DEAD-box RNA helicases and epigenetic control of abiotic stress-responsive gene expression

Abstract

Plant responses to abiotic stresses are controlled by a complex tier of epigenetic, transcriptional and post-transcriptional regulation. We have provided evidence that the DEAD-box RNA helicases, STRESS RESPONSE SUPPRESSOR (STRS) 1 and STRS2 are negative regulators of Arabidopsis thaliana stress-responsive transcription factors. Using GFP-STRS fusion proteins, we have demonstrated that the STRSs are localized to the nucleolus and chromocenters, and are rapidly removed to the nucleoplasm upon application of various abiotic stresses. The STRSs appear to act via RNA-directed DNA methylation to suppress Arabidopsis stress responses; this repressive epigenetic mechanism is abrogated by abiotic stress eventually leading to an open chromatin structure allowing expression of stress-responsive genes.

Keywords:

• abiotic stress
• Arabidopsis thaliana
• DEAD-box RNA helicases
• epigenetic gene silencing
• nucleolus
• RNA-directed DNA methylation

Abbreviations

ABA	=	abscisic acid
STRS	=	STRESS RESPONSE SUPPRESSOR
RdDM	=	RNA-directed DNA methylation

Plants responses to abiotic stresses involve a plethora of physiological and biochemical changes that are activated and integrated via global changes in gene expression. Reprogramming of gene expression in response to stress involves a complex network of regulatory proteins controlled at the epigenetic, transcriptional and post-transcriptional levels.^1-3

In a systems biology-based screen for novel abiotic stress regulatory proteins,^4,5 we identified genes encoding two DEAD-box RNA helicases that were rapidly down-regulated by multiple stresses and by abscisic acid (ABA).⁶ Mutations in either gene caused increased tolerance to salt, osmotic and heat stresses, as well as enhancement of stress-induced gene expression, whereas overexpression yielded the opposite effect.⁷ We therefore designated the genes, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2. We further demonstrated that STRS1 and STRS2 are part of the ABA-independent and ABA-dependent stress regulatory networks, and function to attenuate the stress-induced expression of transcriptional activator genes.

DEAD-box RNA helicases comprise the largest family of RNA helicases, with 58 members having been identified in the Arabidopsis thaliana genome.^8-10 Their name derives from one of 12 helicase core motifs that contains the characteristic Asp–Glu–Ala–Asp (DEAD) sequence. RNA helicases are major regulators of gene expression in prokaryotes and eukaryotes via their role in almost all aspects of RNA metabolism. They are often constituent parts of supramolecular complexes where they function in remodeling RNA and in assembly of ribonucleoprotein structures. RNA helicases possess RNA duplex unwinding activity and can promote duplex formation as well as displacement of proteins from RNA. They may also provide nucleation centers by acting as RNA clamps for assembly of large ribonucleoprotein complexes. In plants, DEAD-box RNA helicases have been associated with diverse molecular functions such as siRNA-mediated gene silencing, pre-mRNA splicing, chloroplast/mitochondrial intron splicing, nonsense-mediated mRNA decay, mRNA export, ribosome biogenesis and exon-junction complex functions.^11-22 Furthermore, it has been shown that these helicases play roles in various biological processes including abiotic stress responses.^{6,12,15,19,23-29}

To further explore the function of the STRS DEAD-box RNA helicases in regulating abiotic stress-responsive gene expression, we examined STRS sub-cellular localization.⁷ GFP-STRS fusion proteins localized to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. Chromocenters are regions of heterochromatic DNA that are generally transcriptionally inactive,³⁰ and this observation led us to hypothesize that the STRSs may be involved in gene silencing consistent with their role as negative regulators of gene expression. Accordingly, the STRSs were mislocalized in specific gene silencing mutants; STRS2 displayed mislocalization in three mutants of the RNA-directed DNA methylation (RdDM) pathway, rdr2, dcl3 and drd1, while STRS1 was mislocalized in the hd2c mutant. HD2C, is a histone deacetylase that interacts with HDA6, a nuclear histone deacetylase that has been implicated in RdDM, is important for establishment of transcriptionally repressive CG methylation and regulates locus-directed heterochromatin silencing in cooperation with MET1.^31-35 Importantly, both HD2C and HDA6 are involved in ABA and salt stress responses.^35,36 Notably, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, the evidence suggests that the STRSs are involved in RdDM-mediated epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response (Fig. 1).

