings. All samples were measured in triplicate. Statistical significance was determined by Student’s t test. **P<0.01 shown above the bar reprent results significantly different from wild type control.

Figure 6. Stress or ABA sensitivity of wild type and transgenic rice seedlings.

(A) Southern hybridization analysis of transgenic plants. M, molecular marker; WT, wild type; L10, L20, L30, three transgenic lines. (B) Expression level of OsLEA3-2 in transgenic rice, Zhonghua 11 was used as control; (C) photographs were taken after 5 d or 2 weeks of growth in water control. Photographs were taken after 5 d (D) or 2 w (E) of growth in water containing 10% or 20% PEG, 100 or 200 mM NaCl, or 10 µM ABA. Effect of 2 weeks abiotic-stress on root (F) and shoot (G) length of rice. For each treatment, 8 seedlings were measured. *P<0.05 and **P<0.01 shown above the bar reprent results significantly different from wild type control.

Figure 7. Drought-treatment assay of wild type and transgenic rice plants.

One month old seedlings of the wild type cultivar and transgenic plants (A) were treated with drought stress (without irrigation) for 20 days (B), then irrigated with water and grown for 10 days (C), and one month (D). 1, wild type; 2, transgenic line 10; 3, transgenic line 30. Each container had six plants.

Figure 8. Effect of drought-stress on wild type and transgenic rice plants.

One month old seedlings of the wild type cultivar and transgenic plants (A) were treated with drought stress (without irrigation) for 18 days (B), then irrigated with water and grown for 10 days (C). (D) Spikes from survived ZH11 and transgenic line L10. (E) Survival ratio of the rice plants. (F) Statistic analysis of the grains. Three and ten spikes were analyzed for ZH11 and transgenic line L10, respectively. **P<0.01 shown above the bar reprent results significantly different from wild type. Effect of drought-stress was assayed in triplicate.

Figure 9. Protection of LDH by protein protectants.

(A) SDS-PAGE analysis of OsLEA3-2 protein expression and purification. M, protein molecular weight marker; 1, 3, uninduced precipitant and soluble supernatant of BL21 (DE3) cells, respectively; 2, 4, induced precipitant and soluble supernatant, respectively; 5, OsLEA3-2 protein after purification. (B) Protection of LDH by protein protectants from aggregation due to freezing and thawing. LDH aggregation on repeated freezing and thawing is indicated by light scattering at A₃₄₀. Results are shown for LDH alone (open bar), for LDH in the presence of BSA (black bar) and for LDH together with OsLEA3-2 (grey bar). *P<0.05 and **P<0.01 shown above the bar reprent results significantly different from those for LDH alone. (C) Effect of desiccation on LDH activity. LDH activity after vacuum drying (open bar) in the presence of BSA (black bar) or OsLEA3-2 (grey bar). All samples were assayed in triplicate.