Overexpression of a microRNA-targeted NAC transcription factor improves drought and salt tolerance in Rice via ABA-mediated pathways

Abstract

Background: The NAC (NAM, AFAT, and CUC) transcription factors play critical roles in rice (Oryza sativa) development and stress regulation. Overexpressing a microRNA (miR164b)-resistant OsNAC2 mutant gene, which generates transcripts that cannot be targeted by miR164b, improves rice plant architecture and yield; however, the performance of these mOsNAC2-overexpressing lines, named ZUOErN3 and ZUOErN4, under abiotic stress conditions such as drought have not yet been fully characterized.

Results: In this study, we showed that the germination of ZUOErN3 and ZUOErN4 seeds was delayed in comparison with the wild-type (WT) seeds, although the final germination rates of all lines were over 95%. The quantification of the endogenous ABA levels revealed that the germinating mOsNAC2-overexpressing seeds had elevated ABA levels, which resulted in their slower germination. The mOsNAC2-overexpressing plants were significantly more drought tolerance than the WT plants, with the survival rate increasing from 11.2% in the WT to nearly 70% in ZUOErN3 and ZUOErN4 plants after a drought treatment. Salt (NaCl) tolerance was also increased in the ZUOErN3 and ZUOErN4 plants due to significantly increased ABA levels. A reverse transcription quantitative PCR (RT-qPCR) analysis showed a significant increase in the expression of the ABA biosynthesis genes OsNCED1 and OsNCED3 in the mOsNAC2-overexpressing lines, and the expression levels of the stress-responsive genes OsP5CS1, OsLEA3, and OsRab16 were significantly increased in these plants. Moreover, OsNAC2 directly interacted with the promoters of OsLEA3 and OsNCED3 in yeast one-hybrid assays.

Conclusions: Taken together, our results show that OsNAC2 plays a positive regulatory role in drought and salt tolerance in rice through ABA-mediated pathways.

Keywords: ABA; Drought tolerance; OsNAC2; Rice (Oryza sativa); Salt tolerance; microRNA.

Fig. 1

mOsNAC2-overexpressing seeds germinate more slowly than those of WT. The seeds were presoaked with distilled water for 48 h and then germinated at 25/23 °C (day/night) in petri dishes. The germination percentage was calculated at different times after imbibition. The images were taken at 3 d (a), 7 d (b), and 9 d (c) after imbibition in water. (d) Germination percentage after imbibition in water. WT: wild-type seeds; ZUOErN3 and ZUOErN4: OsNAC2 overexpression lines; HAI: hours after imbibition. Error bars represent SD of three biological replicates (n > 200). ** P < 0.01 (t-test)

Fig. 2

ABA levels are elevated in transgenic seeds and drought-treated seedlings. ABA levels in rice seeds (a) and seedlings with PEG treatment (b). Seedlings were cultured in hydroponic solution with 10% PEG 6000 to simulate drought. Error bars show the SD of three biological replicates. ** P < 0.01 (t-test)

Fig. 3

Drought tolerance of WT and OsNAC2 overexpression plants. Seeds were germinated in water and transferred to Kimura B nutrient solution. Two-week-old seedlings were treated in Kimura B nutrient solution containing 10% PEG 6000. Plants before treatment (a, e), 10% PEG-treated for 3 d (b, f), and after 8 d treatment and then recovery for 8 d (c, g), are shown. d: The survival rate of WT and transgenic lines treated with 10% PEG 6000 in c and g. Error bars show the SD of three biological replicates (n = 60). ** P < 0.01 (t-test). h: The phenotype of WT and OsNAC2 overexpression line ZUOErN3 plants treated with 15% PEG 6000 for 5 d and allowed to recover for 7 d. Three independent experiments were performed. ZUONrE3, ZUOErN4: OsNAC2 overexpression plants

Fig. 4

Salt tolerance of WT and OsNAC2 overexpression plants. Seeds were germinated in water and transferred to Kimura B nutrient solution. Two-week-old seedlings were treated in Kimura B nutrient solution with 50 mM sodium chloride. WT and OsNAC2 overexpression plants (ZUONrE3 and ZUOErN4) before treatment (a, d), 50 mM sodium chloride treated for 8 d (b, e), and then recovery for 8 d (c, f) are shown

Fig. 5

Expression levels of ABA biosynthesis-related genes. The relative expression levels of OsNCED1 (a), OsNCED3 (b), OsABA2 (c), and OsZEP1 (d) were assessed using RT-qPCR with leaves at the tillering stage. The data were normalized using the rice UBI gene and are shown relative to WT. Error bars represent SD of three biological replicates. ** P < 0.01 (t-test)

Fig. 6

Expression level of abiotic stress-related genes. The relative expression levels of OsLEA3, encoding a group 3 late-embryogenesis abundant protein; OsP5CS, involved in biosynthesis of proline, encoding Δ1-pyrroline-5-carboxylate synthetase; OsProt, encoding a proline transporter; and OsRab16, encoding a small GTP-binding protein, were assayed using RT-qPCR with seedlings. The data were normalized using the rice UBI gene and are shown relative to WT. Error bars represent SD of three biological replicates. ** P < 0.01 (t-test)

Fig. 7

OsLEA3 and OsNCED3 are direct target genes of OsNAC2. The full-length OsNAC2 cDNA was fused to the GLA4 activation domain in the prey vector. The promoter sequence regions of different lengths were fused to an AbAi reporter gene in the bait vector. For yeast one-hybrid assay, Yeast cells were cotransformed with a bait vector and a prey vector and grown in liquid medium and diluted in a 10× dilution series (from 10⁰ to 10^− 3). Each dilution was spotted on both SD/−leu and SD/−leu with 300 ng/ml AbA to suppress the background and to test the strength of the interaction. L3, partial OsLEA3 promoter sequence; N6 and N5, partial OsNCED3 promoter sequences