New Phytologist

Arbuscular mycorrhizal fungi (AMF) are widespread plant symbionts that enhance nutrient acquisition, growth, and stress tolerance, yet plant responsiveness to AMF varies substantially and remains difficult to predict. We developed Trait2Myco, a Random Forest based machine-learning framework that integrates plant functional traits and environmental variables to predict mycorrhizal growth response (MGR). The framework was trained using 80 observations spanning four grass species grown under four precipitation regimes. Predictor variables included leaf and root economic traits together with precipitation, while total MGR served as the response variable. A full model incorporating ten functional traits and precipitation was trained using 80% of observations and evaluated on an independent testing dataset (20%, n = 16). The full model explained 41% of variation in MGR (R2 = 0.41), whereas a reduced ecological model containing only specific leaf area, specific root area, root carbon concentration, and precipitation improved predictive performance and explained 51.7% of variation (R2 = 0.517). Ten-fold cross-validation of the full model yielded an R2 of 0.983, RMSE of 0.046, and MAE of 0.036. Variable importance analysis identified specific leaf area, precipitation, root carbon concentration, and specific root area as the strongest predictors of responsiveness. Partial dependence analysis revealed nonlinear relationships between these predictors and MGR, while principal component analysis showed that highly responsive plants occupied distinct regions of functional trait space characterized by resource-acquisitive trait syndromes. Leaf and root traits contributed nearly equally to model performance, accounting for 41.9% and 41.3% of cumulative importance, respectively, while environmental factors contributed 16.8%. An exploratory analysis using an independent AMF colonization dataset produced similarly strong predictive relationships, suggesting that trait-based machine-learning approaches may generalize across multiple AMF-associated responses. Trait2Myco provides a reproducible framework for identifying plant trait syndromes associated with responsiveness to AMF and offers a practical tool for predicting mycorrhizal benefits across environmental gradients.

Background and aims: Perennial root systems develop over decades and exhibit continuous turnover of structural and absorptive components, yet most root architecture models treat woody roots as permanent and neglect root age structure. We developed an empirically constrained modelling framework that explicitly represents age-structured demography, growth regulation and dormancy in perennial root systems. Methods: We extended the functional-structural plant model CPlantBox to simulate perennial root development over decades by integrating age-dependent survival, secondary radial growth and climate-driven dormancy. The model was parameterised using unique three-dimensional root system data from young and mature grapevines, together with dendrochronological root age measurements, and coupled to root hydraulic properties. Key results: Empirical analyses revealed multi-cohort age distributions and substantial turnover of lignified structural roots, contradicting the assumption of a static woody scaffold. The model reproduced age distributions, order-specific survival probabilities and fine-to-structural root length ratios, and generated realistic, genotype-specific three-dimensional architectures. Simulated uptake profiles and hydraulic scenarios showed strong age- and architecture-driven effects. Conclusions: Explicitly representing woody root age distributions and age-dependent processes may fundamentally change predictions of perennial root system architecture and function. The framework advances root modelling beyond annual paradigms and provides a transferable tool for analysing long-term architectural and hydraulic consequences of genotype x climate interactions in perennial crops.

Photosynthesis is a key determinant of crop productivity, yet conventional gas-exchange measurements are labor-intensive and unsuitable for high-throughput assessment of dynamic photosynthetic acclimation across large genotype panels. Consequently, genetic variation in photosynthetic acclimation remains poorly characterized and difficult to exploit in breeding programs. Here we present a computational pipeline that integrates rapid optical sensing (chlorophyll meters and hyperspectral reflectance) with a mechanistic model of photosynthetic protein-turnover to estimate dynamic nitrogen allocation among photosynthetic components. The pipeline was demonstrated using 60 winter wheat (Triticum aestivum L.) cultivars sampled at 10 time points spanning leaf emergence to senescence. Uncertainties associated with each pipeline component were quantified and benchmarked against gas-exchange ground-truth measurements under three controlled-environment light and temperature regimes. Dynamic nitrogen allocation to light harvesting, electron transport, and carboxylation was characterized using three biologically interpretable parameters: maximum synthesis rate, degradation rate, and age-dependent decline in synthesis. Although hyperspectral models showed moderate predictive accuracy for photosynthetic capacity, prediction errors were predominantly random, allowing robust parameter estimation when observations across the leaf lifespan were integrated. The pipeline successfully resolved genotype-by-environment interactions in photosynthetic acclimation, with environmental and interaction effects contributing more strongly than genotype main effects to nitrogen dynamics. These results demonstrate a scalable, uncertainty-aware sensor-to-trait architecture for dynamic physiological phenotyping that enables high-throughput germplasm screening across diverse environments and provides novel physiological selection targets for improving crop adaptation under variable climates.

