The Arabidopsis NAC transcription factor ANAC096 cooperates with bZIP-type transcription factors in dehydration and osmotic stress responses

Abstract

Multiple transcription factors (TFs) play essential roles in plants under abiotic stress, but how these multiple TFs cooperate in abiotic stress responses remains largely unknown. In this study, we provide evidence that the NAC (for NAM, ATAF1/2, and CUC2) TF ANAC096 cooperates with the bZIP-type TFs ABRE binding factor and ABRE binding protein (ABF/AREB) to help plants survive under dehydration and osmotic stress conditions. ANAC096 directly interacts with ABF2 and ABF4, but not with ABF3, both in vitro and in vivo. ANAC096 and ABF2 synergistically activate RD29A transcription. Our genome-wide gene expression analysis revealed that a major proportion of abscisic acid (ABA)-responsive genes are under the transcriptional regulation of ANAC096. We found that the Arabidopsis thaliana anac096 mutant is hyposensitive to exogenous ABA and shows impaired ABA-induced stomatal closure and increased water loss under dehydration stress conditions. Furthermore, we found the anac096 abf2 abf4 triple mutant is much more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant. Based on these results, we propose that ANAC096 is involved in a synergistic relationship with a subset of ABFs for the transcriptional activation of ABA-inducible genes in response to dehydration and osmotic stresses.

Figure 1.

Phenotype of anac096 Mutants and ANAC096OX Transgenic Plants. (A) and (B) Effect of exogenous ABA on seed germination. Wild-type (WT) plants, two independent alleles of anac096 mutants, F1 progeny from a cross between anac096-1 and anac096-2 mutants, and two independent ANAC096OX lines were planted on one-half-strength MS plates supplemented with DMSO or ABA, and the germination rates were examined. Plant images were obtained at two different magnifications at 4 d (A) and 5 d (B) after planting on half-strength MS plates supplemented with DMSO or 1 μM ABA. (C) The germination rates were scored 5 d after planting on the plates supplemented with the indicated ABA concentrations. Three independent experiments were performed with 100 plants or seeds per experiment. Error bars indicate sd (n = 3). Statistical analysis was performed between the wild type and anac096 mutants or between the wild type and ANAC096OXs. *P value < 0.01 (Student’s t test). (D) to (G) Effect of exogenous ABA on vegetative growth. Plants grown on half-strength MS plates for 3 d were transferred vertically to half-strength MS plates supplemented with DMSO or 10 μM ABA. (D) and (E) Images of the indicated plants were taken at 12 d after transfer. White underbars indicate the root tip region. (F) and (G) To assess vegetative growth quantitatively, the primary root length (F) and the fresh weight of whole plants (G) were measured at 12 d after transfer. Three independent experiments were performed using 20 plants per experiment. Error bars indicate sd (n = 3). Vertical bars = 2 cm. Statistical analysis was performed between the wild type and anac096 mutants or between the wild type and ANAC096OX. *P value < 0.01 (Student’s t test).

Figure 2.

ANAC096 Plays a Positive Role in Both Stomatal Closure and Water Loss under Dehydration Stress Conditions. (A) Role of ANAC096 in ABA-induced stomata closure. Epidermal peels from wild-type (WT), anac096, and ANAC096OX leaf tissues were exposed to high-light conditions for 3 h to induce full opening of the stomata and subsequently treated with 0.5 or 1 μM ABA. To quantify the stomatal closure, the width of stomatal opening was measured in a triplicate experiment with 120 pairs of guard cells per experiment. Error bars indicate sd (n = 3). Statistical analysis was performed by comparing with the wild type. *P < 0.01 (Student’s t test). (B) Water loss from wild-type, anac096, and ANAC096OX plants. The aerial parts of Columbia-0 (WT), anac096 mutants, and ANAC096OX transgenic plants grown on plates for 10 d were excised and exposed to 30% relative humidity condition. To quantify water loss, fresh weight of the excised plant tissues was measured at the indicated time points. Error bars indicate sd (n = 3). (C) and (D) Dehydration stress sensitivity of wild-type, anac096 mutants, and ANAC096OX plants. (C) Wild-type, anac096, and ANAC096OX plants grown for 9 d in soil under normal growth conditions (watered) were kept in a greenhouse without watering for 14 d (dehydration), and the images of surviving plants were taken at 3 d after rewatering (rehydration). (D) To quantify the survival rate, 16 plants of each plant type were used in each experiment, and five independent experiments were performed. Error bars indicate sd (n = 5). Statistical analysis was performed between the wild type and anac096 mutants or between the wild type and ANAC096OXs. *P value < 0.01 (Student’s t test).

