The role of the Elongator complex in plants

Abstract

The multi-subunit complex Elongator interacts with elongating RNA polymerase II (RNAPII) and is thought to facilitate transcription through histone acetylation. Elongator is conserved in eukaryotes, yet functions in diverse kingdom-specific processes. In this mini-review, we discuss the known functions of Elongator in plants, including its roles in development and responses to biotic and abiotic stresses. We propose that Elongator functions in these processes by accelerating gene induction in response to changing cellular and environmental conditions.

The histone acetyltransferase (HAT) Elongator was identified as an interactor of hyperphosphorylated (elongating) RNA polymerase II (RNAPII) in yeast,1 and later isolated from animals and plants.2,3 Elongator is composed of six subunits (ELP1–6). ELP1–3 forms the core Elongator subcomplex, while ELP4–6 forms the accessory subcomplex that interacts preferentially with non-RNAPII-interacting core Elongator.4,5 All six subunits are evolutionarily conserved in both sequence and their interaction with other subunits.6–10 ELP1 and ELP2 are WD40 proteins that act as scaffolds for complex assembly. ELP1 also contains a functional nuclear localization sequence that is essential for Elongator function.11 ELP3 is the catalytic subunit of Elongator, and encodes a histone acetyltransferase.12 Other than their requirement for ELP3 HAT activity, the function of the accessory complex is unknown, but is postulated to regulate core Elongator by mediating its interaction with hyperphosphorylated RNAPII.9

Yeast Elongator was shown to directly acetylate H3K14 and possibly H4K8 in vitro, and levels of multiply acetylated H3 and H4 are decreased in yeast elp mutants.12 These histone modifications are generally associated with actively transcribed euchromatic DNA.13,14 Consistently, Elongator requires the acetyl donor Acetyl-CoA to facilitate transcription through chromatin.15 Loss of both Elongator function and that of the HAT complex SAGA (Spt-Ada-Gcn5-Acetyltransferase) results in severe growth defects compared to single mutants, while disruption of Elongator and H3 or H4 N-terminal tails causes synthetic lethality.16 In plants and in mammalian cells, genes involved in Elongator-dependent functions are hypoacetylated and downregulated in elp mutants.3,17 Taken together, these results suggest Elongator's HAT activity is important for its function. ELP3 also contains a putative radical S-adenosyl methionine (SAM)-binding domain which was postulated to carry out demethylation of histone residues.18 Although the activity and substrate(s) of the domain are unknown, it was shown to be essential for paternal DNA demethylation in mouse zygotes, while the HAT domain is not required.19

Although Elongator certainly plays a distinct role in transcription, it also functions in translation.20 Elongator is essential for the 5-methoxycarbonylmethyl (mcm) and 5-carbamoylmethyl (ncm) modifications on uridines at the wobble position in several tRNAs.10,20,22 These modifications are conserved in eukaryotes,23 and are required for the accurate decoding of NNR codons (with N being any nucleotide and R either A or G).24

Although its transcriptional and translational roles seem to be conserved, it is becoming clear that Elongator has multiple kingdom-specific functions in diverse organisms. For example, Elongator functions in zymocin toxicity and exocytosis in yeast,25,26 and in α-tubulin acetylation in cortical neurons, where Elongator deficiency results in defective neuron development, manifested as the human disease familial dysautonomia.27 Recent genetic studies performed in Arabidopsis have demonstrated that Elongator functions in plant development and in responses to biotic and abiotic stresses. In this short review, the role of Elongator in plants is discussed.

Growth and Development

At the macroscopic level, Elongator mutants have narrow and elongated leaves and petioles, reduced primary root growth and hypocotyl elongation, aberrant inflorescence and flower architecture, a disorganized shoot apical meristem, delayed seedling growth, reduced germination frequency, delayed flowering and reduced fertility.6,28,29 Mutation of DRL1, a putative interactor of Elongator, results in a similar phenotype. DRL1 requires Ca²⁺ to bind calmodulin, has an ATP/GTP binding domain, and is proposed to regulate Elongator activity.28,29 Disruption of each Elongator subunit results in similar phenotypes, and double and triple mutants resemble single mutants, suggesting that plant Elongator subunits share a common function.6,30

