Engineered poplar for bioproduction of the triterpene squalene.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

Rapid response of nonstructural carbohydrate allocation and photosynthesis to short photoperiod, low temperature, or elevated CO2 in Pinus strobus.

During autumn, decreasing photoperiod and temperature temporarily perturb the balance between carbon uptake and carbon demand in overwintering plants, requiring coordinated adjustments in photosynthesis and carbon allocation to re-establish homeostasis. Here we examined adjustments of photosynthesis and allocation of nonstructural carbohydrates (NSCs) following a sudden shift to short photoperiod, low temperature, and/or elevated CO2 in Pinus strobus seedlings. Seedlings were initially acclimated to 14 h photoperiod (22/15°C day/night) and ambient CO2 (400 ppm) or elevated CO2 (800 ppm). Seedlings were then shifted to 8 h photoperiod for one of three treatments: no temperature change at ambient CO2 (22/15°C, 400 ppm), low temperature at ambient CO2 (12/5°C, 400 ppm), or no temperature change at elevated CO2 (22/15°C, 800 ppm). Short photoperiod caused all seedlings to exhibit partial nighttime depletion of starch. Short photoperiod alone did not affect photosynthesis. Short photoperiod combined with low temperature caused hexose accumulation and repression of photosynthesis within 24 h, followed by a transient increase in nonphotochemical quenching (NPQ). Under long photoperiod, plants grown under elevated CO2 exhibited significantly higher NSCs and photosynthesis compared to ambient CO2 plants, but carbon uptake exceeded sink capacity, leading to elevated NPQ; carbon sink capacity was restored and NPQ relaxed within 24 h after shift to short photoperiod. Our findings indicate that P. strobus rapidly adjusts NSC allocation, not photosynthesis, to accommodate short photoperiod. However, the combination of short photoperiod and low temperature, or long photoperiod and elevated CO2 disrupts the balance between photosynthesis and carbon sink capacity, resulting in increased NPQ to alleviate excess energy.

Evaluation of engineered low-lignin poplar for conversion into advanced bioproducts.

BACKGROUND: Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. RESULTS: The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. CONCLUSION: Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

A new approach to zip-lignin: 3,4-dihydroxybenzoate is compatible with lignification.

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. This study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of 'zip-lignin' derived from the incorporation of DHB into poplar lignin.

Evolutionary patterns in chemical composition and biomechanics of articulated coralline algae.

Seaweeds inhabiting wave-battered coastlines are generally flexible, bending with the waves to adopt more streamlined shapes and reduce drag. Coralline algae, however, are firmly calcified, existing largely as crusts that avoid drag altogether or as upright branched forms with uncalcified joints (genicula) that confer flexibility to otherwise rigid thalli. Upright corallines have evolved from crustose ancestors independently multiple times, and the repeated evolution of genicula has contributed to the ecological success of articulated corallines worldwide. Structure and development of genicula are significantly different across evolutionary lineages, and yet biomechanical performance is broadly similar. Because chemical composition plays a central role in both calcification and biomechanics, we explored evolutionary trends in cell wall chemistry across crustose and articulated taxa. We compared the carbohydrate content of genicula across convergently-evolved articulated species, as well as the carbohydrate content of calcified tissues from articulated and crustose species, to search for phylogenetic trends in cell wall chemistry during the repeated evolution of articulated taxa. We also analysed the carbohydrate content of one crustose coralline species that evolved from articulated ancestors, allowing us to examine trends in chemistry during this evolutionary reversal and loss of genicula. We found several key differences in carbohydrate content between calcified and uncalcified coralline tissues, though the significance of these differences in relation to the calcification process requires more investigation. Comparisons across a range of articulated and crustose species indicated that carbohydrate chemistry of calcified tissues was generally similar, regardless of morphology or phylogeny; conversely, chemical composition of genicular tissues was different across articulated lineages, suggesting that significantly different biochemical trajectories have led to remarkably similar biomechanical innovations.