Figure 3.

ANAC096 Induced by ABA and Dehydration Stress Plays a Positive Role in the Expression of ABA-Inducible Genes. (A) and (B) Induction of the ANAC096 expression by exogenous ABA and different abiotic stresses. Total RNA from wild-type plants treated with exogenous ABA (10 μM), dehydration stress (30% relative humidity), mannitol (300 mM), or cold stress (4°C) at the indicated time points were used for qRT-PCR analysis of ANAC096 (A) and RD29A (B). RD29A was used as a positive control for the treatments. ACT2 was used as an internal control for qRT-PCR. Error bars indicate sd (n = 3). (C) Defect in induction of ABA-responsive genes in anac096 mutants. Total RNA from two alleles of anac096 mutant plants as well as wild-type (WT) plants that had been treated with exogenous ABA (100 μM) for 0.5 or 1 h was used for qRT-PCR analysis of RD29A, RD29B, and COR47 transcript levels. ACT2 was used as an internal control for qRT-PCR. Error bars indicate sd (n = 3).

Figure 4.

ANAC096 Regulates Positively a Major Proportion of ABA-Responsive Genes. (A) Venn diagram of ANAC096 and ABA upregulated genes (P = 2.49 × 10⁻³²¹ by Fisher’s exact test; see Supplemental Figure 5 online). (B) Hierarchical clustering of ANAC096-induced genes in ANAC096-induced (ANAC096) and ABA-treated (ABA) conditions. E1 and E2 represent each sample in the duplicates. C1 and C2 represent the genes upregulated in both conditions and only ANAC096-induced condition, respectively. Heat color gradation in red denotes the increase in log₂ fold change. (C) GOBPs enriched by the genes in C1 and C2 (P < 0.05). The color bar represents the gradient of −log₁₀(P), where P is the enrichment of a P value computed by DAVID. (D) qRT-PCR analysis of ABA-responsive genes. Total RNA from pTA, pTA-ANAC096, and anac096-1 plants that had been treated with 30 μM Dex only for 1 h (Dex) or with 30 μM Dex only for 0.5 h followed by additional 0.5-h incubation with both 2 μM ABA and 30 μM Dex (Dex + ABA) were used for qRT-PCR analyses. ACT2 was used as an internal control for qRT-PCR. Error bars indicate sd (n = 3).

Figure 5.

ANAC096 Activates Transcription by Binding to the Consensus Core cis-Acting Elements at the RD29A Promoter. (A) Nucleotide sequence containing the NACRS at the promoter region of RD29A from −99 to −50 bp. The letters in bold indicate the consensus sequences of the NACRS at the promoter region of RD29A. The two consensus binding sites were mutated either individually (1A, 2A) or simultaneously (1A/2A). The bases in lowercase letters indicate the mutated sequences in the RD29A promoter. WT, wild-type sequence. (B) Binding of ANAC096 to NACRSs at the RD29A promoter. Purified GST-ANAC096N (1 μg) was incubated with 0.025 pmol ³²P-labeled wild-type or mutant fragments. GST alone was included as a negative control. Samples were analyzed by EMSA on 4 to 16% gradient gels, and the gels were exposed to x-ray film. (C) Dimer formation of ANAC096. GST-ANAC096 or GST alone (immobilized on glutathione-agarose beads) was incubated with His-ANAC096N, His-ANAC096N(R11A/E18A), or His-ANAC096N(d2-10), and proteins bound to glutathione-agarose beads were precipitated. The precipitates were analyzed by immunoblotting using an anti-His antibody. Subsequently, the membrane was stained with Coomassie blue. (D) The role of dimerization in the DNA binding. Purified His-ANAC096N, His-ANAC096N(R11A/E18A), or His-ANAC096N(d2-10) (1 μg) was incubated with 0.025 pmol ³²P-labeled 1A probe. Probe alone was used as a negative control. Samples were analyzed by EMSA on 4 to 16% gradient gels, and the gels were exposed to x-ray film. (E) Schematic representation of the constructs used for transient expression. Intact and mutated RD29A promoter fragments were inserted into the upstream region of the LUC reporter gene. The GUS gene, which was used to normalize transformation efficiency, was placed under the control of the UBQ10 promoter. ANAC096 was placed under the Dex-inducible promoter in the pTA vector. NOS-T, Nos terminator. (F) Transcriptional activation of the RD29A promoter by ANAC096 through recognition of the consensus NACRSs. Protoplasts from transgenic plants harboring pTA-ANAC096 or empty vector were cotransformed with reporter and normalizing plasmids, incubated for 23 h, and then incubated an additional 1 h with 30 μM Dex. Total RNA from transformed protoplasts was used for qRT-PCR. The transcript level of LUC was normalized using that of GUS. Error bars indicate sd (n = 3). Statistical analysis was performed. *P value < 0.01 (Student’s t test).

