HD2C interacts with HDA6 and is involved in ABA and salt stress response in Arabidopsis

Abstract

HD2 proteins are plant specific histone deacetylases. Four HD2 proteins, HD2A, HD2B, HD2C, and HD2D, have been identified in Arabidopsis. It was found that the expression of HD2A, HD2B, HD2C, and HD2D was repressed by ABA and NaCl. To investigate the function of HD2 proteins further, two HD2C T-DNA insertion lines of Arabidopsis, hd2c-1 and hd2c-3 were identified. Compared with wild-type plants, hd2c-1 and hd2c-3 plants displayed increased sensitivity to ABA and NaCl during germination and decreased tolerance to salt stress. These observations support a role of HD2C in the ABA and salt-stress response in Arabidopsis. Moreover, it was demonstrated that HD2C interacted physically with a RPD3-type histone deacetylase, HDA6, and bound to histone H3. The expression of ABA-responsive genes, ABI1 and ABI2, was increased in hda6, hd2c, and hda6/hd2c-1 double mutant plants, which was associated with increased histone H3K9K14 acetylation and decreased histone H3K9 dimethylation. Taken together, our results suggested that HD2C functionally associates with HDA6 and regulates gene expression through histone modifications.

Fig. 1.

Expression of HD2A, HD2B, HD2C, and HD2D in response to ABA and NaCl. 2-week-old plants were treated with 100 μM of ABA (A) or 250 mM NaCl (B). Relative mRNA levels were determined by quantitative RT-PCR analysis. Two biological replicates were carried out and gave similar results. Asterisks mark values that are significantly different from the wild type (t test, *P <0.05, **P <0.01).

Fig. 2.

Identification of T-DNA insertion mutants of HD2C. (A) Schematic structure of HD2C and T-DNA insertion sites of hd2c-1 and hd2c-3. The relative locations of PCR primers were indicated by the arrows. (a) LBa1 (5′-GTTCACGTAGTGGGCCATCG-3′), (b) HD2C-RT1 (5′-TGACGCTGACGGTAGTGAAG-3′), and (c) HD2C pr4 (5′-AATTAGATCTGCACTGTGTTTGGCCTTTG-3′). (B) The HD2C wild-type allele was detected in the wild type using HD2C-RT1 and HD2C pr4 primers, whereas T-DNA alleles were detected in hd2c-1 and hd2c-3 using LBa1 and HD2C pr4 primers. (C) RT-PCR analysis indicated that the HD2C transcript was detected in wild-type plants, but absent in hd2c-1 and hd2c-3.

Fig. 3.

Seed germination rates of hd2c-1 and hd2c-3 plants treated with ABA and NaCl. (A) Germination rates of 2-d-old wild-type (WT), hd2c-1, and hd2c-3 seedlings treated with ABA. (B) Germination rates of 2-d-old wild-type (WT), hd2c-1, and hd2c-3 seedlings treated with NaCl. Three biological replicas were performed with three technical replicates for each treatment (n ≥100), Asterisks mark values that are significantly different from the wild type (t test, *P <0.05, **P <0.01).

Fig. 4.

Phenotype comparison of hd2c-1 and hd2c-3 plants in response to salt stress. (A) 5-d-old seedlings of the wild type (WT), hd2c-1, and hd2c-3 were transferred to a medium containing 150 mM NaCl for 5 d. (B) 5-d-old seedlings were transferred to a medium containing 150 mM NaCl, and the percentage survival of seedlings was measured after 5 d. Three biological replicas were performed with three technical replicates for each treatment (n ≥100). Asterisks mark values that are significantly different from the wild type (t test, *P <0.05).

Fig. 5.

HD2C interacted with HDA6. (A) BiFC assays. HD2C and HDA6 fused with N-terminal (YN) or C-terminal (YC) of YFP were co-transfected into protoplasts, and visualized using confocal microscope. The nucleolus is indicated by white triangle. (B) In vitro pull-down assays. Purified His-HDA6 recombinant protein was incubated with GST or GST-HD2C protein. After extensive washing, the pulled-down proteins were eluted and the retention of HDA6-His proteins by GST-HD2C was detected by Western blotting using an anti-His antibody. (C) Co-immunoprecipitation analysis of HD2C interaction with HDA6 in N. benthamiana transient expression system. Agrobacterium cultures carrying 35S-HDA6-Myc and 35S:HD2C-HA were co-infiltrated into tobacco leaves. Crude extracts (input) were immunopreciped (IP) with an anti-Myc antibody and analysed by Western blotting.

Fig. 6.

Seed germination rates of hda6, hd2c-1, and hda6/hd2c-1 plants treated with ABA and NaCl. (A) Germination rates of 2-d-old wild-type (WT), hda6, hd2c-1, and hda6/hd2c-1 seedlings treated with ABA. (B) Germination rates of 2-d-old wild-type (WT), hda6, hd2c-1, and hda6/hd2c-1 seedlings treated with NaCl. Asterisks mark values that are significantly different from the wild type (t test, *P <0.05, **P <0.01). Three biological replicas were performed with three technical replicates for each treatment (n ≥100).

Fig. 7.

Expression of ABI1, ABI2, and AtERF4 in hda6, hd2c-1, and hda6/hd2c-1 double mutant plants. Total RNA was isolated from leaf tissues and the expression of ABI1, ABI2, and AtERF4 was determined by real-time RT-PCR. Asterisks mark values that are significantly different from the wild type (t test, **P <0.01, *P<0.05). The experiment was repeated three times with similar results.

Fig. 8.

Histone acetylation and methylation of genes up-regulated in hda6, hd2c-1, and hda6/hd2c-1 double mutant plants. Relative levels of H3K9K14Ac (A) and H3K9Me2 (B) in ABI1, ABI2, and AtERF4 promoter and first exon regions were determined. The amounts of DNA after ChIP were quantified and normalized to an internal control ACTIN2 for H3K9K14Ac or Ta3 for H3K9Me2. Error bars represent standard errors. Asterisks mark values that are significantly different from the wild type (t test, **P <0.01, *P<0.05). The experiment was repeated three times with similar results.

Fig. 9.

Levels of histone H3K9K14Ac, H3K4Me3, and H3K9Me2 in hda6, hd2c-1, and hda6/hd2c-1 plants. (A) The H3 was pulled down by GST-HD2C and GST-FKBP53 and analysed by Western blotting using anti-H3 antibody. (B) The levels of histone H3K9K14Ac, H3K4Me3, and H3K9Me2 in Col wild-type, hda6, hd2c-1, and hda6/hd2c-1 plants were determined by Western blot analysis. The levels of H3 were shown as a loading control.