Elongator and its epigenetic role in plant development and responses to abiotic and biotic stresses

Elongator, a six-subunit protein complex, was initially isolated as an interactor of hyperphosphorylated RNA polymerase II in yeast, and was subsequently identified in animals and plants. Elongator has been implicated in multiple cellular activities or biological processes including tRNA modification, histone modification, DNA demethylation or methylation, tubulin acetylation, and exocytosis. Studies in the model plant Arabidopsis thaliana suggest that the structure of Elongator and its functions are highly conserved between plants and yeast. Disruption of the Elongator complex in plants leads to aberrant growth and development, resistance to abiotic stresses, and susceptibility to plant pathogens. The morphological and physiological phenotypes of Arabidopsis Elongator mutants are associated with decreased histone acetylation and/or altered DNA methylation. This review summarizes recent findings related to the epigenetic function of Elongator in plant development and responses to abiotic and biotic stresses.


Introduction
Elongator was first identified as an elongating RNA polymerase II (RNAP II)-associated protein complex in yeast Wittschieben et al., 1999), and was later found to be highly conserved in eukaryotes (Versées et al., 2010). This complex consists of six subunits (ELP1-ELP6) with ELP1-3 forming the core subcomplex and ELP4-ELP6 the accessory subcomplex (Li et al., 2001;Winkler et al., 2001). Deletion of any of the six subunits results in almost identical phenotypes, suggesting that all six subunits are required for Elongator's cellular functions (Krogan and Greenblatt, 2001;Nelissen et al., 2005Nelissen et al., , 2010Mehlgarten et al., 2010;Glatt et al., 2012). ELP1 has a nuclear localization sequence essential for Elongator function and WD40 repeats that possibly function together with the WD40-containing subunit ELP2 as scaffolds for complex assembly (Fichtner et al., 2003). ELP3 is the catalytic subunit containing a C-terminal GNAT-type histone acetyltransferase (HAT) domain and an N-terminal iron-sulfur (Fe-S) radical S-adenosylmethionine (SAM) domain (Wittschieben et al., 1999;Chinenov, 2002). ELP4, ELP5, and ELP6 each form a RecA-ATPase-like fold and together assemble into a hexameric ring-shaped structure (Glatt et al., 2012;Lin et al., 2012).
The presence of a conserved HAT domain in ELP3 and the co-purification of Elongator with elongating RNAP II led to the initial assumption that Elongator might facilitate transcription elongation via histone acetylation Wittschieben et al., 1999). Indeed, ELP3 is capable of acetylating all four core histones in vitro (Wittschieben et al., 1999), and highly purified holo-Elongator has a dominant preference for lysine-14 of histone H3 and to a small extent for lysine-8 of Histone H4 (Winkler et al., 2002;Li et al., 2009). Consistently, yeast, human, and plant Elongator mutants contain reduced levels of acetylated histone H3 and H4 (Kim et al., 2002;Winkler et al., 2002;Close et al., 2006;Nelissen et al., 2010). Although chromatin immunoprecipitation (ChIP) failed to detect enrichment of Elongator at actively transcribed genomic regions in yeast (Pokholok et al., 2002), RNA immunoprecipitation (RIP) showed that Elongator interacts with nascent mRNA during transcription elongation (Gilbert et al., 2004). Conversely, in humans and plants, ChIP experiments detected association of Elongator with gene promoters and/or coding regions (Kim et al., 2002;Close et al., 2006;. Furthermore, it was recently reported that the ELP4-ELP6 accessory subcomplex assembles into a hexameric ring-shaped structure that is important for recognizing histone H3 (Lin et al., 2012). These results, together with the finding that Elongator facilitates RNAP II transcription through chromatin in an acetyl-CoAdependent manner (Kim et al., 2002), support that Elongator assists RNAP II during transcription elongation via chromatin remodeling.
ELP3 also contains a putative SAM-binding domain, which was hypothesized to function catalytically in histone demethylation (Chinenov, 2002). In yeast, the radical SAM domain of ELP3 was shown to be a motif required for the structural integrity of Elongator (Greenwood et al., 2009). In contrast, the archaea Methanocaldococcus jannaschii ELP3 SAM-binding motif might have a catalytic role, since it binds and cleaves SAM (Paraskevopoulou et al., 2006). Interestingly, a recent study in mouse indicated that Elongator is required for zygotic paternal genome demethylation, which is mediated by the ELP3 radical SAM domain rather than the HAT domain (Okada et al., 2010).
