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. 2022 Nov 22;18(11):e1010473.
doi: 10.1371/journal.pgen.1010473. eCollection 2022 Nov.

MYB44-ENAP1/2 restricts HDT4 to regulate drought tolerance in Arabidopsis

Bo Zhao  1 , Zhengyao Shao  1 , Likai Wang  1 , Fan Zhang  1 , Daveraj Chakravarty  1 , Wei Zong  1 , Juan Dong  2 , Liang Song  3 , Hong Qiao  1   4
Affiliations

MYB44-ENAP1/2 restricts HDT4 to regulate drought tolerance in Arabidopsis

Bo Zhao et al. PLoS Genet. .

Abstract

Histone acetylation has been shown to involve in stress responses. However, the detailed molecular mechanisms that how histone deacetylases and transcription factors function in drought stress response remain to be understood. In this research, we show that ENAP1 and ENAP2 are positive regulators of drought tolerance in plants, and the enap1enap2 double mutant is more sensitive to drought stress. Both ENAP1 and ENAP2 interact with MYB44, a transcription factor that interacts with histone deacetylase HDT4. Genetics data show that myb44 null mutation enhances the sensitivity of enap1enap2 to drought stress. Whereas, HDT4 negatively regulates plant drought response, the hdt4 mutant represses enap1enap2myb44 drought sensitive phenotype. In the normal condition, ENAP1/2 and MYB44 counteract the HDT4 function for the regulation of H3K27ac. Upon drought stress, the accumulation of MYB44 and reduction of HDT4 leads to the enrichment of H3K27ac and the activation of target gene expression. Overall, this research provides a novel molecular mechanism by which ENAP1, ENAP2 and MYB44 form a complex to restrict the function of HDT4 in the normal condition; under drought condition, accumulated MYB44 and reduced HDT4 lead to the elevation of H3K27ac and the expression of drought responsive genes, as a result, plants are drought tolerant.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. ENAP1 and ENAP2 positively regulates plant response to drought stress.
(A) Drought phenotype of Col-0, enap1-1enap2 and enap1-2enap2. Watering of 1-week-old plants was suspended until 49 days of growth and resumed. Survival rates (%) represented the percentage of surviving plants out of total plants in 6 independent replicates and are indicated under each plant line. (B—C) Western-blot to show the protein levels of ENAP1 (B) and ENAP2 (C) under dehydration conditions at 0, 1, 2 and 4 h. Total proteins were extracted from 10-day-old seedlings dehydrated for the indicated time. Ponceau S staining and Coomassie Brilliant Blue staining show the loading control. (D) Water loss assay of Col-0, enap1-1enap2 and enap1-2enap2. Water loss was measured in detached rosette leaves from 3-week-old plants. Data are shown as mean ± SD of three replicates. (E—F) Analysis of ABA-induced stomatal closure in Col-0, enap1-1enap2 and enap1-2enap2. Representative stomata (E) and the statistical results (F) are shown. 3-week-old leaves were soaked in the MES buffer under light to open the stomata, and then were transferred to 1 μM ABA solution for 2 h before being photographed. Stomata aperture before and after ABA treatment was measured with ImageJ. Data are shown as mean ± SE of three replicates (40 stomata per replicate). Stomatal aperture of enap1-1enap2 and enap1-2enap2 was compared to Col-0 with the unpaired and two- tailed t-test. **** P < 0.0001. Bars, 10 μm.
Fig 2
Fig 2. Transcriptome analysis in enap1-1enap2 under the dehydration condition.
(A) Principal component analysis of RNA-seq in Col-0 and enap1-1enap2 under mock (½ MS) or dehydration (½ MS + 25% PEG8000) conditions. Color codes are shown. Each dot represents one sample. (B) Venn diagram to show the number of differentially regulated genes in Col-0 and enap1-1enap2 under dehydration vs. mock treatment. (C) Dot plot to show the expression of dehydration induced genes in Col-0 and enap1-1enap2. The y-axis represents the expression levels of genes induced by dehydration in Col-0, and the x-axis represents the expression levels of these genes induced by dehydration in enap1-1enap2. 30% log2(FC) change of those genes in enap1-1enap2 compared to Col-0 was used as the cutoff for classification. Color codes are shown. (D) Regulatory network of ABA responsive transcription factors. The big dots on the circle represent ABA responsive transcription factors, and the unidirectional lines show their target genes. The small dots inside the circle represent the genes that are less induced at least 30% by dehydration in enap1-1enap2 than in Col-0. The dot color shows the log2(Fold Change) in Col-0 under dehydration vs. mock treatment. The graph is generated by Cytoscape.
Fig 3
Fig 3. ENAP1 and ENAP2 interact with MYB44.
(A) Yeast two-hybrid assay to show the interaction between ENAP1 and MYB44. The indicated constructs were co-transformed into yeast. Yeasts were grown on Leu (L), Trp (W) and His (H) drop-out medium (SD/-L-W-H) with 3’AT is to evaluate protein-protein interaction (left panel). Yeasts were grown on the Leu and Trp drop-out medium (SD/-L-W) served as the loading control (right panel). (B—C) Pull-down assay to show the interaction between ENAP1 and MYB44 (B), and the interaction between ENAP2 and MYB44 (C). GST was used as the control and GST-MYB44 was used as the bait protein. (D—E) Co-IP assay to show ENAP1 interacts with MYB44 (D), and ENAP2 interacts with MYB44 (E) in vivo. Detached leaves of three-week plants expressing MYB44-3xMyc and ENAP-YFP-HA were air-dried for 4 h (Dehydration) or untreated (Control) and subjected for total proteins extraction. Total proteins were immunoprecipitated with GFP trap or the empty trap (IgG). MYB44 was probed with anti-Myc and ENAP1 or ENAP2 was probed with anti-HA.
