The gaseous plant hormone ethylene stimulates cambial division and modifies xylem differentiation in trees, but the molecular chain linking ethylene to wood formation is only partially described. A 2018 expression-network study of the aspen (Populus) wood-forming zone mapped all ethylene-biosynthesis and signaling genes across xylem development, identified the transcription factors EIN3D and eleven ERFs as network hubs, and left a precise set of unresolved mechanistic questions: how the ethylene precursor ACC is distributed laterally through woody tissues and whether transport can explain the widespread ethylene-related transcriptional activity; the in-vivo functional relevance of the unusual split Populus CTR1 family; the direct role, downstream targets, and protein-level mode of action of EIN3D during wood formation; the functional contributions of the ERF hubs to xylem expansion and secondary cell wall formation, including the division of labor between EIN3- and ERF-mediated regulation; and whether these transcription factors act during normal growth, under stress, or both. No resolution of these specific questions is known in the literature to date.

Background

Wood (secondary xylem) of trees is produced by the vascular cambium, a cylindrical meristem whose periclinal divisions generate xylem inward and phloem outward. Differentiating xylem cells pass through spatially ordered phases — cell division, cell expansion, secondary cell wall (SCW) thickening (deposition of cellulose, hemicellulose and lignin), and programmed cell death — and the high-spatial-resolution AspWood transcriptome database, built from a cryosection series across the wood-forming zone of aspen (Populus tremula) stems, resolves the gene-expression profile of each phase (Sundell et al., 2017).

Ethylene is a gaseous phytohormone that controls many aspects of plant growth and stress responses. Its biosynthesis converts methionine via S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS), and ACC to ethylene by ACC oxidase (ACO); ACC itself is a transported metabolite, moved between cells by amino-acid transporters of the LHT (lysine histidine transporter) family identified in Arabidopsis (Shin et al., 2015). Ethylene is perceived by ER-membrane receptors (ETR family); in the absence of ethylene, the receptor-associated kinase CTR1 (Constitutive Triple Response 1) suppresses signaling, while ethylene binding relieves this repression, allowing cleavage of the EIN2 protein and stabilization of EIN3/EIL transcription factors, which directly activate ethylene-response genes, including genes encoding ERF (ethylene response factor) transcription factors that carry the transcriptional reprogramming further. In seedlings, sustained ethylene produces the 'triple response' (shortened hypocotyl and root, exaggerated apical hook), a standard assay for ethylene signaling; the ein3-1 mutant of Arabidopsis lacks this response and can be complemented by functional EIN3 proteins.

Ethylene acts as an endogenous stimulator of cambial cell division in Populus (Love et al., 2009), and ethylene biosynthesis is spatially regulated during tension wood formation — the special corrective xylem that displaced stems form on their upper side, characterized by cellulose-rich gelatinous (G-)fibers (Andersson-Gunnerås et al., 2003). Notably, ACC was reported to be distributed laterally in an almost ubiquitous pattern across Populus woody tissues, whereas the expression of ACS genes is confined to specific zones, raising the question of how ACC spreads.

Using the AspWood resource, Seyfferth et al. (2018) charted the whole ethylene pathway in Populus. Their findings define the open mechanistic questions. First, the potentially enzymatically active ACS genes (ACS3, ACS4) are expressed mainly in expanding xylem and late xylem maturation, while ACO transcripts occur across all zones, so ubiquitous ethylene synthesis would require lateral ACC movement whose mechanism (e.g. via the seven Populus LHT genes expressed in the wood-forming zones) is untested. Second, the Populus CTR1 family is expanded to five members, including CTR1D, encoding only the receptor-interacting N-terminus, and CTR1E, encoding only the kinase-containing C-terminus — a 'bifurcation' whose functional consequence in vivo is unknown, given that both termini are required for CTR1's suppressor function in Arabidopsis. Third, network-centrality analysis of the wood transcriptome (betweenness and closeness of co-expressed genes) identified EIN3D and eleven ERFs as hub transcription factors (the eight best-connected hubs being ERF27, ERF49, ERF75, ERF76, ERF83, ERF87, ERF118 and ERF119), with ERF118 connected to xylem cell expansion and ERF119 to SCW formation. EIN3D could restore the ethylene triple response of the Arabidopsis ein3-1 mutant, proving it is a functional EIN3, but its role in Populus wood itself is untested. At the time, the only functional evidence touching the hubs was for an ERF118 homolog (PsnSHN2, which modulates SCW-related transcription factors and wall properties; Liu et al., 2017) and for ERF76, which confers salt tolerance in transgenic tobacco (Yao et al., 2016) — evidence outside normal wood development. A prior genome-wide screen had also identified ethylene-induced ERFs that modify stem growth and wood properties when overexpressed (Vahala et al., 2013), without resolving their endogenous functions or targets. Finally, co-expression modules suggested that EIN3 control of ERFs might be prominent mainly under stress, when ethylene levels rise, leaving the growth-versus-stress context of the ethylene-responsive wood regulators undecided.

Problem Statement

Establish the mechanistic basis of ethylene action during wood formation in Populus, by resolving the linked open questions posed by Seyfferth et al. (2018): (i) by what mechanism is ACC distributed laterally through woody tissues, and are ACC transport processes sufficient to account for the apparently ubiquitous ethylene-related transcriptional activity across all zones of wood formation, given the zone-restricted expression of ACC synthase genes and the near-ubiquitous distribution of ACC and of ACO transcripts? (ii) What is the in-vivo functional relevance of the bifurcated Populus CTR1 family, in which CTR1D and CTR1E encode only the N-terminal or C-terminal portion of the kinase, and does this bifurcation occur and matter in other species? (iii) What is the direct role of EIN3D during Populus wood formation — its downstream targets and its protein-level mode of action — beyond its demonstrated capacity to complement the Arabidopsis ein3-1 triple-response defect? (iv) What are the specific functional contributions of the ERF hub transcription factors — the eight best-connected hubs ERF27, ERF49, ERF75, ERF76, ERF83, ERF87, ERF118 and ERF119, which constitute the complete required hub set — to xylem cell expansion, secondary cell wall formation and defense-associated transcription during normal wood development, and how is regulation divided between EIN3-mediated and ERF-mediated branches? (v) Do these ethylene-responsive transcription factors act primarily during normal growth, under stress conditions such as tension wood induction, or in both contexts?

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