Shaping Ethylene Response: The Role of EIN3/EIL1 Transcription Factors

1Plant hormone ethylene coordinates numerous developmental processes (including germination, soil emergence, seedling growth, fruit ripening, senescence, abscission, etc.), as well as diverse biotic and abiotic stress responses. Ethylene has also been shown to induce typical morphological changes in dark-grown seedlings (inhibition of hypocotyl and root elongation, radial swelling of hypocotyl, and exaggeration of apical hook) known as “the triple response”. Ethylene is produced from L-methionine, which is consequently converted to S-adenosyl-L-methionine (by SAM-synthetases), 1-aminocyclopropane-1-carboxylic acid (ACC) (by ACC synthases), and ethylene (by ACC oxidases) (reviewed in ). Ethylene is perceived by a family of receptors (ETHYLENE RESPONSE 1, ETR1; ETHYLENE RESPONSE SENSOR 1, ERS1; ETR2, ETHYLENE INSENSITIVE 4, EIN4; and ERS2 in Arabidopsis) localized in the endoplasmic reticulum (ER) membrane (reviewed in ). Upon binding, ethylene inactivates them and thereby blocks the serine–threonine protein kinase CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) activity promoting the cleavage of ER-anchored EIN2 protein (reviewed in; ). EIN2 C-terminal domain (EIN2-C) released upon cleavage indirectly triggers EIN3 and EIN3-Like (EIL) transcription factors (TFs) that are considered the key transcriptional regulators of ethylene response ( Figure 1 ). Noteworthy, these TFs function as a hub that integrates and processes different cues to “shape” ethylene response in accordance with spatiotemporal and environmental conditions. Below, we will focus on the nuclear events that conduct EIN3/EIL activation and set their functional output.

2EIL is a small family of plant-specific proteins. There are six genes encoding the members of this family in Arabidopsis thaliana genome (EIN3, EIL1-5). They harbor a conservative N-terminal DNA-binding domain with a unique fold structure. EIN3, EIL1, and EIL2 represent functionally homologous proteins involved in the regulation of ethylene-responsive genes. The most closely related EIN3 and EIL1 are considered the major regulators since ein3 eil1 double mutants show complete ethylene insensitivity in terms of the triple response, pathogen resistance, and the ability to fully suppress ctr1 mutation (reviewed in; ). Two paralogs differentially regulate ethylene response in the seedlings (EIN3) and in adult leaves and stems (EIL1). Yet, a minor, EIL2 role in the regulation of ethylene response is supported by its capability to complement ein3 mutation when overexpressed. In Figure 2, we visualized tissue-specific expression levels of EIL genes based on publicly available data on transcriptome profiling in different Arabidopsis tissues retrieved from ThaleMine v1.10.4 (https://apps.araport.org/thalemine/; ). Unlike EIN3 and EIL1, EIL2 transcripts level is low throughout plant tissues; moderate EIL2 expression is restricted to root apical meristem and pollen ( Figure 2 ). Therefore, EIL2 function could be limited to specific spatiotemporal conditions. EIL3/SLIM1 does not function in ethylene pathway but regulates sulfur deficiency response; no defined roles of EIL4 and EIL5 have been reported to date (reviewed in; ).

3EIN3 and EIL1 activation in response to ethylene is the target for complex regulation. EIN3 and EIL1 are short-living proteins that undergo ubiquitination and proteasomal degradation driven by ubiquitin-ligases EIN3 BINDING F-BOX1 (EBF1) and EBF2 ( Figures 1 and 3A ). Stabilization of EIN3/EIL1 upon ethylene release plays a pivotal role in triggering ethylene-directed gene expression. Ethylene dampens EBF1/2 levels via i) translational repression of EBF1/2 mRNA in the cytosol promoted by EIN2-C, and ii) EIN2-dependent proteasomal degradation of EBF1/2 proteins ( Figure 1 ). Stabilized EIN3/EIL1 accumulate in the nucleus.

