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. 2016 Jan 22:7:4.
doi: 10.3389/fpls.2016.00004. eCollection 2016.

Overexpression of a Stress-Responsive NAC Transcription Factor Gene ONAC022 Improves Drought and Salt Tolerance in Rice

Yongbo Hong  1 , Huijuan Zhang  1 , Lei Huang  1 , Dayong Li  1 , Fengming Song  1
Affiliations

Overexpression of a Stress-Responsive NAC Transcription Factor Gene ONAC022 Improves Drought and Salt Tolerance in Rice

Yongbo Hong et al. Front Plant Sci. .

Abstract

The NAC transcription factors play critical roles in regulating stress responses in plants. However, the functions for many of the NAC family members in rice are yet to be identified. In the present study, a novel stress-responsive rice NAC gene, ONAC022, was identified. Expression of ONAC022 was induced by drought, high salinity, and abscisic acid (ABA). The ONAC022 protein was found to bind specifically to a canonical NAC recognition cis-element sequence and showed transactivation activity at its C-terminus in yeast. The ONAC022 protein was localized to nucleus when transiently expressed in Nicotiana benthamiana. Three independent transgenic rice lines with overexpression of ONAC022 were generated and used to explore the function of ONAC022 in drought and salt stress tolerance. Under drought stress condition in greenhouse, soil-grown ONAC022-overexpressing (N22oe) transgenic rice plants showed an increased drought tolerance, leading to higher survival ratios and better growth than wild-type (WT) plants. When grown hydroponically in Hogland solution supplemented with 150 mM NaCl, the N22oe plants displayed an enhanced salt tolerance and accumulated less Na(+) in roots and shoots as compared to WT plants. Under drought stress condition, the N22oe plants exhibited decreased rates of water loss and transpiration, reduced percentage of open stomata and increased contents of proline and soluble sugars. However, the N22oe lines showed increased sensitivity to exogenous ABA at seed germination and seedling growth stages but contained higher level of endogenous ABA. Expression of some ABA biosynthetic genes (OsNCEDs and OsPSY), signaling and regulatory genes (OsPP2C02, OsPP2C49, OsPP2C68, OsbZIP23, OsAP37, OsDREB2a, and OsMYB2), and late stress-responsive genes (OsRAB21, OsLEA3, and OsP5CS1) was upregulated in N22oe plants. Our data demonstrate that ONAC022 functions as a stress-responsive NAC with transcriptional activator activity and plays a positive role in drought and salt stress tolerance through modulating an ABA-mediated pathway.

Keywords: NAC transcription factor; ONA022; abscisic acid (ABA); drought tolerance; rice (Oryza sativa L.); salt tolerance.

