Plant Communications

Achieving high-throughput and precise phenotypic quantification and imaging modalities of stomatal and epidermal cells across diverse species remains a primary bottleneck in elucidating the mechanisms of stomatal dynamics, epidermal patterning, and environmental adaptation of plants. Here, we developed EpiReasoner, an artificial intelligence framework comprising a vision module, EpiVision, and a knowledge-based reasoning module, EpiBrain, for the quantitative phenotypic analysis and domain-specific knowledge reasoning of stomatal complexes and pavement cells in plants. Operating across bright-field, scanning electron microscopy, and differential interference contrast modalities, EpiVision achieves precise instance segmentation in various monocotyledonous, dicotyledonous, and fern species. Its performance significantly surpasses current state-of-the-art models. Moreover, we defined 23 quantitative indices describing stomatal cell morphology and spatial distribution. For domain-specific tasks such as phenotype prediction, genotype deduction, and molecular mechanism reasoning, EpiBrain demonstrates a human preference rate significantly higher than that of general-purpose large language models, including GPT-5 and Claude Sonnet 4. The application of EpiReasoner to phenotypic data of stomatal density derived from a tomato natural population of 170 accessions successfully identified a major quantitative trait locus on chromosome 8. The candidate gene, SKP1-interaction partner 19L (SKIP19L), encoding an F-box family protein, exhibited severe allele frequency drift during tomato domestication, which is highly consistent with the adaptive trend of reduced stomatal density under artificial selection. EpiReasoner provides a novel paradigm that unifies visual phenomics and knowledge-driven reasoning for the biology of stomata and pavement cells, thereby significantly accelerating scientific discovery in plant science.

Understanding how gene families evolve can offer great insight into adaptation at the phenotypic and ecological levels. This is particularly true in plants, where transcription factor gene families are often targeted for breeding programs to improve the agronomic traits of economically important crops. While recent advances in next generation sequencing have accelerated the wealth of genomics data, there remains a lack of accessible and reproducible genome mining pipelines tailored for gene family characterisation. Here, we address this gap by developing GENE-FAM, an automated, scalable and open-source pipeline designed to mine and predict gene families based on conserved domains and motifs. To illustrate its application, we apply GENE-FAM to annotate MADS-box transcription factor genes across multiple Cannabis sativa genomes. A comprehensive set of MADS-box genes was identified across three C. sativa cultivars, including both previously annotated and newly predicted genes. Through phylogenetic analyses, we confirm that all type II MADS-box gene subfamilies represented in flowering plants are present in C. sativa. Comparing our annotations with those of Arabidopsis thaliana and Solanum lycopersicum revealed that while most MADS type II families are highly conserved, SEPALLATA-like genes have undergone diversification in C. sativa. Together, these results demonstrate the application of GENE-FAM for genome-wide identification and characterisation of gene families in non-model species, revealing novel insights into MADS-box gene family evolution in C. sativa.

RNA-seq datasets from medicinal yews are crucial for studying paclitaxel biosynthesis. However, cross-study data analyses are hindered by pronounced batch effects. Here, we compiled 45 RNA-seq samples from three studies across four tissues (bark, leaf, root, stem) and assessed 35 preprocessing pipelines combining six normalization strategies with five batch-effect correction approaches. Unsupervised clustering (HCA, k-means, Grade-of-Membership), evaluated using Jaccard and Adjusted Rand indices, revealed significant variability in batch effect removal. Supervised classification of tissue and project labels (Random Forest and linear/radial SVM) demonstrated improved accuracy in tissue type prediction, highlighting the effectiveness of correction methods. The processed data facilitated the identification of 189 putative ABC transporters across samples, six of which showing a strong correlation to the gene encoding 10-deacetylbaccatin-III-10{beta}-O-acetyltransferase, a key biosynthetic enzyme in the taxol pathway. High expression levels in leaf and bark further support their role in taxane intermediates trafficking in taxol biosynthesis. Structural analysis and molecular docking further supported the selection of these candidates, and the agreement between transcriptomic ranking and docking-based prioritization suggests that these transporters may participate in taxane intermediate recognition, trafficking, or export. These findings demonstrate the importance of normalization and batch effect correction in RNA-seq analysis to advance gene discovery in Taxus species and, more broadly, in plant research. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/723993v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1469162org.highwire.dtl.DTLVardef@1f2c4deorg.highwire.dtl.DTLVardef@15ad821org.highwire.dtl.DTLVardef@123676d_HPS_FORMAT_FIGEXP M_FIG C_FIG