Understanding how crop wild relatives (CWR) adapt to diverse environments is important both for evolutionary biology and for the development of climate-resilient agriculture. CWR represent an important reservoir of adaptive variation that can be exploited for future crop improvement. To investigate how natural selection has shaped adaptation in these species, we analysed whole-transcriptome data from four diploid wild wheat relatives with contrasting mating systems: the outcrossing species Aegilops speltoides and Aegilops mutica, and the selfing species Aegilops tauschii and Triticum urartu. We combined the imputed McDonald--Kreitman test (MKT) with lineage-specific estimates of divergence obtained from branch-specific models of molecular evolution. This framework reduces biases caused by slightly deleterious mutations and enables adaptive substitutions to be assigned to individual evolutionary lineages. We detected marked differences among species in the prevalence of positive selection. Evidence of adaptive evolution was concentrated in the outcrossing species, whereas few positively selected genes were identified in the selfing species. Comparisons between standard and branch-specific MKT approaches showed that some signals initially inferred as shared among species were reassigned to individual lineages when divergence was estimated on a branch-specific basis, emphasizing the importance of accurately localizing adaptive substitutions. Functional annotation revealed several candidate genes containing domains associated with abiotic and biotic stress responses. Our study provides a framework for detecting positive selection in wild wheat relatives and identifies candidate genes that may contribute to adaptation and represent valuable targets for future functional analyses and wheat improvement programs.

Pigment composition and regulation are critical for efficient photosynthesis in the ocean, where light intensity decreases and the spectrum narrows with depth. Fucoxanthin (Fx), the main carotenoid of several microalgal lineages, harvests the blue-green light prevailing in the deep euphotic zone. In pelagophytes, the acylated derivative 19'-butanoyloxyfucoxanthin (19'-BFx) is abundant, yet its function and regulation remain unclear. Here, we investigated how irradiance and spectral quality shape photoacclimation in Pelagomonas calceolata, an abundant and cosmopolitan low-light pelagophyte. Cultures were grown under blue or white light across 2-60 umol photons m^-2 s^-1. We analysed growth, PSII photophysiology, pigment composition, gene expression levels, and the impact of spectral shifts on the 19'-BFx/Fx ratio. Pelagomonas calceolata grew optimally under dim blue light and displayed low non-photochemical quenching (NPQ) under assay conditions. Transcriptomes revealed broad remodelling driven mainly by irradiance and modulated by light colour, including carotenoid-related processes. Dim blue light progressively increased the 19'-BFx/Fx ratio, whereas white light kept it low and reversed the response after a blue-to-white shift. These results identify reversible, spectrum-dependent 19'-BFx accumulation as a key component of photoacclimation in P. calceolata and separate irradiance-driven from spectrum-dependent cellular responses. This plasticity may explain the ecological success of P. calceolata in low-light oceans.

