Comparative regulomics across six tree species (three dicots and three conifers, spanning roughly 300 million years of divergence) predicts that conifer wood formation differs from the dicot program at two linked molecular levels: cell-wall polysaccharide acetylation and transcriptional regulation of secondary-cell-wall formation. Conifer genomes lack the dicot xyloglucan- and xylan-related TBL O-acetyltransferases but retain co-expressed pectin- and glucomannan-related TBLs whose activity in conifer walls has never been biochemically verified, and xyloglucan acetylation was recently shown to be almost absent from embryonic Norway spruce tissue, with an unexplained trace signal. The single-copy NST master regulator, which tops the dicot secondary-wall cascade, shows an expression dip during secondary-wall formation in conifers, and the inferred spruce regulatory network is flatter, with MYB83 as the leading predicted regulator of cellulose-synthase genes. The open problem is the experimental determination of these predicted divergences: which polysaccharide acetylations actually occur in conifer wood and which enzymes catalyze them, whether NSTs have lost their regulatory role in conifers — and if so, which transcription factors regulate secondary-wall genes in their place, MYB83 being the leading predicted candidate — and whether bottom-layer MYBs coordinate secondary-wall formation with programmed cell death, as the aspen candidate network predicts.

Background

Wood, botanically the secondary xylem, is the lignified tissue trees lay down each year from the vascular cambium, a cylindrical stem-cell zone in the stem. Cambial daughter cells expand, deposit a thick secondary cell wall (SCW) rich in cellulose, hemicelluloses and the aromatic polymer lignin, and then die by programmed cell death (PCD), leaving hollow water-conducting cells. Dicot (hardwood) trees form both wide vessels and supporting fibers, whereas conifers (softwoods) form only tracheids, cells that combine conductance and mechanical support; the two lineages diverged about 300 million years ago.

The polysaccharide matrix of the wall differs between the lineages. In dicots the dominant hemicellulose is glucuronoxylan, which is O-acetylated, accompanied by smaller amounts of glucomannan and xyloglucan; in conifers the main hemicellulose is acetylated (galacto)glucomannan, while the less abundant arabinoglucuronoxylan is non-acetylated. O-acetyl ester groups on wall polysaccharides tune hydration, porosity, enzymatic accessibility and interactions with cellulose. The enzymes that add these groups are Golgi-localized proteins of the DUF231/trichome birefringence-like (TBL) family, each with narrow substrate specificity. In Arabidopsis thaliana, AXY4 and AXY4L acetylate xyloglucan, ESK1/TBL29 and relatives acetylate xylan, TBL25 acetylates mannan, and PMR5/TBL44 together with the POAT suite (biochemically characterized only in 2024) acetylates the pectins homogalacturonan (HG) and rhamnogalacturonan-I (RG-I), the two major pectin classes of primary walls. All of this enzyme biochemistry was established in angiosperms or, for evolutionary work, in charophyte algae and liverwort; no conifer TBL protein has been characterized, and whether pectin or residual xyloglucan acetylation occurs in conifer wood at all has, per the source study, not previously been analyzed.

Transcriptional control of SCW formation was also established in Arabidopsis as a three-layer cascade: top-layer NAC-domain master switches — VASCULAR-RELATED NAC-DOMAIN proteins (VND1–7) acting in vessels and NAC SECONDARY WALL THICKENING PROMOTING FACTORs (NST1–3) acting in fibers — activate middle-layer R2R3-MYB factors MYB46 and MYB83, which in turn activate bottom-layer MYBs and the SCW biosynthetic genes such as the cellulose synthases (CesAs). Whether this circuit operates in conifer wood has been an open evolutionary question: earlier high-resolution expression resources for aspen (AspWood) and Norway spruce (NorWood) already noted that the spruce NST ortholog does not peak during SCW formation.

The source study — Rodriguez et al., first posted as a preprint in June 2025 and published in Nature Communications in July 2026 — provides the first high-spatial-resolution comparative wood transcriptomes of three dicots (aspen, birch, cherry) and three conifers (Norway spruce, Scots pine, lodgepole pine), analyzed through orthology-aware co-expression networks (so-called co-expressologs, pairs of orthologous genes with conserved co-expression context), Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq, which maps open chromatin) in aspen and spruce, and inferred gene regulatory networks. It reports that TBLs responsible for xylan and xyloglucan acetylation lack conifer orthologs while pectin-related TBLs (TBL12, PMR5/TBL44) and glucomannan TBL25 are conserved and co-expressed; that the single-copy conifer NST shows an expression dip during SCW formation; that VND/NST binding motifs are absent from the regulatory regions of spruce MYB46/83; that the spruce network is flatter, with MYB83 the strongest predicted regulator of SCW CesA genes; and that three bottom-layer MYBs are predicted in aspen to also regulate the mature-xylem module containing PCD markers. A new enzymatic-fingerprinting experiment (LC–MS of xyloglucanase-released oligosaccharides) showed acetylated xyloglucan fragments abundant in aspen but almost absent from embryonic spruce tissue, with only a trace of one acetylated fragment. The authors themselves classify the regulatory networks as species-specific candidate networks, and every finding above on the conifer side remains a prediction awaiting direct biochemical or genetic validation.

Problem Statement

Determine experimentally, in wood-forming tissue, the molecular basis of the divergences that comparative regulomics predicted between conifer and dicot wood formation. The source study presents three separate open items — needs for further analysis and candidate-network predictions awaiting experimental validation — whose common objective is to establish which cell-wall chemistry and which transcriptional circuit actually operate in conifer secondary xylem, replacing the predicted (candidate) picture with direct evidence. (1) Polysaccharide acetylation: establish which cell-wall polysaccharide O-acetylations occur in conifer wood — in particular whether pectin (HG and RG-I) acetylation occurs, and whether any residual xyloglucan acetylation beyond the trace detected in embryonic Norway spruce tissue exists in secondary xylem — and identify the enzymes that catalyze them; the conifer TBL candidates (the TBL12, TBL25 and PMR5/TBL44 orthologs and any others) are present and co-expressed but biochemically uncharacterized. (2) Master-regulator status: establish whether the NST-clade NAC master regulators have lost their role in controlling secondary-cell-wall formation in conifers, and test which transcription factor(s) regulate spruce SCW CesA and other secondary-wall genes in their place, for which MYB83 is the leading network-predicted candidate. (3) SCW–cell-death coordination: establish whether bottom-layer MYB transcription factors directly coordinate secondary-cell-wall formation with programmed cell death and lignification, as predicted by the aspen (Populus tremula) candidate network, in which three bottom-layer MYBs are predicted to regulate the mature-xylem module containing the cell-death marker gene; this item is validated where the source makes the prediction — in aspen (Populus tremula) wood-forming tissue — with no conifer arm, since the source makes no corresponding prediction for spruce, where the secondary-cell-wall and cell-death modules are merged.

References

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