OsDIRP1, a Putative RING E3 Ligase, Plays an Opposite Role in Drought and Cold Stress Responses as a Negative and Positive Factor, Respectively, in Rice (Oryza sativa L.)

Abstract

As higher plants are sessile organisms, they are unable to move to more favorable places; thus, they have developed the ability to survive under potentially detrimental conditions. Ubiquitination is a crucial post-translational protein modification and participates in abiotic stress responses in higher plants. In this study, we identified and characterized OsDIRP1 (Oryza sativa Drought-Induced RING Protein 1), a nuclear-localized putative RING E3 ubiquitin (Ub) ligase in rice (Oryza sativa L.). OsDIRP1 expression was induced by drought, high salinity, and abscisic acid (ABA) treatment, but not by low temperature (4°C) stress, suggesting that OsDIRP1 is differentially regulated by different abiotic stresses. To investigate its possible role in abiotic stress responses, OsDIRP1-overexpressing transgenic rice plants (Ubi:OsDIRP1-sGFP) were generated, and their phenotypes were analyzed. The T4 Ubi:OsDIRP1-sGFP lines showed decreased tolerance to drought and salt stress as compared to wild-type rice plants. Moreover, Ubi:OsDIRP1-sGFP progeny were less sensitive to ABA than the wild-type during both germination and post-germination growth. In contrast, Ubi:OsDIRP1-sGFP plants exhibited markedly higher tolerance to prolonged cold (4°C) treatment. These results suggest that OsDIRP1 acts as a negative regulator during drought and salt stress, whereas it functions as a positive factor during the cold stress response in rice.

Keywords: RING E3 Ub ligase; abscisic acid; cold stress; drought stress; opposite response; rice (Oryza sativa).

FIGURE 1

Identification and characterization of OsDIRP1 in rice. (A) (Upper) Schematic diagram of full-length OsDIRP1 cDNA. The solid bar depicts the coding region. The solid lines represent the 5’- and 3’-untranslated regions. (Lower) Schematic structure of OsDIRP1. The putative beta-ketoacyl synthase active site, nuclear localization sequence (NLS), and RING motif are shown as dark gray bars. (B) RT-PCR analysis of OsDIRP1 in different tissues of rice plants. Total RNA was isolated from various rice tissues as indicated and analyzed by RT-PCR. OsUbiquitin was used as an equal loading control. (C) Expression patterns of OsDIRP1 in response to various abiotic stresses in rice plants. Light-grown, 10-day-old wild-type seedlings were subjected to drought, salt, cold, and ABA treatments at different time points as indicated. OsUbiquitin was used as an internal control for all the RT-PCR analyses. OsRab16b was used as a positive control for drought, salt, and ABA treatments, whereas OsDREB1A was used as a positive control for cold stress. (D) Subcellular localization of OsDIRP1. A 35S:OsDIRP1-sGFP fusion construct was transfected into wild-type rice protoplasts, and the fluorescent signals of the expressed proteins were visualized by fluorescence microscopy under dark-field conditions. sGFP and NLS-mRFP were used as cytosol- and nucleus-localized marker proteins, respectively. Bars = 5 μm.

FIGURE 2

Molecular characterization of OsDIRP1-overexpressing and RNAi-mediated knock-down transgenic rice plants. (A) Morphology of 2-month-old wild-type (WT), T4 Ubi:OsDIRP1-sGFP, and T4 Ubi:RNAi-OsDIRP1 rice plants grown under long-day conditions (16 h light and 8 h dark). (B) Genomic Southern blot analysis. Total leaf genomic DNA was isolated from wild-type (WT), T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3), and T4 Ubi:RNAi-OsDIRP1 (lines #1, #2, and #3) rice plants. The DNA was digested with HindIII and hybridized to a ³²P-labeled hygromycin B phosphotransferase (Hph) probe under high stringency conditions. (C) RT-PCR analysis of the wild-type (WT) and T4 Ubi:OsDIRP1-sGFP (independent lines #1, #2, and #3) transgenic rice plants to examine OsDIRP1 transcript levels. OsUbiquitin was used as a loading control. (D) Immunoblot analysis of wild-type (WT) and T4 Ubi:OsDIRP1-sGFP plants. Total proteins were isolated using 2x SDS sample buffer and immunoblotted with anti-GFP antibody. Rubisco was used as an equal loading control. (E) RT-PCR analysis of the wild-type (WT) and T4 Ubi:RNAi-OsDIRP1 plants. RNA was isolated from whole seedlings of non-drought-treated (0 h) and drought-treated (4 h) wild-type (WT) and Ubi:RNAi-OsDIRP1 (lines #1, #2, and #3) plants. OsUbiquitin was used as a loading control.

FIGURE 3

Decreased tolerance of Ubi:OsDIRP1-sGFP transgenic rice plants to drought stress. (A) Drought stress phenotypes of wild-type and T4 Ubi:OsDIRP1-sGFP transgenic rice plants. Light-grown, 5-week-old wild-type (WT) and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) plants were grown for 9 days without watering (drought stress). After 9 days of dehydration, these plants were re-watered, and their growth patterns were monitored for 15–20 days after re-watering. OE represents OsDIRP1-overexpressing transgenic rice plants. (B) Survival rates of the wild-type (WT) and T4 Ubi:OsDIRP1-sGFP plants in response to drought stress. Data are means ± SE (n ≥ 6 independent biological experiments; >30 plants were used in each assay, ^∗∗P < 0.01, Student’s t-test). (C) Total leaf chlorophyll content of wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) plants before and after drought treatment. The chlorophyll content of mock-treated (before drought) and drought-treated plants was measured after 1 month of rehydration. Data are means ± SE (n ≥ 3 independent biological experiments; >30 plants were used in each assay, ^∗∗P < 0.01, Student’s t-test). (D) Water loss rates of detached leaves. The leaves of 5-week-old wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) plants were detached, and their fresh weights were measured at the indicated time points. Data are means ± SD (n = 3 independent biological experiments; >6 plants of each genotype were used in each experiment).

