Involvement of Arabidopsis histone deacetylase HDA6 in ABA and salt stress response

Abstract

Histone modifications play an important role in the epigenetic regulation of gene expression. All histone modifications are reversible, which may therefore provide a flexible way for regulating gene expression during the plant's development and during the plant response to environmental stimuli. The reversible acetylation and deacetylation of specific lysine residues on core histones are catalysed by histone acetyltransferases and histone deacetylases (HDAs). HDA6 is an RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering plants displayed a phenotype that was hypersensitive to ABA and salt stress. Compared with wild-type plants, the expression of the ABA and abiotic stress-responsive genes, ABI1, ABI2, KAT1, KAT2, DREB2A, RD29A, and RD29B, was decreased in axe1-5 and HDA6 RNA-interfering plants when treated with ABA or salt stress. It was found that both ABA and salt stress could enrich the gene activation markers, histone H3K9K14 acetylation, and H3K4 trimethylation, but decrease the gene repression marker, H3K9 dimethylation, of the ABA and abiotic stress-responsive genes. Our study indicates that HDA6-involved histone modifications modulate seed germination and the salt stress response, as well as ABA- and salt stress-induced gene expression in Arabidopsis.

Fig. 1.

Seed germination rates of axe1-5 and HAD6-RNAi plants treated with ABA and NaCl. (A) Germination rates of 2-d-old Col wild-type, axe1-5, Ws wild-type, and HDA6-RNAi (CS24039) seedlings treated with ABA. Each data point represents the mean of a single experiment performed in triplicate (n ≥100). (B) Germination rates of 2-d-old Col wild-type, axe1-5, Ws wild-type, and HDA6-RNAi (CS24039) seedlings treated with NaCl. Each data point represents the mean of a single experiment performed in triplicate (n ≥100). Asterisks mark values that are significantly different from the wild type (t test, *P <0.05, **P <0.01).

Fig. 2.

Phenotype comparison of axe1-5 and HAD6-RNAi plants in response to salt stress. (A) 5-d-old seedlings of Col wild-type, axe1-5, Ws wild-type, and HDA6-RNAi (CS24039) were transferred to a medium containing 125 mM NaCl, and the pictures were taken after 5 d. (B) 5-d-old seedlings were transferred to a medium containing 125 mM NaCl, and the percentage survival of seedlings was measured after 5 d. Each data point represents the mean of a single experiment performed in triplicate (n ≥100). Asterisks mark values that are significantly different from the wild type (t test, *P <0.05, **P <0.01).

Fig. 3.

RT-PCR analysis of ABA-responsive gene expression in axe1-5 and HDA6-RNAi plants. 2-week-old Col wild-type, axe1-5, Ws wild-type, and HDA6-RNAi (CS24039) plants were treated with 100 μM of ABA for 3 h (Ws and CS24039) or 12 h (Col and axe1-5). Total RNA for RT-PCR analysis was isolated from leaf tissues. Ubiquitin (UBQ) was used as an internal control.

Fig. 4.

RT-PCR analysis of abiotic stress-responsive gene expression in axe1-5 and HDA6-RNAi plants. 2-week-old Col wild-type, axe1-5, Ws wild-type, and HDA6-RNAi (CS24039) plants were treated with 250 mM NaCl for 6 h. Total RNA for RT-PCR analysis was isolated from leaf tissues. Ubiquitin (UBQ) was used as an internal control.

Fig. 5.

Relative positions of the amplified fragments of ABA- and salt-inducible genes in the ChIP assay. The positions and sizes of the amplified fragments from promoter (P) and exon (E) areas are indicated. The black boxes correspond to exons and white boxes to 5′ or 3′ UTR.

Fig. 6.

H3K9K14 acetylation, H3K4 trimethylation, and H3K9 dimethylation of ABA-inducible genes. Relative H3K9K14 acetylation and H3K4 trimethylation were determined by ChIP assays and normalized to an internal control (Tubulin 2). Relative H3K9 dimethylation levels were determined by ChIP assays and normalized to the input DNA. The value of Col wild type without treatment was arbitrarily given as 1. Data are the average of three biological replicates. Error bars represent standard errors. For ABA treatment, 2-week-old plants were treated with 100 μM ABA for 3 h. P indicates the promoter and E indicates the first exon. Asterisks mark values that are significantly different from the wild type without treatment (t test, *P <0.05, **P <0.01). The experiment was repeated three times with similar results.

Fig. 7.

H3K9K14 acetylation, H3K4 trimethylation, and H3K9 dimethylation of abiotic stress-inducible genes. Relative H3K9K14 acetylation and H3K4 trimethylation were determined by ChIP assays and normalized to an internal control (Tubulin 2). Relative H3K9 dimethylation levels were determined by ChIP assays and normalized to the input DNA. The value of Col wild type without treatment was arbitrarily given as 1. Data are the average of three biological replicates. Error bars represent standard errors. For NaCl treatment, 2-week-old plants were treated with 250 mM NaCl for 6 h. P indicates the promoter and E indicates the first exon. Asterisks mark values that are significantly different from the wild type without treatment (t test, *P <0.05, **P <0.01). The experiment was repeated three times with similar results.