A stress-responsive NAC transcription factor SNAC3 confers heat and drought tolerance through modulation of reactive oxygen species in rice

Abstract

Adverse environmental conditions such as high temperature and drought stress greatly limit the growth and production of crops worldwide. Several NAC (NAM, ATAF1/2, and CUC2) proteins have been documented as important regulators in stress responses, but the molecular mechanisms are largely unknown. Here, a stress-responsive NAC gene, SNAC3 (ONAC003, LOC_Os01g09550), conferring drought and heat tolerance in rice is reported. SNAC3 was ubiquitously expressed and its transcript level was induced by drought, high temperature, salinity stress, and abscisic acid (ABA) treatment. Overexpression (OE) of SNAC3 in rice resulted in enhanced tolerance to high temperature, drought, and oxidative stress caused by methyl viologen (MV), whereas suppression of SNAC3 by RNAi resulted in increased sensitivity to these stresses. The SNAC3-OE transgenic plants exhibited significantly lower levels of H2O2, malondiadehyde (MDA), and relative electrolyte leakage than the wild-type control under heat stress conditions, implying that SNAC3 may confer stress tolerance by modulating reactive oxygen species (ROS) homeostasis. Quantitative PCR experiments showed that the expression of a large number of ROS-scavenging genes was dramatically increased in the SNAC3-OE plants, but significantly decreased in the SNAC3-RNAi transgenic plants. Five ROS-associated genes which were up-regulated in SNAC3-OE plants showed co-expression patterns with SNAC3, and three of the co-expressed ROS-associated enzyme genes were verified to be direct target genes of SNAC3. These results suggest that SNAC3 plays important roles in stress responses, and it is likely to be useful for engineering crops with improved tolerance to heat and drought stress.

Keywords: Abiotic stress; NAC; Oryza sativa; ROS; transcription factors..

Fig. 1.

Expression pattern analysis of SNAC3. (A) Expression level of SNAC3 under various abiotic stresses and ABA treatment. Four-leaf stage seedlings were subjected to drought, salt (200 mmol l^–1 NaCl), heat (42 °C), cold (4 °C), flood, wounding, H₂O₂ (1% H₂O₂), and ABA treatment (100 mmol l^–1 ABA). The relative expression level of SNAC3 was detected by qPCR at the indicated times. Error bars indicated the SE based on three replicates. (B) Expression pattern of the GUS reporter gene driven by the SNAC3 promoter in transgenic resistant callus under normal conditions (control), drought, heat, and MV stress. (C) GUS activity quantification in P _SNAC3:GUS transgenic resistant callus under normal conditions, drought, heat, and MV stress. GUS activity was defined in picomoles of 4-MU generated per minute per microgram of protein. (D) Detection of SNAC3 expression in various tissues and organs using qPCR. Error bars indicate the SE based on three technical replicates. (E) Expression pattern under normal conditions. (a–k) GUS staining of tissues and organs from P _SNAC3:GUS transgenic plants; (l–v) GUS staining of tissues and organs from ZH11 plants. (a and l) callus; (b and m) regenerated seedling; (c and n) leaf tip; (d and o), leaf blade; (e and p) ligule, collar, and auricle; (f and q) leaf sheath; (g and r) cross-section of the leaf sheath; (h and s) root; (i and t) hull; (j and u) stamen and pistil; (k and v) seed.

Fig. 2.

Enhanced heat tolerance of the SNAC3-OE transgenic plants at the seedling stage. (A) Phenotype of the SNAC3-OE transgenic plants under heat stress conditions. Four-leaf stage plants were subjected to 42 °C heat stress in a growth chamber (14h light/10h dark) for 1–2 d, and then transferred to normal growth conditions. (B) Survival rate of SNAC3-OE and ZH11 after heat stress treatment. Data represent the mean ±SE (n=3). *P<0.05, t-test; **P<0.01, t-test. (C) MDA content of SNAC3-OE and ZH11 seedlings under normal and heat stress conditions. Data represent the mean ±SE (n=3). *P<0.05, t-test; **P<0.01, t-test. (D) Relative electrolyte leakage of the leaves from SNAC3-OE and ZH11 seedlings under normal and heat stress conditions. Data represent the mean ±SE (n=3). *P<0.05, t-test; **P<0.01, t-test.

Fig. 3.