Figure 6.

Interaction of ANAC096 with ABF2 and ABF4 in Vitro and in Vivo. (A) Interaction of ANAC096 with ABF2 and ABF4, but not with ABF3, in vitro. GST-ANAC096 or GST alone (immobilized on glutathione-agarose beads) was incubated with MBP-ABF2, MBP-ABF3, and MBP-ABF4, and the proteins were precipitated. The precipitates were analyzed by immunoblotting with an anti-MBP antibody. Subsequently, the membrane was stained with Coomassie blue. Asterisk indicates a nonspecific band that was often copurified with GST-ANAC096 depending on the growth conditions of the E. coli. (B) Interaction of ANAC096 with ABF2 and ABF4, but not with ABF3, in vivo. Protoplasts were transformed with the indicated constructs and BiFC signals were observed under a fluorescence microscope. NLS-RFP was included as a marker for the nucleus. YFP, yellow fluorescent protein. Bars = 20 μm. (C) Expression of proteins from the BiFC constructs. Protein extracts from protoplasts transformed with the indicated BiFC constructs were analyzed by protein gel blotting using anti-HA and anti-Myc antibodies.

Figure 7.

Synergistic Activation of RD29A Expression in Protoplasts by ANAC096 and ABF2. (A) and (B) The effect of coexpressing ANAC096 or ANAC019 with ABF2 on the induction of RD29A. Protoplasts from wild-type plants were transformed with ANAC096 and ABF2 (A) or ANAC019 and ABF2 (B), and total RNA from the transformed protoplasts was used to qRT-PCR to measure the transcript levels of RD29A. ACT2 was used as an internal control for qRT-PCR. Error bars indicate sd (n = 3). (C) Effect of the abf2 abf4 mutations on ANAC096-mediated transcriptional activation of RD29A. Protoplasts from wild-type (WT), abf2 abf4, ANAC096/abf2 abf4 (pTA-ANAC096 crossed with abf2 abf4), and ANAC096 (pTA-ANAC096) plants were cotransformed with RD29A_pro:LUC and UBQ10_pro:GUS and incubated in the presence of DMSO or Dex (30 μM) for 1 h. Total RNA from the protoplasts was subjected to qRT-PCR analysis to measure the transcript levels of LUC. The expression level was normalized using the transcript level of GUS. Error bars indicate sd (n = 3).

Figure 8.

The anac096 Mutation Enhances the Phenotype of the abf2 abf4 Double Mutant under Conditions of Exogenous ABA Application and Osmotic Stress. (A) Phenotype of the indicated mutant plants. The seedlings grown on half-strength MS plates for 3 d were transferred onto MS plates supplemented with DMSO, 10 μM ABA, or 130 mM mannitol and grown vertically for 12 d. White underbars indicate the root tip region. Vertical bars = 2 cm. WT, the wild type. (B) Quantification of the mutant phenotypes. To quantify the phenotypes, root length was measured in a triplicate experiment with 20 plants per experiment. Error bar indicate sd (n = 3). Statistical analysis was performed between wild-type and anac096 plants, between wild-type and abf2 abf4 plants, or between wild-type and anac096-1 abf2 abf4 triple mutant plants. *P value < 0.01 (Student’s t test).