Versatility of HDA19-deficiency in increasing the tolerance of Arabidopsis to different environmental stresses

ABSTRACT

Histone acetylation is controlled by HATs and HDACs, which are essential epigenetic elements that regulate plant response to environmental stresses. A previous study revealed that a deficiency in an HDAC isoform (HDA19) increases tolerance to high salinity stress in the Arabidopsis wild-type Col-0 background. Here, the increased tolerance of hda19 to drought and heat stresses is demonstrated. Results indicate that hda19 plants have greater tolerance than wild-type plants to stress conditions. The data indicate that the stress response pathway coordinated by HDA19 plays a pivotal role in increasing tolerance to a variety of different abiotic stresses in Arabidopsis, including salinity, drought, and heat. The greater level of tolerance of hda19 plants to several different environmental stresses suggests that HDA19 represents a promising target for pharmacological manipulation in order to enhance abiotic stress tolerance in plants.

Abbreviations: HAT, histone acetyltransferase; HDAC, histone deacetylase; HSF, heat shock transcription factor; RPD3, reduced potassium dependency 3; SIRT, Silent Information Regulator 2

The diverse types of histone modifications, such as acetylation, methylation, and phosphorylation, are considered to reflect a homeostasis that is constantly changing.^1-3 Recent studies have demonstrated the role of histone modifications in plant response to various environmental stresses. More specifically, the mechanism by which histone acetylation optimizes plant response to environmental stresses through HDACs has been elucidated.^4,5 Three types of HDAC proteins have been recognized in plants, RPD3-like, SIRT, and HD-tuins.⁶ The Arabidopsis genome encodes 18 genes that belong to three HDAC families: 12 RPD3-like family proteins; 2 sirtuin family proteins; and 4 HD-tuin family proteins.⁶ The RPD3-like HDACs are further divided into three sub-classes (I, II, and IV). Collectively, functional analyses of both hdac mutants and HDAC overexpression lines revealed roles of HDAC genes in salinity stress responses. For example, Arabidopsis plants (Col-0 background) deficient in HDA19 (class I RPD3-like HDAC) exhibit tolerance to salinity stress.⁷ Additionally, hda9 (class I RPD3-like HDAC) mutants.⁸ and HD2D (HD-tuins) overexpression lines⁹ are salinity stress tolerant. In contrast, however, hda6 mutants (class I RPD3-like HDAC),¹⁰ quadruple class II HDACs mutants (hda5/14/15/18),⁷ and hd2c¹¹ mutants were all sensitive to salinity stress. When HD2C was overexpressed, transgenic plants were less sensitive to NaCl during germination.¹² Taken together, these functional analyses suggest that HDACs elicit positive and negative regulation to salinity stress tolerance. Furthermore, HDAC forms a multi-complex with proteins, and the complex formation is considered to be essential for fine-tuning HDAC activity as an adaptive mechanism to variations of environmental conditions.¹³ For example, abscisic acid (ABA) is a phytohormone which plays a pivotal role in integrating various stress signals and controlling downstream stress responses. Precise control of endogenous ABA levels is critical to enable plants to adapt to the changing physiological and environmental conditions in their surrounding environments.¹⁴ Actually, HDC1 and MSI1, which form a complex with HDA19, modulate the sensitivity to ABA through the transcription of ABA signaling related genes.^15,16

The mRNA expression levels of ABA signaling-related genes, such as ABI5 and NAC019, in hda19 plants are strongly induced⁷, suggesting that these plants, in addition to increased salinity tolerance, might also have greater tolerance to drought and heat stress. This hypothesis is supported by the observation that upregulation of NAC019 increases tolerance to both heat and drought stress^17-19. In the present study, we in fact report greater tolerance of hda19 plants to drought and heat stresses, and discuss the potential use of HDAC inhibitors to enhance abiotic stress tolerance.

The present investigation determined if hda19 mutants (hda19-3 and hda19-5) exhibited greater tolerance than wild-type plants to drought stress in two independent experiments. Plants were subjected to drought stress, as described by Matsui et al. (2014) with minor modifications²⁰. Two-week-old plants were subjected to a water depletion treatment for two weeks, simply by withholding water. After the water depletion treatment, the watering of plants was resumed (details of the protocol are presented in the Figure 1 legend). Results indicated that both hda19-3 (P < 0.01, χ2 test, 20 pots) and hda19-5 (P < 0.05, χ2 test, 20 pots) plants exhibited a significantly higher survival ratio than wild-type plants in response to the drought stress conditions used in the experiments (Figure 1).

Figure 1.

HDA19 deficiency enhances tolerance to drought stress.