Forests regulate climate and sustain biodiversity, but their resilience is increasingly threatened by drought-induced hydraulic failure, when xylem water transport is impaired by embolism. A critical but poorly constrained determinant of this risk largely due to limited observations of deep root water uptake is the amount of root-accessible (sub)soil water storage (SR), which governs land-atmosphere exchanges during prolonged dry periods. While ecohydrological theory has long suggested a balance between soil water storage in the rooting zone, vegetation growth and drought tolerance, this concept has not yet been integrated into predictive models of hydraulic failure. Here we propose a novel, process-based inversion framework that integrates ecohydrological optimality theory with principles of plant hydraulic to infer SR. Using the SurEau plant hydraulic model, we estimate the SR value that balances the costs of soil exploration with the avoidance of drought-induced hydraulic damage. Applied first to a well-instrumented Mediterranean Quercus ilex forest, the inferred SR closely matched independent neutron probe and eddy covariance measurements and accurately reproduced observed drought responses, including leaf water potential and sap flow dynamics. We then scaled the approach across European forests remotely-sensed data, to assess spatially explicit SR estimates and associated hydraulic failure risk. Across more than 20 species and sites, inferred SR and drought stress metrics aligned with field observations and outperformed estimates based on conventional soil databases or remote-sensing-only approaches. By linking plant physiology and ecohydrological theory within a scalable inversion framework, this approach improves predictions of forest drought risk under climate change.

Whether phenotypes and environments predict performance under stress is central to ecology and evolution and can reveal species' vulnerability to global change. We sampled Mimulus guttatus range-wide to test whether reproductive, morphological, and physiological traits predict drought responses. Surprisingly, we find that stomatal response, a key mediator of water balance, did not change as drought intensified. Climate, however, predicted survival after turgor loss: plants from regions with large daily relative to seasonal temperature ranges survived wilting longer. Stress responses also depended on life history: where summers are dry, perennials withstood lower water potentials, whereas annuals increased leaf cooling as they wilted. Known trait associations were not fixed: although many baseline traits tracked climate, none predicted drought responses. Drought performance therefore cannot be inferred from traits measured under well-watered conditions. Lacking stomatal regulation, riparian drought escapers like M. guttatus may be vulnerable if shorter growing seasons bring drought before reproduction.

Flower colour is an important component of floral signaling, yet the genetic basis of naturally occurring colour variation remains poorly understood in many non-model plant groups. Here, we compare genome sequences of a normally pigmented and a largely unpigmented individual of Muscari commutatum, a species typically characterized by very dark violet flowers. Both assemblies were approximately 3.5 Gbp in size, with contig N50 values exceeding 130 Mbp and BUSCO completeness of about 99%. Most structural genes of the flavonoid biosynthesis and the corresponding transcriptional regulators were identified in both plants. Strikingly, no flavanone 3-hydroxylase (F3H) gene was detected in the genome sequence of the unpigmented plant, and additional manual searches provided no evidence for a bona fide F3H copy. Comparison of the two genome sequences revealed a lack of local synteny around the F3H locus of the anthocyanin-pigmented plant, suggesting that F3H might have been lost in the unpigmented plant in association with a genomic rearrangement.

The xylem safety-efficiency trade-off is a central paradigm in plant hydraulics, yet empirical support remains inconsistent. In this study, we highlight how this inconsistency may arise from variation in environmental and evolutionary constraints and from differences in observational scale. We analysed 38 diffuse-porous angiosperm and 24 conifer species grown in a common garden experiment to quantify the relationship between embolism resistance (P50) and specific hydraulic conductivity (Ks) across and within species, and its differences between three species groups differing in evolutionary ancestry and eco-hydrological strategy. When pooling over all species, we found evidence for a safety-efficiency trade-off both across and within species. However, this pattern was driven almost exclusively by the mesic broadleaved species. In contrast, the across-species pattern was suppressed in coniferous species due to the evolutionary constraints on efficiency posed by their tracheid-based wood anatomy, while there was no evidence for a trade-off on either level in hydrophilic broadleaved species, which evolved under conditions that pose reduced selective pressure on embolism resistance. Our results demonstrate that the safety-efficiency trade-off is not universal, but emerges only in specific ecological and evolutionary contexts, highlighting the importance of multi-scale analyses and constraint-based frameworks for interpreting plant hydraulic strategies.