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Figures

FIGURE 1
FIGURE 1
Characterization of rice ONAC022 protein and gene. (A) Alignment of ONAC022 with rice ONAC095, SNAC1 (OsNAC9), and SNAC2 (OsNAC6) and Arabidopsis ANAC036. Identical amino acids are shaded in black and the conserved NAC domain is boxed. Black arrowed lines indicate the locations of the five highly conserved subdomains A–E, while red arrowed lines show the locations of the newly identified C1 and C2 domains. (B) Phylogenetic tree analysis of ONAC022 with other known stress-responsive rice NAC proteins. Sequence alignment was performed using Clustal X1.81 program and phylogenic tree was created and visualized using MEGA 5.05. Protein sequences used for alignment are as follow: ONAC012 (Os05g37080), ONAC022 (Os03g04070), ONAC017 (Os11g05614), ONAC049 (Os08g02160), ONAC059/ENAC1 (Os01g64310), ONAC063 (Os08g33910), ONAC066 (Os03g56580), ONAC075 (Os01g66490), ONAC087 (Os05g34600), ONAC095 (Os06g51070), ONAC096 (Os07g04560), ONAC134 (Os12g05990), ONAC140 (Os12g43530), ONAC002/SNAC1/OsNAC9 (Os03g60080), ONAC048/SNAC2/OsNAC6 (Os01g66120), ONAC058/OsNAP (Os03g21060), ONAC122/OsNAC10 (Os11g03300), ONAC131 (Os12g03040), ONAC068/OsNAC4 (Os01g60020), ONAC009/OsNAC5 (Os11g08210), ONAC054/RIM1 (Os03g02800), ONAC045 (Os11g03370). Reported names and functions in stress response of the known stress-responsive NAC genes were given in parentheses in the tree and listed at right of the tree, respectively. (C) Distribution of major stress-related cis-elements in the promoter region of the ONAC022 gene.
FIGURE 2
FIGURE 2
Induction of ONAC022 expression by drought, salt, and abscisic acid (ABA). Two-week-old seedlings were exposed to drought (A), 150 mM NaCl (B), or treated by foliar spraying with 100 μM ABA (C) and leaf samples were collected at indicated time points for analyses of gene expression by qRT-PCR. Relative expression levels as compared to those of the actin gene at each time point are presented as the means ± SD from three independent experiments. ∗Above the columns indicate significant differences at p ≤ 0.05 level with corresponding controls.
FIGURE 3
FIGURE 3
Subcellular localization and transactivation activity of the ONAC022 protein. (A) ONAC022 is localized in nucleus. Agrobacteria carrying pFGC-Egfp-ONAC022 or pFGC-Egfp empty vector were infiltrated into leaves of Nicotiana benthamiana plants expressing a red nucleus marker protein RFP–H2B and leaf samples were collected at 24 h after infiltration for observation under a confocal laser scanning microscope. Images were taken in dark field for green fluorescence (left) and red fluorescence (middle right), white field for cell morphology (middle left) and in combination (right), respectively. (B) DNA binding activity of ONAC022. Wild type version of the cis-element sequence (wNACRS) and a mutated version (mNACRS) were used. Electrophoretic mobility shift assays were performed using the recombinant GST-fused ONAC022 protein. Biotin-labeled wNACRS and mNACRS probe and biotin-labeled wNACRS probe in combination with unlabeled wNACRS or mNACRS probe were incubated with GST-fused ONAC022 protein or a purified GST preparation as a negative control. Specific DNA-protein complexes and free probes are indicated by the arrowheads on left. (C) ONAC022 has transactivation activity. Yeast cells carrying pBD-ONAC022, pBD-ONAC022ΔC, pBD-ONAC022ΔN or pBD empty vector (as a negative control) were streaked on SD/Trp- plates (left) or SD/Trp-His- plates supplemented with x-α-gal (right) for 3 days at 28°C.
FIGURE 4
FIGURE 4
Characterization of the ONAC022-overexpressing transgenic rice lines and their agronomic traits. (A) Schematic diagram of the overexpression construct used for rice transformation. HptII, hygromycin (Hgr) phosphotransferase II; LB, left border; RB, right border; Ubi, maize ubiquitin promoter; 35S, CaMV 35S promoter. (B) Southern blot analysis of copy number of the transgene in T4 generation of the N22oe lines. Genomic DNA (∼50 μg) extracted from the N22oe and wild-type (WT) plants was digested with EcoRI and probed with a DIG-labeled fragment of the HptII gene. (C) Expression levels of the ONAC022 gene in the N22oe lines. (D) Growth phenotypes of the N22oe plants at heading stage under normal watered condition in greenhouse. (E) Reduced plant height of the N22oe plants grown under normal watered condition in greenhouse. (F) Comparison of the panicles between the N22oe and WT plants grown under normal watered condition in greenhouse. (G) Numbers of grains per panicle between the N22oe and WT plants grown under normal watered condition in greenhouse. (H) Weights of 1000-grain from the N22oe and WT plants grown under normal watered condition in greenhouse. Data presented (C,E,G,H) are the mean ± SD from three independent experiments and different letters above the columns indicate significant differences at p ≤ 0.05 level with corresponding WT.
