H3K4 demethylase SsJMJ11 negatively regulates drought-tolerance responses in sugarcane

Abstract

Background: Drought-induced gene alteration is usually associated with changes of histone H3K4me3 in plants. Histone methylation homeostasis relies on the coordinated activity of methyltransferases and demethylases. We previously demonstrated that SsJMJ11 is an H3K4me3 demethylase in Saccharum spontaneum and participates in regulating flowering time. However, the role of H3K4me3 regulators in regulating drought-stress responses in sugarcane (Saccharum spp.) remains elusive.

Results: We show that SsJMJ11 negatively regulates drought-stress responses by acting as an H3K4me3 demethylase. Ectopic overexpression of SsJMJ11 in Arabidopsis thaliana resulted in a hypersensitivity to soil drought stress as well as abscisic acid (ABA) and mannitol. Meanwhile, the drought-induced expression of AtRD20 and AtDREB2A, two well-known positive regulators of drought tolerance, was repressed by SsJMJ11 overexpression. In S. spontaneum, the ABA- and dehydration-induced transcription of SsRD20 and SsDREB2A was associated with increased H3K4me3 levels at these loci. Furthermore, transient overexpression of SsJMJ11 in S. spontaneum protoplasts reduced the ABA-induced transcription of SsRD20 and SsDREB2A, paralleling reduced H3K4me3 levels at these loci.

Conclusions: Our results suggest that SsJMJ11-mediated dynamic deposition of H3K4me3 is required for proper adaptation to drought stress in sugarcane.

Keywords: Saccharum spontaneum; Drought stress; H3K4me3; Histone demethylase; JmjC protein.

Fig. 1

The expression of SsJMJ11 and SsJMJ26 genes in response to drought stress. (a) The phenotype of S. spontaneum watered normally or suffered from drought stress (RSWC, 10%). Bar = 5 cm. (b) RT-qPCR analysis of the SsJMJ11 and SsJMJ26 expression in leaf, root and stem of S. spontaneum in response to drought stress. (c, d) RT-qPCR analysis of the expression of SsJMJ11 and SsJMJ26 genes in response to NaCl, MeJA, Cold, Heat, ABA, PEG and Mannitol treatment. Four-week-old S. spontaneum seedlings were treated with 100 µM ABA, 200 mM NaCl, 100 mM Mannitol, 30% PEG, Cold (4℃), Heat (38℃), 100 µM MeJA for the indicated time. Both SseEF and SsACTIN2 were used as internal controls. As similar results were obtained using different reference genes, only the results based on SseEF are presented. Data are presented as means ± SD (n = 3). Different letters denote significance difference between samples within each treatment (ANOVO with Turkey’ post hoc test, P < 0.05)

Fig. 2

Overexpression of SsJMJ11 and SsJMJ26 in Arabidopsis enhance sensitivity to drought stress. (a) Drought sensitivity of EV, SsJMJ11 OE and SsJMJ26 OE transgenic Arabidopsis grown in soil. (b) Survival rates of EV, SsJMJ11 OE and SsJMJ26 OE plants after re-watering. Data represent means ± SD (n = 6). (c) Water loss rate of EV, SsJMJ11 OE and SsJMJ26 OE detached leaves. Data represent means ± SD (n = 4). All different letters represent statistically significant differences between them (ANOVO with Turkey’ post hoc test, P < 0.05)

Fig. 3

SsJMJ11 and SsJMJ26 negatively regulate stomatal closure. (a) Stomatal density and (b) Stomatal index in abaxial epidermis of leaves from three-week-old EV, SsJMJ11 OE and SsJMJ26 OE plants. Data shown as means ± SD (n = 10). (c) The stomatal aperture phenotype of EV, SsJMJ11 OE and SsJMJ26 OE plants in response to ABA and Ca²⁺. Bar, 10 µm. (c) Quantization of EV, SsJMJ11 OE and SsJMJ26 OE plants response to ABA- and Ca²⁺-induced stomatal closure. Data represent means ± SD (n = 60). All different letters represent statistically significant differences between genotypes within each treatment (ANOVO with Turkey’ post hoc test, P < 0.05)