BackgroundN6-Methyladenosine (m6A) regulates RNA fate in flowering plants, but the repertoire and expression patterns of m6A-related genes remain incompletely characterized in liverworts. Here, we characterized these gene families in the liverwort Marchantia polymorpha within a comparative framework spanning diverse plant lineages. ResultsA comparative survey of 119 plant genomes using sequence similarity and conserved-domain criteria identified 18 candidate m6A-related loci in M. polymorpha, comprising seven writer-related genes, five AlkB homolog genes, and six YTH- or CPSF30-related genes. Across the surveyed genomes, canonical writer components generally had low copy numbers, whereas AlkB homolog and reader-related families showed greater copy-number variation. Phylogenetic and domain analyses supported the family assignments of the M. polymorpha candidates. Eight of the 18 candidate loci showed detectable collinearity with Marchantia quadrata. Putative promoter analysis identified motifs annotated as hormone-, light-, and stress-responsive, with hormone-responsive motifs accounting for the largest share of predicted sites. Public transcriptome analysis revealed variation in candidate gene expression across developmental stages and tissues. Quantitative reverse-transcription PCR showed higher transcript abundance for all six YTH- or CPSF30-related genes in thalli than in gemmae or gemma cups. Cold, heat, osmotic, salt, and dark treatments elicited gene-specific expression responses. Darkness increased transcript abundance of several candidates, including MpMTA, MpALKBH1A, and MpECT2. A predicted protein-association network suggested links between subsets of the candidates and RNA-processing proteins. ConclusionsThese findings define a candidate m6A-related gene repertoire in M. polymorpha and identify tissue- and stress-responsive genes for targeted functional studies of RNA methylation in this liverwort.

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

- C2 photosynthesis reduces photorespiratory carbon loss by shuttling glycine from mesophyll (M) to bundle sheath (BS) tissues, where glycine decarboxylase (GDC) releases CO2 that accumulates around rubisco. C2 photosynthesis has been considered a constitutive character, but as a metabolic trait, it may acclimate to the rate of photorespiratory glycine production. - We grew eleven species from two eudicot and two grass lineages spanning C3 to C2 phenotypes at 32 {degrees}C days and 200 or 800 {micro}mol mol-1 CO2. Acclimation was assessed using leaf gas exchange, GDC P-protein immunolocalization, M and BS ultrastructure, photosynthetic enzyme activity, and photosynthesis modelling. - Low relative to high growth CO2 enhanced C2-related phenotypes in sub-C2 and C2 species but not C3 or proto-Kranz species. Sub-C2 species positioned chloroplasts closer to mitochondria and had lower modelled BS conductance. C2 species increased rubisco capacity and GDC allocation to the BS. Aspartate aminotransferase activity increased in both sub-C2 and C2 species. - Acclimation to greater photorespiratory conditions strengthens C2 photosynthesis, but only in species whose BS has crossed a threshold of organelle and GDC investment. We hypothesize that acclimation could expose C2-related traits to selection and facilitate the origin of C4 photosynthesis.

Ordinal scoring of plant disease severity by human raters compresses variation in lesion color, size, and number. Inter-rater variability further complicates comparisons and integrated analyses across environments. We developed a low-cost portable imaging chamber to rapidly image large numbers of leaves under standardized lighting, orientation, and backdrops in the field and employed this system to image more than 11,000 leaves across three states. Embeddings from vision encoders predicted human-assigned disease severity scores. No significant GWAS hits were identified using human-assigned or vegetation-index-based disease severity scores, but GWAS using embeddings identified twelve genomic hotspots controlling leaf appearance. Nine were linked to variation in disease symptom severity. Five hotspots corresponded to previously characterized sorghum genes: all three hotspots not linked to disease and two of the nine that were. Roughly one-third of tested embedding--hotspot associations replicated across at least two states, and twenty replicated across all three. eQTL, PheWAS, and large-effect variant analyses identified single candidate genes with plausible mechanistic links to disease symptom severity for six of the seven hotspots not mapping to characterized genes. These results demonstrate the power of combining scalable, standardized leaf imaging with pretrained image encoders to capture genetically controlled variation in diverse disease symptoms that human ordinal scoring misses.