Accumulating evidence suggests that Elongator also plays a role in formation of the 5-methoxycarbonylmethyl (mcm 5 ) and 5-carbamoylmethyl (ncm 5 ) side chains on uridines at the wobble position in tRNAs (Karlsborn et al., 2015). Yeast Elongator mutants lack tRNA modifications at wobble uridines or thiouridines at position 34 of the anticodon (Huang et al., 2005). Interestingly, elevated levels of two tRNA species rescue the defects of transcription and exocytosis in yeast Elongator mutants (Esberg et al., 2006), and overexpression of tRNA Lys UUU complements the stress-related phenotypes of the yeast sin3/elp3 mutant cells (Fernández-Vázquez et al., 2013). These results are in line with the recent finding that the hexameric ELP456 accessory subcomplex specifically recognizes tRNA (Glatt et al., 2012). Therefore, it was proposed that the effects of elp mutations on transcription and secretion might be indirect consequences of inappropriate tRNA modifications (Esberg et al., 2006).
In addition to its functions in histone acetylation and tRNA modification, Elongator has also been implicated in multiple kingdom-specific activities, such as exocytosis in yeast and neuronal development in animals (Rahl et al., 2005;Close et al., 2006). Recent studies performed in the model plant Arabidopsis thaliana have revealed that the structure and function of Elongator are conserved in plants (Figure 1; Table 1; Nelissen et al., 2010;DeFraia and Mou, 2011;Van Lijsebettens et al., 2014;Yan et al., 2014). This review focuses on recent advances in the study of the epigenetic function of Elongator in plant development and responses to biotic and abiotic stresses.

Identification and Characterization of the Elongator Complex in Plants
In a genetic screen for mutants with abnormal shaped leaves in Arabidopsis, four elongata (elo) mutants, elo1, elo2, elo3, and elo4, were isolated (Berná et al., 1999). The elo4 mutant was later found to be allelic to the drl1-2 (deformed roots and leaves1) mutant, which carries a transposed Ds element in the Arabidopsis homolog of the yeast Elongator-associated protein KTI12 (KILLER TOXIN INSENSITIVE12; Fichtner et al., 2002;Nelissen et al., 2003). Further BLAST search indicated that homologs of all six subunits of the yeast Elongator are also present in the Arabidopsis genome (Nelissen et al., 2003). Indeed, the elo1, elo2, and elo3 mutations were identified in the A. thaliana (At) Elongator subunits AtELP4, AtELP1, and AtELP3, respectively, (Nelissen et al., 2005).
To verify the existence and composition of an Elongator complex in plants, tandem affinity purification (TAP) was performed using Arabidopsis cell suspension cultures overexpressing TAP-tagged ELO3/AtELP3, ELO1/AtELP4, or AtELP5 . All six subunits (ELO2/AtELP1, AtELP2, ELO3/AtELP3, ELO1/AtELP4, AtELP5, and AtELP6) were purified in the TAPs with ELO3/AtELP3, ELO1/AtELP4, or AtELP5 as bait, confirming that the Elongator composition is conserved in plants. Moreover, stoichiometric concentrations of ELO2/AtELP1, AtELP2, and ELO3/AtELP3 were found on gels when ELO2/AtELP1 was used as bait, whereas only stoichiometric concentrations of ELO1/AtELP4, AtELP5, and AtELP6 were detected when using ELO1/AtELP4 or AtELP5 as bait, suggesting that the Arabidopsis holo-Elongator is also composed of two distinct subcomplexes. The structural conservation was further supported by the fact that AtELP6 interacts with both ELO1/AtELP4 and AtELP5 in yeast two-hybrid assays .
ELO3/AtELP3 was shown to colocalize with euchromatin and the phosphorylated form of RNAP II, indicating that plant Elongator is also involved in the process of RNAP II transcription elongation . The functional conservation was further corroborated by results from heterologous complementation experiments. Ectopic expression of ELO2/AtELP1 in a yeast elp1 mutant restores sensitivity toward zymocin, a yeast killer toxin complex (Chen et al., 2006), and ELO2/AtELP1 and ELO3/AtELP3 are able to structurally replace the respective yeast Elongator subunits and functionally restore ochre suppression and γ-toxin sensitivity of a yeast elp1 elp3 double mutant by reconstituting U34 tRNA modifications (Mehlgarten et al., 2010). Moreover, tRNA wobble uridine modifications are compromised in the Arabidopsis elo2/Atelp1, elo3/Atelp3, and drl1 mutants (Chen et al., 2010;Mehlgarten et al., 2010). Therefore, the structure of Elongator and its functions in RNAP II transcription elongation as well as tRNA modification are conserved between yeast and plants. The currently available information suggests three possible roles for the Elongator complex in plants. In the nucleus, Elongator is associated with hyperphosphorylated RNA polymerase (RNAP II) and is required for histone acetylation and/or DNA demethylation/methylation at various genetic loci. On the other hand, Elongator may also regulate protein translation through tRNA modification in the cytoplasm.