Fig 4
Fig 4. MYB44 functions synergistically with ENAP1 in response to the drought stress.
(A) Venn diagram to compare the binding targets of MYB44 and ENAP1 identified from ChIP-seq. (B-C) Mean ChIP-seq signals of MYB44 (B) and ENAP1 (C). The ChIP-seq signals (Log2[RPKM(ChIP/Input)]) from 2 kb upstream of TSS to 2 kb downstream of TTS were presented. Target genes were classified as in (A), that is, MYB44 unique binding targets, ENAP1 unique binding targets and MYB44 and ENAP1 co-binding targets. (D—E) Drought phenotype of Col-0, enap1-1enap2, myb44-1 and enap1-1enap2myb44-1 (D) and Col-0, ENAP1ox, myb44-1 and ENAP1ox/myb44-1 (E). 1-week-old plants were exposed to the drought stress by withholding water for indicated days and rewatered thereafter. Survived plants were recorded 3 days after rewatering. Survival rates (%) were calculated from surviving plants out of total plants in 6 independent experiments and were indicated under each plant line. (F—G) Water loss in detached leaves of Col-0 and mutants. Rosette leaves from 3-week-old plants were used. Data represent mean ± SD in triplicate.
Fig 5
Fig 5. HDT4 interacts with MYB44 but negatively regulates drought response.
(A) BiFC assay showing the interaction between MYB44 and HDT4. Agrobacteria containing indicated partner constructs was co-injected into tobacco leaves and the fluorescence was observed two days after infiltration. White arrow indicates the interactive complex. DAPI staining shows the nucleus. Bars, 50 μm. (B—C) Reciprocal pull-down to show MYB44 interacts with HDT4. Recombinant proteins purified from E. coli were used for the pull-down assay. GST-MYB44 (B) and His-HDT4 (C) acted as the bait protein respectively. (D) Drought phenotype of Col-0, enap1-1enap2myb44-1, hdt4-1 and enap1-1enap2myb44-1hdt4-1 (quadruple mutant). 1-week-old seedlings were subjected the drought stress by withholding water until 38 days and rewatered. Survival rates (%) represented the percentage of surviving plants out of total plants in 6 independent replicates and are indicated under each plant line. (E) Water loss of Col-0, enap1-1enap2myb44-1, hdt4-1 and enap1-1enap2myb44-1hdt4-1 (quadruple mutant). Water loss was examined in detached rosette leaves of 3-week-old plants. Data are shown as mean ± SD in triplicate.
Fig 6
Fig 6. MYB44-ENAP1/2 counteracts HDT4 to regulate H3K27ac.
(A—B) MYB44 (A) and HDT4 (B) protein level changes under dehydration condition. Total proteins from 10-day-old seedlings exposed to 25% PEG8000 for 0 h (D0) and 4 h (D4) were used for the western blot. H3 showed the loading. (C) Co-IP to show the interaction between MYB44 and HDT4. Agrobacteria with plasmids encoding HA-HDT4 and MYB44-FLAG was co-injected into 4-6-week tobacco leaves. Two days after the infiltration, the injected leaf was evenly scissored into two pieces. One piece was air dried for 4 h (D4) and the other piece was untreated (D0). Total proteins were immunoprecipitated with FLAG trap and the empty trap (control). Nonspecific bands indicated the loading. (D—F) ChIP-qPCR to examine the enrichment of ENAP1 (D), MYB44 (E) and HDT4 (F) on the promoter of target genes. Chromatin isolated from 10-day-old seedlings subjected for Mock and Dehydration treatments for 4 h was immunoprecipitated with anti-GFP, anti-Myc and anti-FLAG respectively. Data represent mean ± SD of three replicates. The binding of ENAP1, MYB44 and HDT4 under the Dehydration condition was compared to the Mock condition with unpaired and two- tailed t-test. *** P < 0.001, ** P < 0.01, * P < 0.05. (G—I) ChIP-qPCR to show the enrichment of H3K27ac on the promoter of UGT74E2 (G), RD28 (H) and DREB2C (I). Chromatin purified as in (D—F) was immunoprecipitated with anti-H3K27ac. (J—L) qPCR to show the relative expression of UGT74E2 (J), RD28 (K) and DREB2C (L) in the Col-0 and mutants. Total RNA was isolated from seedlings treated as in (D—F). Data represent mean ± SD of three replicates. Different letters represent significant differences with P < 0.05 in the one-way ANOVA test. I: Col-0, II: enap1-1enap2, III: myb44-1, IV: hdt4-1, V: enap1-1enap2myb44-1, VI: enap1-1enap2myb44-1hdt4-1.
Fig 7
Fig 7. A possible model to show MYB44-ENAP1/2 restricts HDT4 to regulate drought response.
Under the normal condition, ENAP1, ENAP2, MYB44 and HDT4 form a complex, and MYB44-ENAP1/2 restricts the function of HDT4 from deacetylating H3K27ac, leading to a basal level expression of target genes (upper panel); under drought stress condition, the up- regulation of MYB44 and the down- regulation of HDT4 lead to elevation of histone acetylation and activation of target genes, resulting in enhanced drought tolerance in plants (lower panel).
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