The functional annotation of plant genes lags significantly behind their genomic annotation. Closing this gap requires thorough cataloging of reported protein activities alongside predictive methods that scale beyond sequence-similarity inference. Focusing on the BAHD acyltransferase enzyme family as a model, we assembled FuncZymeDB-BAHD, a large database of 2,705 LLM-retrieved and curated enzyme-acceptor-donor activities covering 336 BAHDs from 156 plant species, a 2-to-6-fold expansion over Swiss-Prot and prior compilations. We further developed FuncPred-OG, which maps queries to orthologous groups and previously characterized enzymes in FuncZymeDB-BAHD, returning hits with high evidence provenance. FuncPred-OG enabled functional prediction of over half of BAHDs across 85 plant proteomes, of which five novel predictions were validated via in vitro assays and recent studies. For the remaining BAHDs without FuncPred-OG annotation, we developed FuncPred-AI, where logistic-regression classifiers trained on protein language model embeddings achieved high Area-Under-the-Precision-Recall-curve (AUPR) scores and correct-hit rates up to 93%. FuncPred-AI yielded [≥]1 probable donor/acceptor annotation for 99.9% (8894/8897) of BAHDs in our pan-plant dataset. Finally, the FuncPred workflow and datasets were deployed on a web portal for broader utilization, potentially reducing experimentalists efforts for selecting candidates from days to minutes. Overall, this framework provides a generalizable template for functional annotation of entire enzyme families.

The prevalence of naturally occurring C13-acetoxy taxanes, together with the presence of a native C13-acetyltransferase in yew trees, suggests that the natural biosynthetic pathway for paclitaxel may involve a cryptic C13-O-deacetylation step. However, whether a putative taxane C13-O-deacetylase (T13dA) acts in the pathway of paclitaxel biosynthesis remains elusive. Here we functionally characterized two novel taxane C13-O-deacetylases (T13dA1 and T13dA2) from Taxus x media cell cultures, providing experimental evidence for the molecular and biochemical plausibility of C13-O-deacetylation in paclitaxel biosynthesis in Taxus species. Also, we identified a previously uncharacterized bifunctional taxane C7{beta}-O-, C9-O-deacetylase, designated T79dA, which demonstrates the functional promiscuity by enabling stepwise deacetylation at taxane C7{beta} and C9 positions in a single enzymatic reaction. Furthermore, T7dA1, a novel taxane C7{beta}-O-deacetylase with higher activity than the reported T7dA was discovered and characterized here. Moreover, we reconstituted two new pathways (an 18-gene and a 19-gene pathway) enabled by the integration of a C13-O-acetylation-deacetylation module for the de novo biosynthesis of baccatin III in Nicotiana benthamiana leaves. These pathways with the previously established 17-gene baccatin III pathway, further allow paclitaxel biosynthesis to be a network. Our reconstituted 19-gene pathway achieves a baccatin III yield of up to 23 g g-1 dried weight (DW) in N. benthamiana leaves, which is comparable to the yield reported for the 17-gene pathway. This work facilitates a better understanding, elucidation and reconstruction of metabolic network of paclitaxel biosynthetic pathway, and provides new enzymes and strategies for artificial pathway reconstruction and efficiently bio-chemical production of paclitaxel.