Premise of the study. Phenotypic plasticity is advantageous for many plants that experience environmental fluctuations, but strong selection pressures can establish limits to plasticity. In deciduous trees, the ability to time leaf elongation in response to spring temperatures can extend the length of the growing season for a given year's climate. However, emergence timing may have undergone strong selection by factors like cold temperatures, limiting the capacity of locally adapted populations to adjust via plasticity to new conditions. Methods. Using a nine-year dataset of 659 families of Quercus lobata in two common gardens, we tested how climate of origin and annual temperatures at the gardens affected variation in phenology. We then examined whether leaf emergence timing was under selection in the gardens by testing its effects on growth. Finally, we tested whether among-year plasticity in leaf emergence timing conferred a growth benefit. Key results. Year-to-year environmental variation explained more phenological variation than genetic differences associated with climate of origin, yet trees from cooler climates were more constrained in their ability to track temperature than trees from warmer climates. We found evidence of weak stabilizing and directional selection on phenology, suggesting that strong selection in favor of earlier phenology is unlikely. Finally, we found no evidence that selection has favored increased plasticity in Q. lobata. Conclusions. Phenological plasticity remains a key driver of plants' response to changing temperatures, but it is constrained by natural selection. As temperatures rise, these processes may disproportionately limit the ability of trees adapted to cooler climates to shift their phenology to match spring temperatures.

Understanding how carbon-water coupling scales from leaves to ecosystems remains a major challenge because water-use efficiency (WUE) metrics integrate physiological, hydrological, and ecological processes. We integrated tree-ring width, triple stable isotopes ({delta}13C, {delta}18O and {delta}2H), eddy covariance (EC) observations, and a process-based model to investigate the drivers of intrinsic (iWUE) and ecosystem water-use efficiency (WUEeco) across biological scales in a mature Fagus sylvatica forest between 2004 and 2023. Modelled and isotope-derived iWUE were strongly correlated (r = 0.61, p < 0.05) and both indicated an approximately 20% long-term increase, consistent with increasing gross primary productivity. In contrast, WUEeco derived from EC was lower than model estimates and exhibited a positive trend not reproduced by the model. This divergence primarily reflected differences in biological representation and hydrological integration, as the single-species model underestimated ecosystem evapotranspiration by excluding co-occurring deciduous and evergreen species together with understory vegetation. Triple-isotope analyses showed that the long-term increase in iWUE was driven by enhanced photosynthetic assimilation rather than sustained stomatal closure, whereas {delta}18O and {delta}2H variability was mainly controlled by source-water dynamics. These findings provide a mechanistic framework for reconciling leaf- and ecosystem-scale WUE and improving predictions of forest carbon-water coupling under climate change.

Despite the recent cloning of RLK-encoding resistance genes and the identification of a common point of hyphal arrest at the stomatal level in incompatible interactions, the molecular mechanisms leading to the establishment of wheat resistance against Septoria tritici blotch (STB) remain poorly characterized. Here, we use long-read RNA-sequencing data to compare the early transcriptomic responses of two wheat cultivars carrying major, isolate-specific resistances to Zymoseptoria tritici via differential expression, differential transcript usage (DTU), functional- and pathway enrichment analyses. We show that both magnitude and composition of the responses depend strongly on the combination of host cultivar and pathogen isolate and that DTU appears to be uncommon at this infection stage. We do not detect a set of genes exclusively differentially expressed across incompatible interactions, but genes coding for proteins involved in several defense-related processes consistently show higher expression levels in incompatible than compatible interactions, including receptors and ROS homeostatic, antifungal hydrolytic, phenylpropanoid and diterpenoid biosynthetic enzymes. Our results suggest that whilst interaction-specific differences in magnitude and composition may also shape resistance, different major STB resistances may quantitatively enhance overlapping defense machinery, providing a basis for its functional investigation.

Heritable variation in microbiome composition could theoretically evolve in response to selection acting on hosts, but empirical evidence of this is scarce. We tested for effects of allelic variation in Boechera stricta on microbiome composition and downstream consequences for host fitness. In the greenhouse, near-isogenic lines with contrasting phytochemistry alleles differentially conditioned soil microbiota in ways that changed the relative fecundity of host genotypes. In the field, root microbiome differences mediated allelic effects on host fitness. Selection gradients on individual taxa and multivariate axes of root microbiome composition suggest that independent components of the bacterial community favor each allele, especially under drought. These results show that microbiota contribute to natural selection on a major-effect phytochemistry gene, empirically closing the loop between microbiome heritability and host trait evolution.