FIGURE 4

Decreased tolerance of Ubi:OsDIRP1-sGFP transgenic rice plants in response to salt stress. (A) The salt stress phenotypes of wild-type and T4 Ubi:OsDIRP1-sGFP plants. Light-grown, 5-week-old wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) plants were treated with 200 mM NaCl for 16–18 days and then transferred to normal growth conditions with watering for 1 month. OE represents OsDIRP1-overexpressing transgenic rice plants. (B) Survival rates of wild-type (WT) and T4 Ubi:OsDIRP1-sGFP plants in response to salt stress. Data are means ± SE (n ≥ 5 independent biological experiments; >40 plants were used in each assay, ^∗P < 0.05, ^∗∗P < 0.01, Student’s t-test). (C) Leaf disk senescence assays in response to high salinity. Leaf disks (0.5 cm in diameter) were prepared from 5-week-old wild-type and transgenic plants and floated in different concentrations (0, 200, 400, 600, and 800 mM) of NaCl for 3 days. A representative photo was taken after 3 days of incubation. (D) Chlorophyll content in the leaf disk senescence assay. The amounts of chlorophyll (chlorophyll a + chlorophyll b) in the wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) leaf disks were determined 3 days after incubation with different concentrations (0, 200, 400, 600, and 800 mM) of NaCl. Data are means ± SE (n ≥ 4 independent biological experiments, ^∗∗P < 0.01, Student’s t-test).

FIGURE 5

Hyposensitive phenotypes of Ubi:OsDIRP1-sGFP transgenic rice plants in response to ABA. (A) Germination tests of wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) transgenic seeds in response to ABA. Sterilized seeds were germinated on half-strength MS medium in the presence of different concentrations (0, 3, and 5 μM) of ABA. Representative photos were taken at 7 days after germination. (B) Diagram of shoot and root lengths in the germination assays. The shoot and root lengths were measured at 7 days after germination. Data are means ± SE (n ≥ 3 biological independent experiments; >50 plants were used in each assay, ^∗∗P < 0.01, Student’s t-test). (C) Post-germination assays. Wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) seeds were germinated on half-strength MS medium for 2 days, after which germinated seedlings were transferred to half-strength MS medium supplemented with 0, 3, 5, and 10 μM ABA. Representative photos were taken at 6 days after transfer. (D) Diagram of the shoot and root lengths in the post-germination assays. Data are means ± SE (n ≥ 3 independent experiments; >100 plants were used in each assay, ^∗∗P < 0.01, Student’s t-test).

FIGURE 6

Increased tolerance of Ubi:OsDIRP1-sGFP transgenic rice plants to cold stress. (A) Cold stress phenotypes of wild-type and T4 Ubi:OsDIRP1-sGFP transgenic plants. Light-grown, 5-week-old wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) plants were transferred to a cold room at 4°C for 8 days, after which the plants were recovered at 28°C for 27 days. OE represents OsDIRP1-overexpressing transgenic plants. (B) Survival rates of wild-type (WT) and T4 Ubi:OsDIRP1-sGFP plants in response to cold stress. Data are means ± SE (n ≥ 6 independent biological experiments; >30 plants were used in each assay, ^∗∗P < 0.01, Student’s t-test). (C) Electrolyte leakage analysis of wild-type (WT) and T4 Ubi:OsDIRP1-sGFP plants in response to cold stress. Electrolyte leakage analysis was conducted using 8-day-old wild-type and transgenic seedlings at different time points before and after cold (4°C) treatment (0, 5, and 10 days). Data are means ± SD (n = 3 independent biological experiments; 12 plants of each genotype were used in each experiment, ^∗P < 0.05, ^∗∗P < 0.01, Student’s t-test). (D) Total leaf chlorophyll content of wild-type and T4 Ubi:OsDIRP1-sGFP (lines #1, #2, and #3) transgenic rice plants before and after cold treatment. The amounts of leaf chlorophyll (chlorophyll a + chlorophyll b) of mock-treated (before cold) and cold-treated plants were determined 1 month after recovery from cold stress. Data are means ± SE (n ≥ 3 biological independent experiments; 30 plants were used in each assay, ^∗∗P < 0.01, Student’s t-test).

FIGURE 7

Expression analysis of cold stress-inducible genes in wild-type and OsDIRP1-overexpressing transgenic rice plants. Light-grown, 10-day-old wild-type and Ubi:OsDIRP1-sGFP transgenic plants were exposed to cold (4°C) stress for 0 or 24 h. The induction patterns of five stress-responsive genes, OsDREB1A, OsDREB1B, OsDREB1D, GAD, and MRP4, were analyzed by real-time qRT-PCR. The relative expression of each gene was normalized to that of OsActin. Data are means ± SE (^∗P < 0.05, ^∗∗P < 0.01, Student’s t-test) of three independent experiments.