Auxin is a key phytohormone that regulates all aspects of plant growth, development, and environmental responses, making the precise analysis of its distribution and signaling essential for understanding plant adaptation and physiological processes. However, despite the agricultural importance of oilseed rape (Brassica napus), the lack of robust, species-specific molecular tools limits detailed studies of hormone signaling in this crop. Here, we developed and characterized reporter systems for the sensitive visualization and quantification of auxin distribution and signaling in B. napus. The DR5cc auxin signaling reporter and a novel synthetic auxin-responsive reporter, BIP3, assembled from promoter fragments of three oilseed rape IAA genes, were generated to drive GUS expression. In hairy roots, both reporters showed auxin-responsive expression in the root apical meristem that became broader after auxin treatment. In transgenic seedlings, flowers at anthesis, and 12-day-old embryos, DR5cc exhibited a more defined expression pattern than BIP3. To monitor real-time auxin dynamics under abiotic stress, DR5cc fluorescent reporters were employed in hairy roots. Mannitol and NaCl treatments induced a time-dependent increase in fluorescence, peaking at 6-12 h before returning to basal levels after 24 h. Furthermore, dual-reporter assays enabled simultaneous monitoring of auxin and cytokinin signaling, revealing distinct hormone-specific spatial responses in hairy roots. Finally, we established a quantitative DII (qDII) reporter system using degron domains from B. napus Aux/IAA proteins, providing a high-resolution quantitative readout of auxin depletion. Together, these reporter systems enable spatial, temporal, and quantitative analyses of auxin dynamics during development and stress adaptation in oilseed rape.

Legumes include some of the worlds most significant crop species, such as cowpea (Vigna unguiculata), a subsistence crop widely grown in sub-Saharan Africa. Despite their importance, legume crop improvement is hindered by a lack of high-resolution expression data, particularly for reproductive tissues and cell types. Here, we report on VigExp, a tool for visualising cowpea gene expression datasets. We demonstrate its utility across a range of vegetative and reproductive cell types of varieties IT97K-499-35 and IT86D-1010, which exhibit 93.75% protein sequence conservation and are amenable to stable transformation. This includes previously published transcriptomes of vegetative, floral and seed tissues, combined with developmentally staged male and female reproductive tissues. Also integrated are novel transcriptomes of laser-captured cell types covering reproductive development from meiosis to early embryo formation post-fertilisation. Spatial expression patterns and transcript levels can be visualised through an electronic fluorescent pictograph (eFP) browser. Validated by RT-qPCR, in situ hybridisation, transgenic, and CRISPR gene editing analyses, the predictive accuracy of VigExp matches prior cowpea functional study observations. Critical genes for nodule development and regulation were also identified and their expression patterns established in cowpea. Novel reference genes, constitutively expressed gene promoters for visualization makers/gene-editing, and tissue and cell specific gene promoters for targeting these regions, are identified. The A-type cyclin, VuTAM2, was also identified, with a critical role in male meiosis established. Collectively, VigExp represents an adaptable and updatable resource to support crop improvement in cowpea and other legumes, which are often highly syntenic with respect to genome composition.