4EIN3/EIL1 are predominantly transcriptional activators (; reviewed in ). In Arabidopsis, EIN3, EIL1, and EIL2 specifically bind a short DNA sequence referred to as EIN3 binding site (EBS) in gene promoters ( Figure 1 ). EIN3 binds its target loci as a homodimer, and the dimerization is DNA independent. Accordingly, EIN3 demonstrates higher binding affinity to the inverted repeats of EBS compared to the monomeric site in the in vitro experiments. EIN3 binding to the targets is facilitated by elevated levels of H3K14 and non-canonical H3K23 histone acetylation both promoted by a EIN2-C-scaffolded histone acetylation complex, which is triggered upon EIN2-C interaction with a histone binding protein EIN2 NUCLEAR ASSOCIATED PROTEIN 1 (ENAP1) (;; reviewed in ) ( Figure 3 ). Since neither EIN2-C nor ENAP1 possess histone acetyltransferase domains, they might recruit other proteins to promote histone modifications. EIN3 is capable of interacting with ENAP1, too, and it is thought to contribute to ethylene-induced elevation of H3K14 and H3K23 acetylation as well.

5Along with well-known EIN3/EIL1-promoted gene transcriptional activation, ethylene downregulates a considerable set of genes. In a recent work, demonstrated that histone deacetylases SRT1 and SRT2 mediate transcriptional repression in response to ethylene by downregulating the levels of H3K9 acetylation (at least for a particular set of ethylene-repressed genes) ( Figure 3 ). Both deacetylases interact with ENAP1, and the function of SRT2 is EIN2- and EIN3/EIL1-dependent. The mechanism used to distinguish between the activator and repressor pathways as well as the role of EIN3/EIL1 in SRT1/2-mediated gene repression are still unclear and need further investigations.

6Upon DNA binding, EIN3/EIL1 modulate multiple transcriptional cascades. Ethylene-sensitive EIN3 target genes encoding TFs include ERF1, involved in a range of ethylene responses, PIF3, RSL4, ESE1, and CBF1/2/3, the regulators of de-etiolation, root hair development, salt and cold stress responses, correspondingly. To supplement this list, numerous TF-encoding genes comprising representatives of AP2/ERF, WRKY, NAC, and other families were retrieved from whole-genome data on EIN3 binding and ethylene-induced transcriptomes. Besides, EIN3 directly regulates expression of chlorophyll biosynthesis genes PORA/B, the pigment-binding proteins LHC essential for photosynthesis initiation, the immune receptor FLS2, and the apical hook formation regulator HLS1. EIN3/EIL1 affect the pathways of many hormones, including direct regulation of hormones biosynthesis (e.g., salicylic acid biosynthesis gene SID2, ), and signaling (e.g., type-A negative regulators of cytokinin signaling ARR5/7/15, ). To maintain a homeostasis, EIN3 activates a feedback regulatory circuit by inducing transcription of EBF2 and probably some other negative regulators of ethylene signaling.

7To provide a proper phenotypic outcome upon ethylene release, these transcriptional cascades and the downstream growth control pathways should be tightly coordinated, which is supported by data on the dynamic changes of ethylene-induced transcriptomes in etiolated Arabidopsis seedlings where four distinct transcriptional waves are segregated. The observed transcription kinetics may be due to distinct mechanisms of transcriptional control, or the heterogeneity of the ethylene response in different tissues. Transcriptome profiling of Arabidopsis mutants identified large groups of EIN3/EIL1-regulated genes that were co-regulated by the other TFs such as RHD6 (root hair development) and PIFs (light signaling), which implies co-regulation of EIN3/EIL1-triggered transcription by certain developmental and environmental cues. In the following sections, we illustrate that EIN3/EIL1 proteins represent crucial targets for tuning the downstream transcriptional cascades in time and space.