Enhanced drought resistance of the SNAC3-OE transgenic plants. (A) Phenotype of the SNAC3-OE plants under drought stress conditions at the seedling stage. Four-leaf stage plants were growth without water supply for 10 d, followed by rewatering for 7 d. (B) Survival rates of SNAC3-OE and ZH11 after drought stress treatment. Data represent the mean ±SE (n=3). *P<0.05, t-test; **P<0.01, t-test. (C) Water loss rates of detached leaves from the SNAC3-OE and ZH11 plants. Data represent the mean ±SE (n=3). *P<0.05, t-test; **P<0.01, t-test. (D) Enhanced drought resistance of SNAC3-OE transgenic plants at the reproductive stage. (E) Relative spikelet fertility of SNAC3-OE and ZH11 under drought stress treatment at the reproductive stage. Data represent the mean ±SE (n=12). *P<0.05, t-test. (F) Enhanced tolerance of SNAC3-OE plants to osmotic stress conditions. (G) Plant height of SNAC3-OE and ZH11 plants under normal and osmotic stress conditions. Data represent the mean ±SE (n=12). *P<0.05, t-test; **P<0.01, t-test.

Fig. 4.

Phenotype of SNAC3-RNAi transgenic plants under heat and osmotic stresses. (A) Four-leaf stage plants were subjected to 42 °C heat stress in a growth chamber (14h light/10h dark) for 1–2 d, and then transferred to normal growth conditions. (B) Survival rates of SNAC3-RNAi and ZH11 plants after heat stress treatment. Data represent the mean ±SE (n=3). *P<0.05, t-test; **P<0.01, t-test. (C) Enhanced sensitivity of SNAC3-RNAi plants to osmotic stress conditions. (D) Plant height of SNAC3-RNAi and ZH11 under normal and osmotic stress conditions. Data represent the mean ±SE (n=12). **P<0.01, t-test.

Fig. 5.

SNAC3 participated in regulation of ROS metabolism. (A) DAB staining of leaves from SNAC3-OE and ZH11 seedlings under normal conditions, and heat and drought stress treatments. (B) Relative H₂O₂ content in leaves from SNAC3-OE and ZH11 seedlings under heat, drought treatments, and normal conditions. Data are the mean ±SE (n=3). *P<0.05, t-test. (C) Enhanced tolerance of SNAC3-OE plants and enhanced sensitivity of SNAC3-RNAi plants to oxidative stress conditions. (D) Plant height of SNAC3-OE, SNAC3-RNAi, and ZH11 under oxidative stress treatments. Data are the mean ±SE (n=12). **P<0.01, t-test.

Fig. 6.

SNAC3 directly regulates ROS-associated genes. (A) Expression of ROS-associated genes in SNAC3-overexpression materials. Error bars indicated the SE based on three technical replicates. (B) Expression of ROS-associated genes in SNAC3-repression materials. Error bars indicate the SE based on three technical replicates. (C) The schematic structure of the constructs for yeast one-hybrid analysis. (D) pGAD-SNAC3 and each of the reporter constructs were co-transformed into yeast strain Y187 and the transformants were examined by growth performance on SD/-Leu/-Trp medium and on SD/-Leu/-Trp/-His medium containing 30 mmol l^–1 3-AT. pGAD-53 was co-transformed with pHIS2-P₅₃ as a positive control (P), and pGAD-SNAC3 was co-transformed with pHIS2-P₅₃ as a negative control (N). Labels 1 and 2 indicate two independent transformants of each transformation event. (This figure is available in colour at JXB online.)

Fig. 7.

Identification of SNAC3-interacting proteins. (A) Nuclear localization of SNAC3 in tobacco epidermal cells. Infection of tobacco leaves by Agrobacterium containing the 35S promoter-driven SNAC3–GFP fusion expression construct. DAPI staining indicates the nuclear regions. (B) Nuclear localization of SNAC3 in rice protoplasts. Ghd7–CFP and SNAC3–GFP were co-transformed into etiolated shoot protoplasts of rice. Ghd7–CFP was used as a nuclear marker. (C) Confirmation of SNAC3 and SNAC3IP1/SNAC3IP2/SNAC3IP3/SNAC3IP4/SNAC3IP5/SNAC3IP7 interaction using BiFC in rice protoplasts. NC, negative control.

Fig. 8.

SNAC3 regulates stress responses in an ABA-independent manner. (A) No significant difference was observed between the SNAC3-OE, SNAC3-RNAi, and the control (ZH11) under ABA treatment. (B) Plant height of SNAC3-OE, SNAC3-RNAi, and ZH11 under ABA treatment. (C) Endogenous ABA content of SNAC3-OE and ZH11 WT leaves under normal conditions, drought stress, and heat stress conditions. Data represent the mean ±SE (n=3). (D) Expression analysis of SNAC3 in the ABA-deficient mutant phs3 under normal and drought stress conditions. Error bars indicate the SE based on three replicates.