Isoflavone hydroxylases (IFHs, CYP81E) convert isoflavone aglycones into their respective hydroxylated intermediates, which direct legume isoflavones into specialized defense pathways. In soybean, their functions have been studied mostly in the context of the daidzein-derived glyceollin biosynthesis. Here we combine metabolomics-guided feature mining, phylogenetic analysis, heterologous enzymology, structural elucidation, and in planta metabolite validation to determine the functional landscape of the soybean IFH family. Analysis of a soybean isoflavonoid-enriched metabolomic dataset revealed unidentified hydroxyisoflavone features that co-accumulated with glyceollins, indicating branch chemistry that is not well-recognized. The systematic characterization of the repertoire of soybean CYP81E has demonstrated that 9 out of 11 GmIFHs are catalytically active and collectively span both 2'- and 3'- hydroxylation of the major soybean isoflavone aglycones. Among them, GmIFH9A showed broad substrate scope and regioselectivity, yielding canonical and previously unknown hydroxylated isoflavone products. NMR and LC-MS/MS were used to identify and validate the hydroxylated isoflavone products as 2'-hydroxyglycitein and 2'-hydroxyformononetin, whose presence was also confirmed in soybean roots, thus confirming two of the hidden soybean isoflavonoid network metabolites. Kinetic studies also indicated that, although the majority of GmIFHs prefer daidzein and genistein as substrates, a few isoforms are active towards methoxylated isoflavones as well, indicating functional divergence in this expanded family. Our findings collectively redefine soybean IFHs as a multi-functional enzyme module that expands the hydroxyisoflavone chemical space and reveals new biosynthetic entry points beyond canonical glyceollin pathway.

Herbicide resistance threatens effective weed control in modern agriculture, particularly in grass weeds such as Alopecurus myosuroides and Lolium multiflorum. Cinmethylin is a pre-emergence herbicide with a novel mode of action that inhibits plastidial fatty acid thioesterases (FATs), enzymes essential for fatty acid biosynthesis. Although no cases of field resistance to cinmethylin have been reported, its resistance risk has not been fully assessed. In this study, we biochemically characterized defined amino acid substitutions in FAT A and FAT B to evaluate their effects on cinmethylin inhibition profile. Some substitutions in FAT A reduced inhibition in vitro, with mutations at residue R171 causing the largest shifts in sensitivity. However, these highly resistant variants required multiple specific nucleotide polymorphisms and are therefore predicted to be unlikely to arise in weed populations. In FAT B, sensitivity shifts were generally moderate. Importantly, most substitutions that reduced cinmethylin sensitivity also impaired enzymatic activity, suggesting limited viability in planta. Overall, these results indicate that while theoretical target-site resistance mechanisms exist, the practical risk of rapid resistance evolution to cinmethylin is low, supporting its value for integrated grass weed management

Potato crop is highly vulnerable to abiotic stresses like salinity and low nutrient availability. Rapid identification of stress-resilient genotypes is therefore essential for breeding, yet conventional phenotyping is often slow, space-demanding and expensive. We present LOCOPOTS -- a LOw-COst high-throughput screening platform for in vitro POTatoes under abiotic Stress -- which combines individual in vitro plant culture, low-cost RGB imaging and machine-learning-based automatic segmentation using a trained model of a convolutional neural network, based on U-Net architecture. LOCOPOTS enabled the automated extraction of growth, colour, and vegetation-index traits and demonstrated robust performance across independent phenotyping rounds. We screened 30 potato varieties under control, low-nutrient and saltinity conditions, identifying contrasting growth and physiological responses. Integrated traits such as final area and height, Area_AUC and height_AUC, together with GLI, Chol, cive and chlorophyll fluorescence parameters, discriminated genotype performance under stress. Metabolic profiling further revealed genotype-specific reprogramming in carbon and nitrogen metabolism under low nutrition and salt stress, including changes in fructose, myo-inositol, {beta}-aminobutyric acid, {gamma}-aminobutyric acid, proline, and certain polyamines, identifying them as specific chemical biomarkers of plant stress responses. LOCOPOTS provides a scalable, affordable and space-efficient platform for early screening of potato genetic diversity and identification of candidate traits associated with stress resilience.