Monolignol export by diffusion down a polymerization-induced concentration gradient.

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

pHBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar.

Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins.

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Distinct and Overlapping Functions of Miscanthus sinensis MYB Transcription Factors SCM1 and MYB103 in Lignin Biosynthesis.

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

Cannabis Glandular Trichome Cell Walls Undergo Remodeling to Store Specialized Metabolites.

The valuable cannabinoid and terpenoid metabolites of Cannabis sativa L. are produced by floral glandular trichomes. The trichomes consist of secretory disk cells, which produce the abundant lipidic metabolites, and an extracellular storage cavity. The mechanisms of apoplastic cavity formation to accumulate and store metabolites in cannabis glandular trichomes remain wholly unexplored. Here, we identify key wall components and how they change during cannabis trichome development. While glycome and monosaccharide analyses revealed that glandular trichomes have loosely bound xyloglucans and pectic polysaccharides, quantitative immunolabeling with wall-directed antibodies revealed precise spatiotemporal distributions of cell wall epitopes. An epidermal-like identity of early trichome walls matured into specialized wall domains over development. Cavity biogenesis was marked by separation of the subcuticular wall from the underlying surface wall in a homogalacturonan and α-1,5 arabinan epitope-rich zone and was associated with a reduction in fucosylated xyloglucan epitopes. As the cavity filled, a matrix with arabinogalactan and α-1,5 arabinan epitopes enclosed the metabolite droplets. At maturity, the disk cells' apical wall facing the storage cavity accumulated rhamnogalacturonan-I epitopes near the plasma membrane. Together, these data indicate that cannabis glandular trichomes undergo spatiotemporal remodeling at specific wall subdomains to facilitate storage cavity formation and metabolite storage.

Dwarfism of high-monolignol Arabidopsis plants is rescued by ectopic LACCASE overexpression.

Lignin is a key secondary cell wall chemical constituent, and is both a barrier to biomass utilization and a potential source of bioproducts. The Arabidopsis transcription factors MYB58 and MYB63 have been shown to upregulate gene expression of the general phenylpropanoid and monolignol biosynthetic pathways. The overexpression of these genes also results in dwarfism. The vascular integrity, soluble phenolic profiles, cell wall lignin, and transcriptomes associated with these MYB-overexpressing lines were characterized. Plants with high expression of MYB58 and MYB63 had increased ectopic lignin and the xylem vessels were regular and open, suggesting that the stunted growth is not associated with loss of vascular conductivity. MYB58 and MYB63 overexpression lines had characteristic soluble phenolic profiles with large amounts of monolignol glucosides and sinapoyl esters, but decreased flavonoids. Because loss of function lac4 lac17 mutants also accumulate monolignol glucosides, we hypothesized that LACCASE overexpression might decrease monolignol glucoside levels in the MYB-overexpressing plant lines. When laccases related to lignification (LAC4 or LAC17) were co-overexpressed with MYB63 or MYB58, the dwarf phenotype was rescued. Moreover, the overexpression of either LAC4 or LAC17 led to wild-type monolignol glucoside levels, as well as wild-type lignin levels in the rescued plants. Transcriptomes of the rescued double MYB63-OX/LAC17-OX overexpression lines showed elevated, but attenuated, expression of the MYB63 gene itself and the direct transcriptional targets of MYB63. Contrasting the dwarfism from overabundant monolignol production with dwarfism from lignin mutants provides insight into some of the proposed mechanisms of lignin modification-induced dwarfism.

The uncharacterized gene EVE contributes to vessel element dimensions in Populus.