The Epigenetic Function of Elongator in Plant Growth and Development
As elo/Atelp mutants exhibit abnormal shaped leaves (Berná et al., 1999;Nelissen et al., 2003Nelissen et al., , 2005, Elongator clearly plays an important role in plant growth and development. At the macroscopic level, elo/Atelp mutants have narrow and elongated leaves, reduced primary roots and lateral root density, abnormal inflorescence phyllotaxis, delayed seedling growth, and reduced apical dominance (Nelissen et al., 2003(Nelissen et al., , 2005. At the cellular and ultrastructural levels, leaves of elo/Atelp mutants have larger and fewer cells, which show less stacked grana in the chloroplasts, a hypotonic vacuole, and massive presence of Golgi vesicles in the cytoplasm (Falcone et al., 2007).
Since the phytohormone auxin plays a leading role in regulating cell proliferation (Wang and Ruan, 2013), the abnormal phenotypes of elo/Atelp mutants might be due to defects in auxin signaling or distribution. Indeed, a group of auxin-related genes are down-regulated in elo/Atelp mutants . Interestingly, decreased expression of two auxin-related genes, SHY2/IAA3 and LAX2, in the elo3/Atelp3 mutant is correlated with reduced histone H3K14 acetylation at the coding regions and 3 -UTRs (Table 2), suggesting that these two genes might be direct targets for Elongator's HAT activity during RNAP II transcription elongation. On the other hand, H3K14 acetylation levels in several other auxin-related genes, which are also down-regulated in elo3/Atelp3, are not changed, indicating that Elongator likely targets specific genes for histone acetylation rather than affecting overall histone acetylation levels . Additionally, ethylene (ET) and jasmonic acid (JA) signaling and abiotic stress responses are up-regulated in elo/Atelp plants, which might also contribute to their pleiotropic phenotypes (Chen et al., 2006;Zhou et al., 2009;Nelissen et al., 2010).
It is well known that normal cell proliferation is required for the leaf adaxial-abaxial polarity establishment in Arabidopsis (Yuan et al., 2010). The abnormal leaf polarity formation in elo/Atelp mutants is likely due to the defective cell cycle progression caused by aberrant DNA replication and increased DNA damage (Xu et al., 2012). In yeast, Elongator associates with PCNA (proliferating cell nuclear antigen) and functions in maintenance of genome stability (Li et al., 2009). Elongator also interacts with PCNA in Arabidopsis, and the interaction is required for DNA replication and repair. Moreover, ELO3/AtELP3 is required for cellular histone H3 and H4 acetylation and DNA replication-coupled H3K56 and H4K5 acetylation ( Table 2), which are important for DNA replication-coupled chromatin assembly (Xu et al., 2012). Thus, Elongator likely modulates mitotic cell cycle through interacting with PCNA and functioning in histone acetylation. Elongator may also mediate the establishment of leaf adaxialabaxial polarity in Arabidopsis by repressing transcription of abaxial-determinant genes and class 1 KNOX genes (Kojima et al., 2011). This negative role of Elongator in gene expression may be an indirect consequence of positively regulated genes.
Recently, the function of a tomato AtELP2-like gene, SlELP2L, was characterized (Zhu et al., 2015). Similar to Atelp mutants, SlELP2L-RNAi transgenic tomato plants display pleiotropic phenotypes, such as delayed seedling development, reduced leaf growth, rapidly senescing leaves and sepals, and dark-green fruits. A number of ET-and ripening-related genes are downregulated in SlELP2L-silencing plants, whereas several DNA methyltransferase genes are up-regulated. It was therefore proposed that the tomato SlELP2L might regulate plant growth and development by modulating DNA methylation. Additionally, levels of GA and IAA, which have profound effects on plant growth and development (Martí et al., 2006;Zhao, 2010), are reduced in SlELP2L-RNAi plants. Interestingly, some phenotypes of the SlELP2L-RNAi tomato plants are in marked contrast to those of elo/Atelp mutants. For instance, ET signaling is upregulated in elo/Atelp mutants , but down-regulated in SlELP2L-silencing plants (Zhu et al., 2015). Moreover, elo/Atelp mutants accumulate high levels of auxin, whereas SlELP2L-RNAi tomato plants exhibit reduced levels of auxin. These differences suggest that the function of Elongator in different plant species may not be exactly the same.