Iron (Fe) is an essential micronutrient for plant growth, and the hormone auxin is a key regulator of developmental processes, including root gravitropism. Here, we investigated the molecular mechanisms underlying the crosstalk between iron nutrition and auxin-mediated root growth in Arabidopsis thaliana. Phenotypic analysis revealed that iron deficiency strongly shaped root system architecture and root gravitropism, and these phenotypes were exacerbated in the iron uptake mutant irt1-1. Genetic analysis revealed that iron deficiency did not aggravate the gravitropic defect of the pin2 mutant, eir1-4, suggesting that iron availability modulates root gravitropism through a PIN2-dependent pathway. Further transcriptomic analysis confirmed that iron deficiency significantly altered the expression of numerous genes related to the auxin pathway, providing molecular evidence for the observed physiological connection. Collectively, this study revealed that iron availability regulates root gravitropic growth by modulating PIN-mediated auxin transport and distribution, providing insights into how plants integrate nutritional cues with developmental programs. Graphical abstract A brief descriptionIron modulates auxin transport and root tip distribution by regulating PIN2 protein, thereby mediating root gravitropism in Arabidopsis. Public summaryO_LIIron nutrition specifically regulates root gravitropism and architecture in Arabidopsis. C_LIO_LIIron deficiency disrupts local auxin homeostasis in root tips and impairs asymmetric distribution in response to gravity. C_LIO_LIIron deficiency stress significantly reduces the abundance of PIN2 protein in root tip cells and disrupts its polar localization pattern on the plasma membrane, thereby precisely modulating polar auxin transport by interfering with the vesicle trafficking and recycling efficiency of PIN2. C_LIO_LIRNA-seq results showed that iron deficiency induced differential expression of multiple auxin-related genes, indicating that iron nutrition affects root development through the auxin pathway. C_LI

O_LIShoot branching is critical not only in breeding for yield but also for ornamentals architecture. In the ornamental plant poinsettia (Euphorbia pulcherrima), shoot branching has traditionally been induced by phytoplasma (Candidatus Phytoplasma pruni) inoculation. This study aimed to identify regulatory genes that could be leveraged in future breeding-by-genetic engineering efforts to develop phytoplasma-free, branching poinsettia plants. C_LIO_LITo elucidate mechanisms of phytoplasma-induced shoot branching, we performed RNA-sequencing and assembled an axillary bud-specific transcriptome for expression analyses in phytoplasma-infected and -free poinsettia. Phenotyping and RNA-sequencing were also conducted on Arabidopsis mutants and wild-type lines to investigate the transcriptional regulatory effects of candidate genes. C_LIO_LIThe transcription factors EpMAF3 and EpbZIP67 were highly de-regulated in phytoplasma-infected poinsettia. We also found a two-fold increase in primary-stem branching levels of the Arabidopsis maf3 and bzip67 mutants, suggesting the two transcription factors as potential shoot branching suppressors. AtTcp1, a CYC-clade TCP transcription factor, was up-regulated (78x) in leaves of the maf3 mutants. Analyzing previously reported protein-level interactions for the differentially expressed genes (e.g., AtClamt, AtGh3.9/3.15, AtSaur32/36, AtAbi3, AtGamt2, AtTcp3, and AtDwf4) in bzip67 mutants shed light on other shoot branching regulators such as TCPs, PINs, ABIs, DWARF14, and BES1, highlighting two regulatory sub-networks including membrane transport and hormonal signaling. C_LIO_LIThe results open the way to rational engineering of shoot branching in poinsettia by targeted mutagenesis of MAF3 and bZIP67. In that way, the tedious and viral-infection prone process of phytoplasma inoculation can be avoided and poinsettia plants would have more homogenous branching. C_LI One-sentence summaryPhytoplasma infection in poinsettia induces bud-specific repression of the transcription factors EpMaf3 and EpbZip67, consistent with the enhanced stem branching observed in Arabidopsis maf3 and bzip67 mutant lines.