At the cellular level, leaves of elp mutants have larger and fewer cells which exhibit slightly increased ploidy. This suggests that these cells have a reduced cell division rate,6,29 which may be responsible for the decreased leaf and root growth. Furthermore, ultrastructural studies showed that elp leaf cells have fewer stacked grana, a hypotonic vacuole, and relatively high numbers of exocytotic vesicles.31

In addition to the developmental defects mentioned above, elp mutants also exhibit reduced apical dominance, defective phyllotaxis, agravitropic root growth and reduced lateral root density.3 These phenotypes are often associated with aberrant auxin biosynthesis and/or transport. Indeed, auxin accumulation in elp plants is increased.3 Elongator likely controls plant development through its regulation of auxin-responsive genes, as a large number of auxin-responsive genes are differentially expressed in elp plants. Some of these genes are hypoacetylated at H3K14,3 suggesting that Elongator may facilitate the expression of auxin-responsive genes by directly promoting euchromatin formation and transcription via its interaction with RNAPII. Abnormal development may also derive from the upregulated jasmonic acid (JA) and ethylene (ET) signaling pathways observed in elp mutants.3

Abiotic Stress Responses

Elongator has been shown to function in the tolerance of abiotic stresses. Mutations in both the core and accessory Elongator subcomplexes confer resistance to oxidative stress caused by methyl viologen and CsCl. This is accompanied by increased expression of FSD1 (FE SUPEROXIDE DISMUTASE 1), which converts the free radical superoxide into hydrogen peroxide and water and CAT3 (CATALASE 3), which decomposes hydrogen peroxide.6,30 Enhanced breakdown of these harmful reactive oxygen species may contribute to the increased oxidative stress resistance of elp mutants. High light-induced anthocyanin biosynthesis is also increased in these plants.30 The contribution of anthocyanins to the increased oxidative stress resistance and drought tolerance in Elongator mutants requires further investigation.

Elongator also represses drought tolerance and abscisic acid (ABA) sensitivity, but the relationship between these two processes in elp mutants is unclear. ABA triggers stomatal closure during drought conditions, preventing transpirational water loss.32 Disruption of both core and accessory subcomplexes results in the hyperinhibition by ABA of germination and seedling growth. Interestingly, only mutations in core Elongator cause enhanced ABA-induced stomatal closure and water retention.30 This is surprising, given that both core and accessory subcomplexes are essential for every other function of Elongator described to date. Consistently, mutation of ELP1 results in increased drought tolerance. It will be interesting to see if both core and accessory subcomplexes function in drought tolerance. This difference in core and accessory subcomplex function represents a unique opportunity to study the distinct contributions of each subcomplex in plants.

Immune Responses

Defense against many non-necrotizing, biotrophic pathogens is mediated by the signal molecule salicylic acid (SA). SA accumulation occurs after pathogen infection and induces the expression of defense genes and the activation of systemic acquired resistance (SAR), a long-lasting broad-spectrum resistance to secondary infection in distal tissues.33,34 Effective SA-mediated resistance requires the transcriptional co-activator NPR1, which regulates the activity of several transcription factors to modulate defense gene expression.35,36 We recently showed that ELP2 is essential for an effective and timely immune response to the hemibiotrophic pathogen Pseudomonas syringae.37 This immune deficiency may be due to the delayed induction of SA biosynthesis and defense gene expression seen in elp2 plants. Consistently, pre-activation of immunity by SAR activation or treatment with an SA analog is sufficient to restore pathogen resistance to elp2. Other core and accessory subunits are also essential for pathogen resistance (Mou Z, et al. unpublished results). The npr1 mutant plants lack tolerance to the cytotoxic effects of SA.38 Elongator is essential for this SA toxicity in npr1, as disruption of ELP2,37 as well as other subunits and DRL1 (Mou Z, et al. unpublished results), partially restore SA tolerance to npr1. High levels of SA produce ROS and subsequent cellular damage.39 Elongator's repression of oxidative stress resistance and antioxidant genes may be partially responsible for its function in SA toxicity.