8Climacteric fleshy fruits (the ones that demonstrate a respiratory burst at the beginning of ripening) use ethylene as a ripening signal. Mature fruit produces ethylene in an autocatalytic manner (system II) unlike immature fruit and vegetative tissues where self-inhibitory ethylene production (system I) is implemented. Autocatalytic regulation suggests a positive feedback loop controlling ethylene synthesis. Presumably, the corresponding regulatory circuit includes EIN3 triggered transcriptional cascade that finally activates ethylene biosynthesis genes (ACSes and ACOs). To prevent uncontrolled ethylene production, this circuit should be under a tight spatiotemporal regulation.

9Epigenetic modifications often promote spatiotemporal regulation of plant hormone responses (reviewed in ). In Arabidopsis, a repressive mark H3K27me3 regulates expression of a large number of genes. A systematic analysis of epigenome and transcriptome data suggests that climacteric fruits use removal of H3K27me3 to trigger autocatalytic system II ethylene production specifically in the mature fruit. Accordingly, EIN3 targeted promoters—a part of transcriptional feedback circuit controlling climacteric fruit ripening (RIN in tomato, NAC in peach and banana)—are associated with the repressive histone mark H3K27me3 in leaf and immature fruit. They become demethylated and therefore accessible only in the ripening fruit tissues. Presumably, this epigenetic mechanism prevents autocatalytic ethylene production in vegetative and immature fruit tissues.

10Recently, using a systematic analysis of publicly available ChIP-Seq data on EIN3 binding in Arabidopsis, we have demonstrated that EIN3 direct targets are enriched in a chromatin state 4 according to the classification of, which is associated with H3K27me3 repressive mark. Therefore, H3K27me3-associated epigenetic silencing might be a more general mechanism providing spatiotemporal specificity of ethylene response via restriction of EIN3 function.

11Regulation of EIN3/EIL1 levels via the control of the protein stability by EBF1/2 is a pivotal mechanism of EIN3/EIL1 adjustment in ethylene signaling. Simultaneously, it can be affected by environmental factors resulting in a modulation of transcriptional response to ethylene. Plants germinating in the darkness assume a light-regulated developmental program known as skotomorphogenesis, which phenotypically results in rapid hypocotyl elongation, small closed chlorotic cotyledons, and apical hook formation. EIN3/EIL1 and their target genes (e.g., HLS1, ERF1, PIF3, PORA/B) play essential roles in these processes. They contribute in chlorosis and increased apical hook curvature of buried seedlings, induce shortening and thickening of hypocotyl to enhance lifting capacity of the seedling, and finally promote seedlings greening upon light irradiation.

12In the seedlings growing through the soil, EIN3/EIL1 are stabilized by both light signaling and ethylene, which accumulates in response to mechanical pressure. In the former case, E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a central repressor of light signaling, directly targets EBF1/2 for ubiquitination and degradation. As seedlings grow toward the surface, light intensity gradually increases. As a result, COP1 activity, which is negatively regulated by photoreceptors, gradually decreases attenuating ethylene response.

13When the seedling reaches the soil surface, light triggers a dramatic developmental transition known as de-etiolation that leads to immediate termination of ethylene responses. Light-activated photoreceptor phytochrome B (phyB) directly interacts with both EIN3 and EBF1/2 proteins, thereby stimulating robust EIN3 degradation, rapidly turning off ethylene signaling.