O_LIEngineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network C_LIO_LITo identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. C_LIO_LIBfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. C_LIO_LIOur results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems. C_LI

While multi-species genomic language models have advanced biological representation learning, high-quality, single-species foundation models for crops remain scarce. Leveraging recently expanded rice pangenome resources, we introduce OryzaG3, a species-specific DNA language model with 700M parameters. OryzaG3 was pretrained on 59.20 Gb of chromosome-level sequences from 149 high-quality rice genomes using a non-overlapping 3-mer tokenization strategy and a causal language modeling objective, featuring context-length variants up to 32k tokens. On the Plants Genomic Benchmark polyA prediction task, OryzaG3 achieves competitive predictive performance against leading multi-species models while delivering a four-fold increase in inference throughput under identical long-context conditions. Ultimately, OryzaG3 demonstrates that lightweight, single-species foundation models trained on high-quality pangenomes can match multi-species benchmarks while significantly reducing computational overhead. This work provides a scalable framework for rice functional genomics, molecular breeding, and targeted crop foundation model development.

Prenylated isoflavonoids are widely distributed specialized metabolites within the Fabaceae and contribute to various characteristic biological activities for both plants and humans. Several aromatic prenyltransferases (PTs) have been identified in Glycyrrhiza species, which are the most widely consumed crude drugs in traditional Chinese medicine. However, these enzymes do not sufficiently explain the structural diversity of prenylated flavonoids produced in the Glycyrrhiza genus. To identify additional novel PTs, we used elicited cultured Glycyrrhiza glabra roots as source material, in which elicitor treatment of cultured roots increased the accumulation of multiple prenylated flavonoids. To identify the responsible enzyme, PT candidates were screened using G. uralensis transcriptomes, currently the sole publicly available transcriptomic resource within the genus, and a homolog designated GgBSPT1 (BSPT; a broad-substrate prenyltransferase) was subsequently isolated from elicited cultured G. glabra roots. GgBSPT1 differed from previously identified Glycyrrhiza PTs in both amino acid sequence and enzymatic properties. GgBSPT1 catalyzed 3'-prenylation of isoliquiritigenin and 6-prenylation of five flavonoids, i.e., this PT displayed broad substrate acceptance across 20 distinct flavonoid structures. Overall, elicited cultured G. glabra roots enabled the identification of a previously unrecognized PT that is functionally distinct from earlier reported Glycyrrhiza PTs. This study provides a new insight into the metabolic plasticity of Glycyrrhiza species and expands the enzymatic toolkit for future metabolic engineering of prenylated phytochemicals by the unusually broad substrate specificity of GgBSPT1. Main conclusionUsing cultured Glycyrrhiza glabra roots, we identified a new prenyltransferase involved in the formation of a variety of flavonoids, thereby revealing novel prenylated isoflavonoid pathways in licorice.

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that serve as defense compounds in Arabidopsis and other members of the Brassicales. Although the enzymatic pathway that produces GSLs is well-studied, the upstream mechanisms that control their tissue-specific synthesis are poorly understood. We identified a novel repression module that transcriptionally regulates GSL levels in sepals, the modified leaves that protect reproductive tissues within the floral bud. GLABRA2 (GL2) INTERACTING REPRESSOR1 (GIR1) interacts directly with Arabidopsis thaliana MERISTEM LAYER1 (ATML1), an HD-Zip IV transcription factor known to be required for giant cell formation in the sepal epidermis. This interaction requires a predicted Zn finger of GIR1 and the C-terminal START adjacent domain (STAD) of ATML1. The gir1 loss-of-function mutants exhibit excess giant cells, in contrast to atml1 mutants which display fewer giant cells, supporting the role of GIR1 as a negative regulator of ATML1. We confirmed that GIR1 interacts with TOPLESS (TPL) and TOPLESS-RELATED (TPR) corepressors, and coimmunoprecipitation demonstrated that GIR1 acts as an adaptor protein connecting ATML1 and TPL/TPR. RNA sequencing revealed that numerous genes involved in GSL biosynthesis, including the key transcriptional regulator MYB29, are upregulated in gir1 mutants. Consistent with the transcriptomic data, chemical analysis revealed that gir1 mutants display elevated GSL levels in sepals. Mass spectrometry imaging confirmed high GSL accumulation in gir1 sepals compared to wild type and atml1. Overall, our findings uncover a previously unrecognized link between cell expansion and GSL metabolism, suggesting strategies for engineering plants with cell-type specific GSL profiles. Significance StatementPlants belonging to the order Brassicales produce sulfur-containing glucosinolate (GSL) metabolites that serve in defense against herbivory. In cruciferous vegetables such as broccoli and kale, these compounds contribute to their unique flavors and health-promoting attributes. In agriculturally important oilseed crops, they affect the palatability of animal feeds. Here, we identified a novel transcriptional regulatory module that controls GSL biosynthesis in the epidermis of the sepal, the floral organ that protects the reproductive tissues. This regulatory module also controls cell expansion of specific cell types in the sepal, demonstrating a surprising connection between cell growth and a chemical defense pathway in plants. Our results suggest strategies for engineering crops with tissue-specific GSL profiles to fit agronomic needs.