SA cross-talks with several hormones in modulating the immune response to pathogens with various virulence and infection strategies.40 SA mediates resistance to biotrophic pathogens, while JA/ET mediates resistance to necrotrophic pathogens and herbivory.40 Generally, SA antagonizes the JA/ET pathway, and vice versa. Elongator represses JA/ET signaling and promotes the initiation of SA signaling.3,37 It may therefore mediate crosstalk between these pathways, as well as promote susceptibility to necrotrophic pathogens. Further investigation is required to test this hypothesis. SA and ABA signaling are essential for pathogen-induced stomatal closure, which is antagonized by coronatine, a virulence factor and JA mimic.41 Elongator's roles in SA, JA and ABA signaling and its repression of ABA-induced stomatal closure suggest its involvement in pathogen-induced stomatal closure. Once past stomata, P. syringae induces ABA synthesis, which represses SA signaling.42,43 Elongator restrains ABA sensitivity and promotes the initiation of SA signaling,30,37 suggesting that Elongator might modulate crosstalk between these two signaling networks as well.

Auxin signaling also affects plant immunity. Auxin signaling mutants display enhanced pathogen resistance and exogenous auxin application renders plants more susceptible to infection and represses SA-dependent defense gene expression.44–46 Conversely, SA represses auxin responses.47–49 Many pathogens synthesize auxin-like molecules, ostensibly to repress the immune system and/or to increase the availability of nutrients.40 Since Elongator is involved in auxin signaling, the susceptibility of elp mutants may also derive from aberrant auxin biology in these plants. Agrobacterium tumefaciens alters auxin signaling through the insertion of T-DNA into the plant genome. This promotes pathogenesis by increasing nutrient availability and also leads to tumorigenesis.50 ELP3 is essential for efficient T-DNA insertion and tumorigenesis.51 Reduced T-DNA insertion efficiency, possibly caused by an alteration in chromatin condensation, likely underlies this reduction in tumorigenesis. However, it is tempting to speculate that the role of ELP3 in auxin signaling may contribute to its function in tumorigenesis. In summary, hormonal signaling pathways are finely tuned in plants to prioritize defense over growth. Elongator appears to be essential for maintaining hormonal balance, as disruption of Elongator shifts hormonal signaling, resulting in pleiotropic growth and defense phenotypes.

Conclusions and Perspectives

Elongator controls several kingdom-specific processes. Together with its conservation across kingdoms, this suggests that Elongator did not evolve to regulate a specific physiological process. Rather, Elongator may function as an accelerator of gene induction in response to diverse environmental changes, as first suggested by Otero et al.1 It may accomplish this through histone acetylation, which may help overcome transcriptional inhibition by nucleosomes or higher order chromatin compaction, and/or through promoting efficient decoding of NNR codons through its tRNA modification function. Speculating further, Elongator may have evolved as a way to counteract epigenetic inhibition of transcription elongation in eukaryotes.52 Although Elongator functions as an accelerator of gene induction in plant immunity, it is unclear whether it accelerates the induction of auxin- and abiotic stress-responsive genes, as the expression of these genes have not been examined over time in elp mutants.

The aberrant development of elp plants may represent a failure to properly respond to localized developmental signals. Auxinmediated growth and development involves dynamic changes in local auxin concentrations and the establishment of auxin gradients that drive cell division and growth.53 A delayed response to or establishment of these gradients may result in aberrant auxin signaling and abnormal growth and development. It would be interesting to examine Elongator function in cells with specific auxin accumulation patterns over time.

Several major questions about the function of Elongator remain. What are the precise roles of Elongator in transcription and tRNA modification, and how do these functions contribute to physiological processes? What is the importance of Elongator localization, HAT activity, and its putative SAM-binding domain? Is Elongator recruited to genes upon their induction? Perhaps the most controversial question is whether Elongator's HAT and/or tRNA modification activity is essential for Elongator-dependent processes.54 To dissect its function in plants, the transcriptional and translational functions of Elongator will need to be uncoupled in vivo. A better understanding of Elongator will illuminate how the demands of an ever-changing environment are met at the level of gene expression, specifically through epigenetic and post-transcriptional mechanisms.