14In this section, we consider the cross-talk of ethylene signaling pathway with jasmonic acid (JA) and gibberellins (GA) based on an inhibition of EIN3/EIL1 transcriptional activity due to their physical interactions with repressor proteins ( Table 1 ). These protein–protein interactions (PPI) rather prevent EIN3/EIL1 binding to DNA than cause changes in protein stability. JA and ethylene synergistically regulate certain aspects of plant development (such as root hair development and inhibition of root growth) and tolerance to necrotrophic fungi. The transcriptional repressors JASMONATE ZIM-DOMAIN (JAZ) are the master regulators that interact with MYC2, MYC3, and MYC4 TFs and negatively control JA signaling (reviewed in ). JAZ proteins interact with EIN3/EIL1 and enhance EIN3/EIL1 binding to HDA6, an RPD-type histone deacetylase. The resulting complex inhibits EIN3/EIL1-mediated transcription. Upon JA treatment, JAZ degrades, attenuating HDA6-EIN3/EIL1 association and therefore activating EIN3/EIL1. Therefore, pathogenesis-related genes ERF1 and ORA59, directly regulated by EIN3/EIL1, as well as their downstream target PDF1.2, are upregulated in response to JA.

15At the same time, MYC2, MYC3, and MYC4 transcriptional regulators of JA signaling interact with EIN3/EIL1, inhibiting their function. Thus, ERF1, ORA59, and PDF1.2 genes are upregulated in myc2 mutants. This inhibitory mechanism underlies ethylene-JA antagonism. Particularly, JA-directed abolishment of ethylene-promoted apical hook formation proceeds via MYC2-mediated attenuation of HOOKLESS1 (HLS1) expression, which is the key regulator of hook development and a direct EIN3/EIL1 target. Additionally, MYC2 targets EBF1, inducing its expression and therefore promoting EIN3/EIL1 degradation. Noteworthy, the inhibitory effect in the EIN3–MYC2 complex is reciprocal: the interaction suppresses MYC2 activity as well and thereby ethylene attenuates JA-regulated plant defense response against insect attack. Similarly, EIN3 plays an inhibitory role in sulfur deficiency response, forming heterodimers with EIL3/SLIM1 TF and preventing its target gene recognition by EIL3/SLIM1.

16Just as in the case of JA-ethylene synergy, GA enhances apical hook curvature at least partially via a release of EIN3/EIL1 from repressor proteins. DELLA proteins are the main transcriptional repressors of GA responses. Two members of this family (RGA and GAI) are capable of associating with EIN3/EIL1 DNA-binding domain and inhibiting EIN3/EIL1 function. In response to GAs, DELLA proteins rapidly degrade, thereby de-repressing EIN3/EIL1-mediated transcription of at least the HLS1 gene.

17EIN3/EIL1’s capability to function cooperatively with the transcriptional regulators of the other signaling pathways provides another possibility to shape spatiotemporal patterns of ethylene response. This cooperation implies the cross-talk of TFs bound to DNA that goes along with the physical interaction of these TFs ( Table 1 ). In buried seedlings, the chloroplasts’ development is arrested at the etioplast stage, characterized by an immature arrangement of the inner membranes and pigment molecules. EIN3 and PHYTOCHROME INTERACTING FACTOR3 (PIF3), a darkness-stabilized transcriptional regulator of light signaling, form an interdependent module that represses chloroplast development in buried seedlings. Namely, EIN3 and PIF3 directly interact and bind the promoters of LIGHT HARVESTING COMPLEX (LHC) genes in a cooperative manner to synergistically suppress their expression. Upon light exposure, the levels of EIN3 and PIF3 decrease, and activation of LHC expression triggers chloroplast differentiation.

18Interestingly, another TF from PIF family, PIF4, interacts with EIN3 as well, and both TFs target HLS1, the key regulator of apical hook development. However, EIN3 and PIF4 activate HLS1 transcription independently.

19Cooperative regulation also guides ethylene functioning in root hair development. EIN3 promotes root hair elongation by directly activating RHD6-LIKE4 (RSL4) gene. Besides, EIN3 physically interacts with ROOT HAIR DEFECTIVE6 (RHD6), a major regulator of root hair development that targets RSL4 as well. Both EIN3 and RHD6 co-activate RSL4 more efficiently than either of them alone. The role of EIN3–RHD6 cooperative action is most likely not limited to RSL4 regulation, but rather covers a quite extensive set of genes and contributes to ethylene-promoted root hair initiation as well. Similarly, in papaya, EIN3 homolog CpEIN3a interacts with CpNAC2, and both TFs directly activate the transcription of carotenoid biosynthesis-related genes CpPDS4 and CpCHY-b expressed during fruit ripening. Both TFs possess a combinatory effect on the regulation of their targets.