Cytochrome P450 monooxygenases (CYP450s) are key oxidative enzymes that diversify plant specialized metabolites and play a central role in the biosynthesis of bioactive withanolides in Withania somnifera (L.) Dunal. Despite their importance, genome-wide information on CYP450s in W. somnifera has remained elusive. Herein, the first high-quality genome assembly (2.2 Gb, scaffold N50: 47.4 kb) of an Indian W. somnifera cultivar was generated using a hybrid Oxford Nanopore-Illumina sequencing strategy. Comparative analysis with the NCBI reference genome revealed moderate SNP and indel variations, reflecting intraspecific genetic diversity. A comprehensive CYP450 catalog was established and analyzed phylogenomically across nine plant genomes, encompassing both withanolide-producing and non-producing Solanaceae and non-Solanaceae species. Unique CYP families (CYP450A, CYP1194, and CYP705A) were detected exclusively in W. somnifera, suggesting lineage-specific metabolic innovations, while Solanaceae-restricted (CYP82E/M) and absent (CYP81B, CYP6) lineages highlight taxonomic divergence. Across all analyzed genomes, 36 conserved CYP450 subfamilies, including triterpenoid-associated members, were identified, suggesting a shared oxidative framework adaptable to specialized metabolism. Moreover, potential candidate genes in the triterpenoid pathway, including CYP72A692_1, CYP72A560_4, CYP716A48, CYP724B2, and CYP51G1, were identified through phylogenetic integration with functionally validated triterpenoid-modifying enzymes from other plant species. Gene family evolution analysis further revealed contraction of monoterpenoid-related subfamilies (CYP76A), implying a metabolic shift toward triterpenoid specialization. The comprehensive genome assembly and CYPome of W. somnifera offer a valuable resource for functional characterization, evolutionary analysis, and the identification of genes underlying its specialized metabolism. Furthermore, the study advances our understanding of CYP450 diversity and evolution, revealing lineage-specific innovations, conserved subfamilies, and key candidate genes involved in triterpenoid biosynthesis. Together, these findings lay a foundation for future functional studies and pathway engineering aimed at optimizing the metabolic potential of this important medicinal plant.