The radiation of angiosperms led to the emergence of the vast majority of today's plant species and all our major food crops. Their extraordinary diversification occurred in conjunction with the evolution of a more efficient vascular system for the transport of water, composed of vessel elements. The physical dimensions of these water-conducting specialized cells have played a critical role in angiosperm evolution; they determine resistance to water flow, influence photosynthesis rate, and contribute to plant stature. However, the genetic factors that determine their dimensions are unclear. Here we show that a previously uncharacterized gene, ENLARGED VESSEL ELEMENT (EVE), contributes to the dimensions of vessel elements in Populus, impacting hydraulic conductivity. Our data suggest that EVE is localized in the plasma membrane and is involved in potassium uptake of differentiating xylem cells during vessel development. In plants, EVE first emerged in streptophyte algae, but expanded dramatically among vessel-containing angiosperms. The phylogeny, structure and composition of EVE indicates that it may have been involved in an ancient horizontal gene-transfer event.

Learning from methylomes: epigenomic correlates of Populus balsamifera traits based on deep learning models of natural DNA methylation.

Epigenomes have remarkable potential for the estimation of plant traits. This study tested the hypothesis that natural variation in DNA methylation can be used to estimate industrially important traits in a genetically diverse population of Populus balsamifera L. (balsam poplar) trees grown at two common garden sites. Statistical learning experiments enabled by deep learning models revealed that plant traits in novel genotypes can be modelled transparently using small numbers of methylated DNA predictors. Using this approach, tissue type, a nonheritable attribute, from which DNA methylomes were derived was assigned, and provenance, a purely heritable trait and an element of population structure, was determined. Significant proportions of phenotypic variance in quantitative wood traits, including total biomass (57.5%), wood density (40.9%), soluble lignin (25.3%) and cell wall carbohydrate (mannose: 44.8%) contents, were also explained from natural variation in DNA methylation. Modelling plant traits using DNA methylation can capture tissue-specific epigenetic mechanisms underlying plant phenotypes in natural environments. DNA methylation-based models offer new insight into natural epigenetic influence on plants and can be used as a strategy to validate the identity, provenance or quality of agroforestry products.

The in vivo impact of MsLAC1, a Miscanthus laccase isoform, on lignification and lignin composition contrasts with its in vitro substrate preference.

BACKGROUND: Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo. RESULTS: Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4-2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate. CONCLUSIONS: In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.

Analysis of Monosaccharides from Arabidopsis Seed Mucilageand Whole Seeds Using HPAEC-PAD.

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Author Correction: Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Patterned Deposition of Xylan and Lignin is Independent from that of the Secondary Wall Cellulose of Arabidopsis Xylem Vessels.

The secondary cell wall (SCW) of xylem vessel cells provides rigidity and strength that enables efficient water conduction throughout the plant. To gain insight into SCW deposition, we mutagenized Arabidopsis thaliana VASCULAR-RELATED NAC-DOMAIN7-inducible plant lines, in which ectopic protoxylem vessel cell differentiation is synchronously induced. The baculites mutant was isolated based on the absence of helical SCW patterns in ectopically-induced protoxylem vessel cells, and mature baculites plants exhibited an irregular xylem (irx) mutant phenotype in mature plants. A single nucleic acid substitution in the CELLULOSE SYNTHASE SUBUNIT 7 (CESA7) gene in baculites was identified: while the mutation was predicted to produce a C-terminal truncated protein, immunoblot analysis revealed that cesa7bac mutation results in loss of production of CESA7 proteins, indicating that baculites is a novel cesa7 loss-of-function mutant. In cesa7bac , despite a lack of patterned cellulose deposition, the helically-patterned deposition of other SCW components, such as the hemicellulose xylan and the phenolic polymer lignin, was not affected. Similar phenotypes were found in another point mutation mutant cesa7mur10-2 , and an established knock-out mutant, cesa7irx3-4 Taken together, we propose that the spatio-temporal deposition of different SCW components, such as xylan and lignin, is not dependent on cellulose patterning.

Complete substitution of a secondary cell wall with a primary cell wall in Arabidopsis.