Figure 1. Model of STRS function in epigenetic suppression of Arabidopsis responses to abiotic stress. Blue arrows, decrease; TF, transcription factor.

A recent paper has also demonstrated a role for the RdDM pathway in plant abiotic stress responses.³⁷ Arabidopsis mutants defective in various genes encoding RdDM components exhibit reduced tolerance to heat stress. Transcriptome studies of mutants of NRPD2, encoding a subunit of both Pol IV and Pol V complexes, showed that these mutants respond normally to heat stress. However, during recovery from stress, heat stress-induced genes remain at elevated levels indicating an inability of the mutant to exit the heat stress transcriptional program. Intriguingly, the misexpression of several genes in nrpd2 is correlated with defective epigenetic regulation of adjacent transposons. For instance, the expression of At1g34220, which remains up-regulated during recovery in nrpd2, is correlated with reduced DNA methylation of an upstream transposon remnant and with heat-induced read-through transcription originating at the transposon.

It is clear from our findings⁷ as well as those from Popova et al.³⁷ and others that epigenetic silencing of specific genes is a crucial component in regulating plant abiotic stress responses.^2,38 A general theme appears to be promotion of stress-mediated chromatin modifications that are characteristic of open chromatin, linked with increased expression of stress-responsive genes. Thus, in Arabidopsis subjected to dehydration stress, the permissive histone marks, H3K9 acetylation (H3K9ac) and H3K4 trimethylation (H3K4me3), characteristic of open, active chromatin are enriched on the ABA- and drought-inducible RD20, RD29A and AtGOLS2 genes.³⁹ The presence of these marks is reduced during subsequent recovery from stress. The changes in histone marks are correlated with decreased and increased nucleosome density and gene transcription during stress induction and recovery, respectively. Other stresses such as salt stress or application of ABA lead to the enrichment of histone marks associated with open chromatin.^36,40 Further support for the idea of stress-mediated promotion of open, active chromatin comes from priming experiments whereby young Arabidopsis seedlings were exposed to a 24-hour mild salt priming treatment before being returned to control conditions for 10 d⁴¹ Priming leads to a decrease in the repressive H3K27me3 mark that is associated with closed chromatin structure as well as to increased tolerance of primed plants to a subsequent drought treatment. Several genes that display changes in H3K27me3 due to priming also exhibit altered expression in response to the drought treatment.

It is interesting to note that chromatin decondensation as well as other features of stress responses such as disruption of nucleolar structure and a reduction in protein synthesis is characteristic of dedifferentiating cells such as protoplasts.⁴² Indeed, plants exposed to a variety of stresses share a similar profile of altered transcription factor expression to cells undergoing dedifferentiation during protoplasting. We recently proposed therefore that acute stress directs plant cells to undergo dedifferentiation as an adaptive mechanism in preparation for the acquisition of a new cell fate upon perception of a subsequent stimulus.³⁸

How epigenetic silencing mechanisms such as the RdDM pathway remodel chromatin in response to abiotic stress in plants, and how specific components such as the STRS DEAD-box RNA helicases function to suppress the Arabidopsis stress response remains to be elucidated. The epigenetic control of abiotic stress transcriptional networks exemplifies the complex tiers of regulation required for plants to respond appropriately to adverse environmental conditions.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Prof. Gideon Grafi for critical reading and insightful comments.

Additional information

Funding

This work was supported by the Israel Science Foundation (Grant No. 959/11 to S.B.).

Addendum to: Khan A, Garbelli A, Grossi S, Florentin A, Batelli G, Acuna T, Zolla G, Kaye Y, Paul LK, Zhu JK, Maga G, Grafi G, Barak S. The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD-box RNA helicases are nucleolar- and chromocenter-localized proteins that undergo stress-mediated relocalization and are involved in epigenetic gene silencing. Plant J 2014; 79: 28–43.