The Role of Elongator in Plant Responses to Abiotic Stresses
In a genetic screen for Arabidopsis mutants with altered drought sensitivity, a drought-resistant mutant, abo1-1 (ABA-overly sensitive), was identified (Chen et al., 2006). The abo1-1 mutant is hypersensitive to ABA in stomatal closure and seedling growth and carries a mutation in the ELO2/AtELP1 gene. In a separate genetic screen for mutants hypersensitive to ABA in root growth, Atelp2 and Atelp6 were identified . It was found that abo1/elo2/Atelp1, Atelp2, elo1/Atelp4, and Atelp6 are all hypersensitive to ABA in seed germination and seedling growth. Similarly, the tomato SlELP2L-silencing plants exhibit an obvious increase in ABA sensitivity during seedling growth (Zhu et al., 2015). On the other hand, only the core subcomplex mutants abo1/elo2/Atelp1 and Atelp2, but not the accessory subcomplex mutants elo1/Atelp4 and Atelp6, exhibit ABA hypersensitivity in stomatal closure . The different functions of the two subcomplexes in ABA-mediated stomatal movement appear to conflict with the notion that both core and accessory subcomplexes are essential for the function of the holo-Elongator described to date (Svejstrup, 2007;Van Lijsebettens et al., 2014;Karlsborn et al., 2015).
Disruption of the Arabidopsis Elongator complex results in increased resistance to the oxidative stress caused by CsCl and methyl viologen under light, indicating that Elongator functions as a negative modulator of oxidative stress . Consistently, expression of CAT3 (CATALASE 3), which encodes a catalase decomposing hydrogen peroxide in reactive species homeostasis, is up-regulated in elo/Atelp mutants. Elevated expression of CAT3 may contribute to the increased oxidative stress resistance. The elo/Atelp mutants also accumulate high levels of basal and light-induced anthocyanins ). Since anthocyanins function as antioxidants to protect plants from oxidative stress caused by diverse stressors such as drought, salt, and light (Gould, 2004), the elevated levels of anthocyanins in elo/Atelp mutants might also contribute to the enhanced resistance to oxidative stress.

The Epigenetic Function of Elongator in Plant Responses to Biotic Stresses
Salicylic acid (SA) is a key defense signal molecule against biotrophic and hemibiotrophic pathogens in plants (Vlot et al., 2009). SA accumulation occurs after pathogen infection, which is essential for activation of both local and systemic acquired resistance (SAR; Durrant and Dong, 2004). The transcription coactivator NPR1 [NONEXPRESSOR OF PATHOGENESIS-RELATED (PR) GENES] is a master regulator of SA-mediated defense responses (Durrant and Dong, 2004). Mutations in the NPR1 gene significantly compromise basal immunity, effectortriggered immunity (ETI), and SAR (Durrant and Dong, 2004). These mutations also lead to SA hyperaccumulation during pathogen infection (Shah et al., 1997;Wildermuth et al., 2001) and failure of seedling development on Murashige and Skoog (MS) medium containing high concentrations of SA (Cao et al., 1997). In a genetic screen for gns (green npr1 seedling on SA medium) mutants, we found that mutations in AtELP2 and ELO3/AtELP3 restore SA tolerance to npr1 on half-strength MS medium containing 0.5 mM SA and also suppress npr1-mediated SA hyperaccumulation (DeFraia et al., 2010(DeFraia et al., , 2013. Since high levels of SA trigger production of reactive oxygen species and subsequent cellular damage (Rao et al., 1997), Elongator may facilitate SA toxicity by suppressing the expression of antioxidant genes such as CAT3 .