The bulk of a plant's biomass, termed secondary cell walls, accumulates in woody xylem tissues and is largely recalcitrant to biochemical degradation and saccharification1. By contrast, primary cell walls, which are chemically distinct, flexible and generally unlignified2, are easier to deconstruct. Thus, engineering certain primary wall characteristics into xylem secondary walls would be interesting to readily exploit biomass for industrial processing. Here, we demonstrated that by expressing AP2/ERF transcription factors from group IIId and IIIe in xylem fibre cells of mutants lacking secondary walls, we could generate plants with thickened cell wall characteristics of primary cell walls in the place of secondary cell walls. These unique, newly formed walls displayed physicochemical and ultrastructural features consistent with primary walls and had gene expression profiles illustrative of primary wall synthesis. These data indicate that the group IIId and IIIe AP2/ERFs are transcription factors regulating primary cell wall deposition and could form the foundation for exchanging one cell wall type for another in plants.

Overexpression of AtGolS3 and CsRFS in poplar enhances ROS tolerance and represses defense response to leaf rust disease.

Plants respond to pathogens through an orchestration of signaling events that coordinate modifications to transcriptional profiles and physiological processes. Resistance to necrotrophic pathogens often requires jasmonic acid, which antagonizes the salicylic acid dependent biotrophic defense response. Recently, myo-inositol has been shown to negatively impact salicylic acid (SA) levels and signaling, while galactinol enhances jasmonic acid (JA)-dependent induced systemic resistance to necrotrophic pathogens. To investigate the function of these compounds in biotrophic pathogen defense, we characterized the defense response of Populus alba × grandidentata overexpressing Arabidopsis GALACTINOL SYNTHASE3 (AtGolS) and Cucumber sativus RAFFINOSE SYNTHASE (CsRFS) challenged with Melampsora aecidiodes, a causative agent of poplar leaf rust disease. Relative to wild-type leaves, the overexpression of AtGolS3 and CsRFS increased accumulation of galactinol and raffinose and led to increased leaf rust infection. During the resistance response, inoculated wild-type leaves displayed reduced levels of galactinol and repressed transcript abundance of two endogenous GolS genes compared to un-inoculated wild-type leaves prior to the up-regulation of NON-EXPRESSOR OF PR1 and PATHOGENESIS-RELATED1. Transcriptome analysis and qRT-PCR validation also revealed the repression of genes participating in calcium influx, phosphatidic acid biosynthesis and signaling, and salicylic acid signaling in the transgenic lines. In contrast, enhanced tolerance to H2O2 and up-regulation of antioxidant biosynthesis genes were exhibited in the overexpression lines. Thus, we conclude that overexpression of AtGolS and CsRFS antagonizes the defense response to poplar leaf rust disease through repressing reactive oxygen species and attenuating calcium and phosphatidic acid signaling events that lead to SA defense.

Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus †.

The evolution of sexual dimorphism and expansion of sex chromosomes are both driven through sexual conflict, arising from differing fitness optima between males and females. Here, we pair work in poplar (Populus) describing one of the smallest sex-determining regions known thus far in complex eukaryotes (~100 kbp) with comprehensive tests for sexual dimorphism using >1300 individuals from two Populus species and assessing 96 non-reproductive functional traits. Against expectation, we found sexual homomorphism (no non-reproductive trait differences between the sexes), suggesting that gender is functionally neutral with respect to non-reproductive features that affect plant survival and fitness. Combined with a small sex-determining region, we infer that sexual conflict may be effectively stymied or non-existent within these taxa. Both sexual homomorphism and the small sex-determining region occur against a background of strong environmental selection and local adaptation in Populus. This presents a powerful hypothesis for the evolution of dioecious species. Here, we suggest that environmental selection may be sufficient to suppress and stymy sexual conflict if it acts orthogonal to sexual selection, thereby placing limitations on the evolution of sexual dimorphism and genomic expansion of sex chromosomes.