Plastidial phosphorylase (Pho1) regulates starch metabolism and also modulates photosystem I (PSI) activity. In rice, Pho1 contains an 80 amino acid intrinsically disordered region (IDR), L80, that acts as a negative regulator. While total removal of the IDR boosts seedling growth, flowering time, overall biomass, and grain yield and alters PSI redox properties, the precise underlying regulatory basis remains unclear. To pinpoint IDR regulatory elements, four Pho1 partial-IDR deletion variants ({delta}N, {delta}C, {delta}M, and {delta}N{delta}C) were evaluated. Lines carrying {delta}M and {delta}C exhibited enhanced starch metabolism, yielding larger, heavier grains, and flowered 5 to10 days earlier than wild type. Both d{delta}M and {delta}C share an acidic motif, (E/V)SEEV, identifying it as a primary negative regulator of starch synthesis and floral timing. By contrast, no partial deletion fully mirrored the PSI redox modifications of Pho1{delta}L80. These results demonstrate that starch biosynthesis and flowering are governed by one or more discrete sequence motifs within the middle and C-terminal regions, whereas PSI modulation likely requires the structural integrity of the intact IDR.

Fusarium oxysporum f. sp. lactucae (Fola), the cause of lettuce fusarium wilt, poses an increasing threat to global production due to the rapid emergence of resistance breaking isolates. However, the evolutionary origins and mechanisms underpinning this diversification remain poorly understood. We characterised 103 Fola isolates using molecular profiling, selecting ten key isolates for long-read whole-genome sequencing and performed in planta virulence assays for selected isolates. This included the first assemblies for Fola2 and Fola3, recent resistance-breaking variants and a highly divergent isolate from France designated as a putative novel race, FolaX. Phylogenomic analysis revealed that Fola is polyphyletic, with lettuce pathogenicity acquired independently across divergent F. oxysporum clades. Despite this, all lineages share a conserved "PanFola" effector set with near-identical sequences, indicating extensive horizontal gene transfer. Resistance-breaking Fola variants within established races were linked to copy number variations and targeted deletions, such as the loss of the SIX8/PSE1 locus in Fola4+. Conversely, FolaX possessed a divergent core genome and a chimeric accessory genome derived from multiple Fola lineages via segmental chromosome transfer and transposon-mediated reshuffling. Our findings highlight the remarkable evolutionary plasticity of Fola and the need for breeding strategies that address the dynamic nature of effector repertoires.

Abstract: Picrotoxane-type sesquiterpene alkaloids, including dendrobine, are characteristic constituents of Dendrobium nobile, but the enzymes that produce their proposed skeletal precursors remain in completely characterized. Here, we combined tissue metabolite profiling with genome-guided screening and biochemical characterization of terpene synthases (TPSs). GC-MS analysis revealed variation in sesquiterpenoid profiles and dendrobine signals among tissues and developmental stages. Manual curation of the chromosome-level genome yielded 16 full-length TPS-a candidates. Functional screening in an FPP-supplying Escherichia coli system showed that three closely related genes on chromosome 1, DnTPS1~DnTPS3, produced copaborneol as their dominant product. The principal product of DnTPS1 was purified and identified by NMR spectroscopy, and a purified-enzyme assay confirmed its direct conversion of FPP to copaborneol. Fluorescence imaging in rice protoplasts and Nicotiana benthamiana leaves indicated predominantly cytosolic localization of DnTPS1. Guided by sequence comparisons and structural modeling, we tested 12 DnTPS1 variants and found that substitutions at W377 and I295 altered the relative distribution of sesquiterpene products. Phylogenetic and regional gene-order analyses placed DnTPS1~DnTPS3 in the same clade and identified related TPS candidates within corresponding genomic regions of six Dendrobium species. These findings establish that three D. nobile TPSs can form copaborneol and identify DnTPS1 residues that influence product selectivity, providing a basis for testing the proposed connection to dendrobine biosynthesis.