A. Drought stress tolerant phenotype in hda19-3 and hda19-5 plants. Arabidopsis plants were cultivated in pots placed in a tray to receive water. One wild-type Col-0 and hda19 mutant plants were grown in each pot at 22°C under a long-day photoperiod (16-h/8-h light/dark cycle) at 70–90 μE m^{− 2} s^{− 1} of illumination. Plants were grown for two weeks with water supplied to each tray. The drought stress treatment consisted of subjecting two-week-old plants to water depletion by withholding water for ten days. Water content was adjusted in each pot at the onset of the water depletion treatment by adding water to the soil of each pot. The water-depletion period was optimized so that the wild-type plants appeared withered. After the water depletion period, watering was reinitiated. Approximately 50 ml of water was supplied to each pot before and after the drought treatment. Survival rate was determined at 3 days after the reinitiation of watering. Plants with green leaves were counted as having survived the drought stress while plants with bleached leaves were counted as dead. B. Percent survival of wild-type, hda19-3 and hda19-5 plants. Significant differences between the survival rate of wild-type plants and hda19 plants after the drought stress treatment were determined using a chi-square test (* indicates significance at P < 0.05 and ** at P < 0.01, n = 20). “n” corresponds to the number of pots. Each pot contained a single hda19 mutant and wild-type plant. The presented results represent data obtained from two independent experiments.

The expression of genes associated with thermotolerance is primarily regulated by HSFs. Of the HSFs identified to date, HsfA1s are considered to play a principle role in the positive regulation of downstream TFs, including additional HSFs and other signaling components.^17,19 NAC019 participates in the networks governed by HsfA1s, and up-regulates HSFs, such as HsfA1b, HsfA6b, and HsfA7a, via direct binding to DNA sequences within their promoters.¹⁷ The positive role of NAC019 in regulating heat stress-responsive genes, raises the possibility that hda19 mutants may have greater tolerance to heat stress. Therefore, to determine this possibility, plants were subjected to heat stress, as described in Nguyen et al. (2015) with minor modification (details of the protocol are presented in the Figure 2A legend).²¹ Results indicated that both hda19-3 (P < 0.01, Student’s t test) and hda19-5 (P < 0.01, Student’s t test) plants exhibited a significantly higher survival ratio than wild-type plants after being subjected to the heat stress conditions (Figure 2B and C). Although they showed a similar tendency, hda19 mutants exhibited differential survival rations during drought and heat stress tests. It is possible that the mutagenesis to HDA19 by CRISPR/Cas9 might be insufficient to result in a null allele and/or an off-target generated by CRISPR/Cas9 might affect the phenotype.

Figure 2.

HDA19 deficiency enhances thermotolerance.

A. Growth conditions for heat stress test. Wild-type, hda19-3 and hda19-5 seeds were surface sterilized with sodium hypochlorite, followed by two rinses with distilled water. After sterilization, the seeds were placed on agar media (half-strength MS medium with 0.5% (w/v) MES and 0.9% (w/v) purified agar (Nacalai Tesque, Inc.), pH 5.7) in a petri dish at 4°C for 48 h. The seeds were then germinated at 22°C under a long-day photoperiod (16-h/8-h light/dark cycle) at 70–90 μE m^{− 2} s^{− 1} of illumination. Two-week-old plants were exposed to 43.5°C for 4 hours in a MIR-153 Laboratory Incubator (SANYO, Japan). Percent survival was determined one week later. The experiment consisted of four biological replicates which were composed of 10, 11, 13, or 15 plants. The presented data represent the mean ± SD. Evaluation criteria for survival was equivalent to what was used in the drought stress test. B. Thermotolerant phenotype in hda19-3 and hda19-5 plants. C. Percent survival of wild-type, hda19-3 and hda19-5 plants. Significant differences between the survival rate of wild-type and hda19 plants subjected to a heat stress treatment were determined using a Student’s t test (** indicates significant difference at P < 0.01).

Our current study highlights the versatility of HDA19 deficiency in increasing tolerance to drought and heat stress, in addition to the salinity stress tolerance reported in our previous study.⁷ Pharmacogenetic studies targeting HDACs have made significant advances as HDACs represent one of promising targets in the development of new cancer therapies.^22,23 Various HDAC inhibitors are available that are optimized to have an inhibitory effect on specific classes of HDACs. Novel HDAC inhibitors have been developed using plant HDACs (e.g. maize histone deacetylase HD1-A or HD1-B) to improve class selectivity among RPD3-like HDACs.²⁴ Previous studies, however, have mainly focused on the use of HDAC inhibitors for the treatment of cancer. The results of our present study suggest that pharmacologic HDAC inhibitors could potentially be used to enhance the tolerance of plants to a variety of environmental stresses. The use of HDAC inhibitors may have applicability to crops such as cassava and others where there is a critical need for greater tolerance to salinity stress.²⁵

HDA19 appears to regulate mRNA expression associated with ABA signaling pathways through histone deacetylation.⁵ Which transcriptional networks under the control of HDA19 are essential for increasing abiotic stress tolerance, however, is currently unknown. Whether a single pathway or multiple pathways convey increased tolerance to adverse environmental conditions, the extent of the influence of non-histone protein acetylation on the versatility of HDA19-deficiency in increasing stress tolerance, are questions that still need to be addressed. Further studies are required to better understand the linkage between epigenetic regulation and environmental stress responses.

Funding Statement

This work was supported by grants to M.U. and M.S from the RIKEN, and grants to M.S. from Japan Science and Technology Agency (JST) [Core Research for Evolutionary Science and Technology (CREST, Grant Number JPMJCR13B4)], and KAKENHI on Innovative Areas (Grant No. 16H01476, 18H04791, and 18H04705) of the Ministry of Education Culture, Sports and Technology of Japan.

Acknowledgments

The authors would like to show our appreciation to Ms. C. Torii and Ms. K. Mizunashi for their technical support.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.