20Besides, EIN3/EIL1 are capable of binding gene promoters and affecting gene expression indirectly via physical interactions with other TFs and modulation of their activity ( Table 1 ). Increase of auxin biosynthesis in the root tip epidermis via upregulation of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) plays a pivotal role in ethylene-induced inhibition of root growth. EIN3 targets TAA1 promoter through a “piggyback” interaction with RESPONSE REGULATOR 1 (ARR1), a transcriptional regulator of cytokinin signaling, thereby enhancing ARR1 transcriptional activity. Similarly, EIN3/EIL1 interact with FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), a central regulator of Fe acquisition in roots, activating FIT abundance. Moreover, EIN3/EIL1 bridges FIT to the transcriptional Mediator complex to recruit RNA-pol and promote the regulation of iron homeostasis.

21There is growing evidence that despite their essential role, EIN3/EIL1 TFs are not indispensable components of ethylene response. Thus, kinetic studies distinguish two phases of ethylene-induced growth inhibition of the hypocotyl in etiolated Arabidopsis seedlings: a transient phase I (up to 2 h) and a sustained phase II. Both phases require EIN2 function, while only the second requires EIN3/EIL1. Intriguingly, unlike etiolated seedlings, light-grown ein3 eil1 double mutants do not demonstrate the total loss of long-term ethylene responses. Moreover, osmotic stress-induced cell cycle arrest in leaf primordia that coincides with enhanced activation of the ethylene signal is EIN3 independent. These observations favor the existence of an alternative pathway. One possible candidate to promote such regulation is a PAM domain-containing protein EER5. It negatively regulates ethylene signaling during hypocotyl elongation in etiolated seedlings regardless of EIN3 by promoting downregulation of a gene subset upon ethylene treatment. In addition, it physically interacts with EIN2-C. EER5 regulates magnitude of ethylene response via perception of ERS1 signal.

22Ethylene response is a target for a complex regulation, in which EIN3/EIL1 TFs play a crucial role. Recent studies shed light on multiple layers of complexity in tuning EIN3/EIL1 function (including epigenetic gene silencing and modulation of EIN3/EIL1 stability and activity via PPIs) that facilitate the “shaping” of ethylene response according to spatiotemporal and environmental conditions. At the same time, these findings open up new perspectives for further research. Growing evidence of the important role that epigenetic landscape plays in EIN3/EIL1 functioning requires its more detailed characterization. Particularly, the contribution of distinct epigenetic modifications as well as ENAP1 patterns in modulation of EIN3/EIL1 function is of interest. In view of interdependent cooperation of EIN3/EIL1 with some TFs described recently, the detailed analysis of nucleotide context surrounding EIN3 binding sites requires more attention, and genome-wide research appears helpful both to generalize resent findings and to predict new connections. Moreover, it would be interesting to clarify the role of epigenetic regulation and PPIs in suppression of gene expression upon ethylene treatment.

23Yet, despite their essential role, EIN3/EIL1 are not indispensable regulators of ethylene response. To couple the molecular events and phenotypic responses more precisely, EIL2 function in ethylene signaling and EIN3/EIL independent pathways are to be elucidated.

24VAD and EMP performed the literature search and drafted the paper. VAD performed the analysis of the transcriptome datasets. EVZ revised and edited the manuscript. All authors read and approved the final manuscript.

25The work was funded by the Russian Foundation for Basic Research through grant № 18-29-13040. Meta-analysis of the transcriptome datasets was done in the frame of the project supported by the Russian Foundation for Basic Research and the government of Novosibirsk region through grant № 18-44-540039.

26The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.