FIGURE 5
FIGURE 5
Increased drought tolerance in N22oe plants. (A–D) Phenotype of the N22oe and WT seedlings at different stages during the drought stress experiments. The N22oe seedlings were grown in barrels each with WT seedlings as a control. (E) Survival ratios of the N22oe and WT plants at 12 days after re-watering. (F) Growth biomass of the seedlings after drought stress treatment. Data presented in (E,F) are the mean ± SD from three independent experiments and different letters above the columns indicate significant differences at p ≤ 0.05 level.
FIGURE 6
FIGURE 6
Increased salt tolerance in N22oe plants. (A) Growth performance of the N22oe and WT seedlings grown on 1/2 MS medium supplemented with or without 150 mM NaCl. (B,C) Shoot and root length of the N22oe and WT seedlings grown on 1/2 MS medium supplemented with or without 150 mM NaCl. (D) Number of lateral roots of the N22oe and WT seedlings grown on 1/2 MS medium supplemented with or without 150 mM NaCl. (E,F) Na+ contents in roots and shoots of the N22oe and WT seedlings grown in modified Hogland solution with or without 150 mM NaCl. Data presented in (B–F) are the mean ± SD from three independent experiments and different letters above the columns indicate significant differences at p ≤ 0.05 level with corresponding WT.
FIGURE 7
FIGURE 7
Physiological changes inN22oe plants. (A) Proline contents in leaves of the N22oe and WT plants grown under normal and drought stress condition. (B) Soluble sugar contents in leaves of the N22oe and WT plants grown under normal and drought stress condition. Four-week-old plants were subjected for drought stress treatment by stopping watering and leaf samples were collected at 6 days after drought treatment. (C) Rates of water loss in detached leaves of the N22oe and WT plants. Leaves were detached from 4-week-old N22oe and WT plants grown under normal conditions and placed on lab bench for drought treatment. Leaf samples were collected at indicated time points and subjected for measuring water loss. (D) Transpiration rate of the N22oe and WT plants. Transpiration rates in six flag leaves of 3-month-old N22oe and WT plants grown under greenhouse condition were determined by Li-6400 instrument at indicated time points. (E) Stomatal aperture of the N22oe and WT plants grown under normal and drought condition. Scale bar = 50 μM. (F) Percentage of open stomata in leaves of the N22oe and WT plants grown under normal and drought condition. Data presented in (A–D,F) are the mean ± SD from three independent experiments and different letters above the columns indicate significant differences at p ≤ 0.05 level with corresponding WT.
FIGURE 8
FIGURE 8
Increased ABA sensitivity of the N22oe seedlings. (A,C) Growth performance and (B) germination rate of the N22oe and WT seedlings on 1/2 MS medium supplemented with or without different concentrations of ABA. Photos were taken at 6 days after germination. (D,E) Shoot and root lengths of the N22oe and WT seedlings grown on 1/2 MS medium supplemented with or without different concentrations of ABA. The shoot and root lengths were measured at 6 days after germination. Data presented in (D,E) are the mean ± SD from three independent experiments and different letters above the columns indicate significant differences at p ≤ 0.05 level with corresponding WT.
FIGURE 9
FIGURE 9
Increased ABA content and upregulated expression of ABA biosynthetic and stress-responsive genes in N22oe plants. (A) Increased ABA contents in the N22oe plants. (B) Expression of ABA biosynthesis-related genes in the N22oe plants. (C) Expression of stress-responsive genes in the N22oe plants. Leaf samples were collected from 30-day-old seedlings grown under normal condition and subjected for analyses of ABA content and gene expression. ABA in leaf samples was extracted and quantified by HPLC. FW, fresh weight. Expression of the ABA biosynthetic and stress-responsive genes was analyzed by qRT-PCR and relative expression levels were shown as folds of the level of the actin gene. Data presented are the mean ± SD from three independent experiments and different letters above the columns indicate significant differences at p ≤ 0.05 level with corresponding WT.
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