Fig. 4

Overexpression of SsJMJ11 and SsJMJ26 in Arabidopsis enhanced the sensitivity to ABA, NaCl and Mannitol. (a) Photographs of root lengths of EV, SsJMJ11 OE and SsJMJ26 OE plants supplemented with ABA, NaCl and Mannitol. 5 d-old plants with similarly roots length of EV, SsJMJ11 OE and SsJMJ26 OE plants were transferred to 1/2 MS medium (Control), 1/2 MS medium containing 50 µM ABA, 100 mM NaCl and 100 mM Mannitol for growing another 7 d. Bars, 1 cm. (b) Measurements of root lengths of (a). Data represent means ± SD (n = 20). (c-d) Photographs of seed germination of EV, SsJMJ11 OE and SsJMJ26 OE seeds supplemented with ABA, NaCl and Mannitol. EV, SsJMJ11 OE and SsJMJ26 OE seeds germinated on 1/2 MS medium containing 2 µM ABA, 100 mM NaCl and 200 mM Mannitol for 10 days. (d) Quantization of seed germination of (c). Data represent means ± SD (n = 6). All different letters represent statistically significant differences between genotypes within each treatment (ANOVO with Turkey’ post hoc test, P < 0.05)

Fig. 5

SsRD20 and SsDREB2A induced by dehydration associated with the increased H3K4me3 levels in S. spontaneum. (a) RT-qPCR analysis of AtRD20 and AtDREB2A genes in 3-week-old EV and SsJMJ11 overexpression plants subjected to dehydration treatment for 0, 1, and 2 hours. RT-qPCR analysis of SsJMJ11, SsRD20 and SsDREB2A genes in 1-month-old S. spontaneum seedlings subjected to dehydration treatment for 0, 1 hours (b) and 200 µM ABA treatment for 0, 1 and 2 hours (c). (d) ChIP-qPCR analysis of H3K4me3 levels at the SsRD20 and SsDREB2A loci of S. spontaneum seedlings in response to dehydration treatment for 0, 1 hours. Both AtUBC/AtACTIN2 and SseEF/SsACTIN2 were used as the internal controls. As similar results were obtained using different reference genes, only the results based on AtUBC/SseEF are presented. Data are presented as means ± SD (n = 3). Different letters denote statistically significant differences as determined by ANOVO with Turkey’ post hoc test (P < 0.05)

Fig. 6

SsJMJ11 reduces the expression of SsRD20, SsDREB2A through decreasing the H3K4me3 modification levels in S. spontaneum protoplasts. (a) Transient expression of 35S: GFP and 35S: SsJMJ11-GFP in S. spontaneum protoplasts. White arrows indicate protoplasts expressing GFP or SsJMJ11-GFP. Scale bar = 50 µm. (b) RT-qPCR analysis of SsJMJ11, SsRD20, SsDREB2A, and SsACTIN2 expression in S. spontaneum protoplasts expressing GFP or SsJMJ11-GFP. The control group did not receive ABA treatment, whereas the ABA group received 200 µM ABA treatment for 2 h. SseEF served as an internal control. Data are presented as means ± SD (n = 3). Significant differences (ANOVO with Turkey’ post hoc test, P < 0.05) are indicated by different letters. (c) ChIP-qPCR analysis was performed to assess H3K4me3 levels at the SsRD20, SsDREB2A, and SsACTIN2 loci in S. spontaneum protoplasts transfected with either GFP or SsJMJ11-GFP. The data presented are means ± standard deviation from three independent experiments (n = 3). Different letters denote statistically significant differences between groups as determined by ANOVO with Turkey’ post hoc test (P < 0.05)