Mutations in AtELP2 and ELO3/AtELP3 also compromise plant immunity (DeFraia et al., 2010(DeFraia et al., , 2013. While gns1/Atelp2 and gns2/Atelp3 are more susceptible to both virulent and avirulent Pseudomonas syringae, SAR induction in these mutants is normal. Interestingly, simultaneous removal of AtELP2 and NPR1 completely abolishes resistance to two different ETIinducing pathogens, P. syringae pv. tomato (Pst) DC3000/avrRpt2 and Pst DC3000/avrRps4. Microarray analysis revealed that the gns1/Atelp2 mutation has a broader impact than npr1 on Pst DC3000/avrRpt2-induced transcriptome changes, indicating that AtELP2 is a more general modulator of transcription than NPR1. Furthermore, Pst DC3000/avrRpt2-induced expression of a group of SA pathway defense genes is delayed and/or decreased in the gns1/Atelp2 mutant compared to wild type. Similarly, both gns1/Atelp2 and gns2/Atelp3 are significantly more susceptible than wild type to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola and B. cinerea-induced expression of the JA/ET pathway defense genes OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59) and PLANT DEFENSIN1.2 (PDF1.2) is delayed and/or decreased in gns1/Atelp2 plants (Wang and Mou, unpublished data). These results together suggest that AtELP2 is an accelerator of plant defense responses (DeFraia et al., 2010).
Genome-wide DNA methylation analysis revealed that the gns1/Atelp2 mutation increases total number of methylcytosines, decreases average methylation levels of methylcytosines, and alters methylation levels of specific cytosines . Further analysis showed that AtELP2 is required for pathogeninduced dynamic changes in DNA methylation levels of two major defense genes NPR1 and PAD4 (Table 2). On the other hand, histone acetylation assay indicated that histone H3K9/14ac levels in the coding regions of several defense genes, including NPR1, PAD4, EDS1, PR2, PR5, WRK33, ORA59, and PDF1.2, are significantly reduced in gns1/Atelp2 ( Table 2). The reduced histone H3K9/14ac levels are correlated with delayed and/or decreased induction of these defense genes in gns1/Atelp2, suggesting a role for AtELP2 in histone acetylation Wang and Mou, unpublished data). Consistently, mutants of the Arabidopsis Elongator catalytic subunit (ELO3/AtELP3) lacking the conserved residues in either the HAT domain or the radical SAM domain (Figure 2), fail to complement gns2/Atelp3 mutant phenotypes (DeFraia et al., 2013), indicating that both domains are required for Elongator to function in Arabidopsis. Therefore, Elongator likely plays an epigenetic role in response to pathogen infections.

Conclusions and Perspectives
Studies in Arabidopsis indicate that the structure of the Elongator complex and its functions are highly conserved between plants and yeast Winkler et al., 2002;Nelissen et al., 2010). Disruption of the Elongator complex in Arabidopsis leads to pleiotropic growth and defense phenotypes, which could be attributed to delayed and/or decreased expression of some genes involved in these processes (Nelissen et al., 2005;Zhou et al., 2009;DeFraia et al., 2010;Xu et al., 2012). The delayed and/or decreased gene expression in Atelp mutants is associated with reduced histone acetylation and/or altered DNA methylation . Although the intrinsic relationship between histone acetylation and DNA demethylation/methylation in Arabidopsis Elongator mutants remains to be determined, these results suggest that Elongator may epigenetically modulate plant development and responses to abiotic and biotic stresses.
However, a direct role of Elongator in histone acetylation and DNA demethylation or methylation in plants has not been established. Moreover, the enzymatic activities (HAT and DNA demethylase or methyltransferase) of the catalytic subunit ELP3 in plants have not been tested. Without such information, it would be difficult to fully appreciate the epigenetic function of Elongator in plants. Additionally, recent evidence suggests that herbivore and pathogen attack of plants generates defense phenotypes across generations and such transgenerational memory appears to be associated with DNA methylation, histone modifications, and small RNAs (Holeski et al., 2012;Luna et al., 2012;Rasmann et al., 2012;Slaughter et al., 2012). Since Elongator modulates histone acetylation and DNA methylation Xu et al., 2012;, it would be interesting to test whether Elongator is also involved in transgenerational defense induction and epigenetic inheritance in plants.
Accumulating evidence suggests that the yeast Elongator may primarily function in tRNA modification (Karlsborn et al., 2015). Studies in Arabidopsis also indicate that Elongator may play a role in tRNA modification in plants (Chen et al., 2010;Mehlgarten et al., 2010). However, the connection between Elongator's function in tRNA modification and plant development and responses to abiotic and biotic stresses still remains elusive. It would be very interesting to test whether overexpression of certain types of hypomodified tRNAs (tRNA Lys UUU , tRNA Gln UUG , and tRNA Glu UUC ) could rescue some of the plant Elongator mutant phenotypes. Such experiments would help resolve the mystery of the multitasking role of Elongator in plants.

Author Contributions
YD and ZM wrote the manuscript.