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. 2015 Jul 13:6:512.
doi: 10.3389/fpls.2015.00512. eCollection 2015.

Isolation and characterization of an osmotic stress and ABA induced histone deacetylase in Arachis hygogaea

Liang-Chen Su  1 , Bin Deng  1 , Shuai Liu  1 , Li-Mei Li  1 , Bo Hu  1 , Yu-Ting Zhong  1 , Ling Li  1
Affiliations

Isolation and characterization of an osmotic stress and ABA induced histone deacetylase in Arachis hygogaea

Liang-Chen Su et al. Front Plant Sci. .

Abstract

Histone acetylation, which together with histone methylation regulates gene activity in response to stress, is an important epigenetic modification. There is an increasing research focus on histone acetylation in crops, but there is no information to date in peanut (Arachis hypogaea). We showed that osmotic stress and ABA affect the acetylation of histone H3 loci in peanut seedlings by immunoblotting experiments. Using RNA-seq data for peanut, we found a RPD3/HDA1-like superfamily histone deacetylase (HDAC), termed AhHDA1, whose gene is up-regulated by PEG-induced water limitation and ABA signaling. We isolated and characterized AhHDA1 from A. hypogaea, showing that AhHDA1 is very similar to an Arabidopsis HDAC (AtHDA6) and, in recombinant form, possesses HDAC activity. To understand whether and how osmotic stress and ABA mediate the peanut stress response by epigenetics, the expression of AhHDA1 and stress-responsive genes following treatment with PEG, ABA, and the specific HDAC inhibitor trichostatin A (TSA) were analyzed. AhHDA1 transcript levels were enhanced by all three treatments, as was expression of peanut transcription factor genes, indicating that AhHDA1 might be involved in the epigenetic regulation of stress resistance genes that comprise the responses to osmotic stress and ABA.

Keywords: ABA; HDAC; RNA-seq; TSA; acetylation; epigenetics; osmotic stress.

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Figures

Figure 1
Figure 1
Western blot showing the effect of PEG and HBA on histone H3 acetylation status in nuclear proteins from peanut leaves. (A) H3 acetylation status in peanut leaves treated with 20% (w/v) PEG. (B) H3 acetylation status in peanut leaves treated with 100 μM ABC. C, control group; M, Mock plants were placed in an equivalent volume of deionized water as experimental plants; 1–8 h, time point after treatment. The experiments have been carried out at least three times. Each graph displays the mean and SD of three independent experiments. */**, different from control as revealed by t-test, p < 0.05/0.01.
Figure 2
Figure 2
Relatedness of peanut HDACs sequences to counterparts in other plants. (A) Alignment of deduced amino acid sequence of peanut HDACs with other plant HDAC sequences. The degree of similarity of the amino acid residues at each residues at each aligned position is shaded black, red, blue, in decreasing order. GenBank accession numbers for each aligned HDAC protein are indicated in parenthesis. (B) Phylogenetic analysis of amino acid sequences of AhHDA1 and other plant HDACs. A multiple sequence alignment was performed using Clustal W and the phylogenetic tree was constructed via the Neighbor-Joining method in MEGA 4 software. Bootstrap values from 1000 replicates for each branch are shown. GenBank accession numbers: Glycine max HDA6 (XP_003525556.1), Phaseolus vulgaris HAD (XP_007155467.1), Arachis hypogaea HDA1 (JR541338.1), Medicago truncatula HDA (XP_003601202.1), Prunus persica HDA (XP_007209104.1), Populus euphartica HDA (XP_011046214.1). Cucumis melo HDA6 (XP_00864523.1), Cucumis sativas HDA6 (XP_004138094.1), Citrus sinensis HDA6 (XP_006476865.1), Arabidopsis thaliana HA6 (AED97705.1), Gossypium arboretum HDA (KHG12201.1), Theobroma cacao HDA (XP_007036337.1), Arachis hypogaea HDA19-like (AHA85936.1), Arachis hypogaea HDA15-like (AHA85936.1). The scale bar is 0.02.
Figure 3
Figure 3
Quantitative RT-PCR validations of AhHDA1 expression in different peanut tissues. Column chart showing expression of AhHDA1 in PL, plumule; RA, radicle; ME, mesocotyl; L, leaf; S, stem; R, root; and F, flower, respectively. Plumules, radicles, and mesocotyls were taken from peanut embroys which had been cultivated for 7 days germination. Leaves stems and roots were taken from four-leaf stage peanut seedlings. Flowers were taken from peanuts during budding. All plants were grown as started in Materials and Methods. All values ± Standard Error (SE) for n = 3 biological replicates. Each graph displays the mean and SD of three independent experiments. */**, different from control as revealed by t-test, p < 0.05/p < 0.01.
Figure 4
Figure 4
Expression analyses of AhHDA1 and stress resistance genes following ABA treatment by qRT-PCR. Time points of 1, 2, 5, and 8 h were sampled to observe the changing trend. The untreated group was used as the control (no chemical treatment). Each graph shows the mean and SD of three independent experiments. */**, different from control as revealed by t-test, p < 0.0.5/p < 0.01.
Figure 5
Figure 5
Expression analyses of AhHDA1 and stress resistance genes following PEG treatment by qRT-PCR. Time points of 1, 2, 5, and 8 h were sampled to observe the changing trend. The untreated group was used as the control (no chemical treatment). Each graph shows the mean and SD of three independent experiments. */**, different from control as revealed by t-test, p < 0.0.5/p < 0.01.
Figure 6
Figure 6
Expression analyses of AhHDA1 and stress resistance genes following TSA treatment by qRT-PCR. Time points of 1, 2, 5, and 8 h were sampled to observe the changing trend. The untreated group was used as the control (no chemical treatment). Each graph shows the mean and SD of three independent experiments. */**, different from control as revealed by t-test, p < 0.0.5/p < 0.01.
Figure 7
Figure 7
HDAC activity of recombinant AhHDA1 produced in E. coli BL21. (A) SDS-PAGE showing (1) total protein from E. coli cells expressing the recombinant plasmid pPROEX-AhHDA1 before induction by IPTG; (2) total protein from E. coli cells expressing recombinant plasmid pPROEX-AhHDA1 after induction by IPTG; for 20 h; and (3) purified AhHDA1 protein. (B) In vitro HDAC activity assay of the recombinant AhHDA1 protein. 1, positive control, extract from Hela cells; 2, extract from Hela cells treated with 4 μM TSA; 3, negative control, extract form E. coli cells containing plasmid pPROEX; 4, extract from E. coli cells containing plasmid pPROEX-AhHDA1 without IPTG; 5, extract from cells containing plasmid pPROEX after induction by IPTG; 6, extract from cells containing plasmid pPROEX-AhHDA1 after induction by IPTG for 20 h; 7, extract from cells containing plasmid pPROEX-AhHDA1 after induction by IPTG, but treated with 4 μM TSA for 20 h; 8, purified recombinant AhHDA1 protein; 9, purified recombinant AhHDA1 protein treated with 4 μM TSA. Each graph shows the mean and SD of three independent experiments. */**, different from control as revealed by t-test, p < 0.0.5/p < 0.01.
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