Durum wheat (Triticum turgidum subsp. durum) is a globally important crop for pasta and couscous production. Chili and Mahmoudi are historically significant Tunisian landraces valued for exceptional grain quality, high protein content, and adaptation to arid Mediterranean climates. Yet no high-quality reference genome assemblies were available for either variety before this work. We assembled both genomes using publicly available PacBio HiFi long reads and Illumina Hi-C proximity ligation data deposited under NCBI BioProject PRJNA1420514. HiFi reads were assembled with hifiasm v0.25.0 in primary mode, and Hi-C scaffolding was performed with YAHS v1.2a.2 after read alignment with BWA-MEM. Assembly quality was assessed with QUAST v5.3.0 and BUSCO v5.8.0 (embryophyta_odb10 lineage). The Chili assembly spans 10.84 Gbp across 3,472 scaffolds with a scaffold N50 of 844.9 Mbp and BUSCO completeness of 99.4%. The Mahmoudi assembly spans 10.70 Gbp across 3,258 scaffolds with a scaffold N50 of 2,072 Mbp and BUSCO completeness of 99.3%. Merqury v1.3 confirmed high base accuracy (QV 68.0 for Chili, QV 68.3 for Mahmoudi) and k-mer completeness (>98% for both). Independent validation with wfmash confirmed 98.6% mean alignment identity across 11,172 chromosome-to-reference alignments. Post-assembly characterization of GC profiling, centromere architecture, ribosomal DNA arrays, and structural variation revealed extensive genome-level detail. Both assemblies substantially exceed the contiguity of existing durum wheat references and represent the first chromosome-scale-contiguity genomic resources for North African durum wheat landraces. The Mahmoudi accession carried a putative 2B-3B homeologous fusion on chromosome 3B (4,710 Mbp), a structural novelty in an ancient Tunisian landrace. Assembly and Pseudomolecules available from Zenodo (10.5281/zenodo.20366290). The workflow was executed reproducibly on the public Galaxy Europe platform, demonstrating that reference-quality plant genome assembly is achievable without local HPC infrastructure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/727644v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@159c9d4org.highwire.dtl.DTLVardef@1d1a0deorg.highwire.dtl.DTLVardef@19853dborg.highwire.dtl.DTLVardef@1a96018_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst chromosome-scale-contiguity assemblies for Chili and Mahmoudi landraces C_LIO_LIChili genome: 10.84 Gbp, scaffold N50 844.9 Mbp, BUSCO 99.4% C_LIO_LIMahmoudi genome: 10.70 Gbp, scaffold N50 2,072 Mbp, BUSCO 99.3% C_LIO_LIMerqury QV ~68 confirms base accuracy, >98% k-mer completeness C_LIO_LI>140-fold scaffold N50 improvement over Svevo v1 reference C_LI

Heavy metal-associated isoprenylated plant proteins (HIPPs) are encoded by large gene families, which have diversified specifically in vascular plants. Their physiological functions and molecular mode of activity are currently largely unknown. In this study, we characterize a group of phylogenetically closely related genes HIPP32, HIPP33, and HIPP34 in Arabidopsis thaliana, revealing their essential roles in controlling diverse developmental pathways. Through comprehensive genetic analyses, we demonstrate that these genes exhibit partially overlapping pleiotropic functions, influencing multiple aspects of plant growth such as embryogenesis, maintenance of apical meristems, root architecture, shoot branching, leaf morphogenesis and floral organ formation. Transcriptomic profiling of hipp mutants identified significant deregulation in several regulatory pathways involved in plant hormone responses, with a specific impact on auxin signaling processes. Interestingly, we show that the analyzed HIPP proteins localize very specifically to plasmodesmata, suggesting their potential function in regulating intercellular communication in shaping plant development.

Cytochrome P450 enzymes (CYPs) are the primary drivers of chemical diversification in plant secondary metabolism; however, fewer than 10% of the superfamily members have been functionally characterized. Computational docking provides a scalable strategy to prioritize candidates for experimental validation, yet prevailing workflows are poorly adapted to plant P450s because AlphaFold-predicted structures lack the essential heme cofactor and conventional flexible-residue selection relies on subjective geometric cutoffs. To address these limitations, we developed an automated pipeline--PlantP450Dock--that unifies heme cofactor implantation, molecular dynamics-based conformational sampling, data-driven flexible residue selection, and semi-flexible docking within a single integrated workflow. The heme is transferred from a crystallographic template to the AlphaFold model via a local coordinate transformation algorithm, achieving a positional deviation of less than 0.2 [A] relative to the experimentally determined CYP73A33 structure (PDB: 6VBY). Subsequent 100 ns molecular dynamics simulations confirmed faithful preservation of the Fe-S coordination geometry (2.61 {+/-} 0.08 [A]) across all trajectory frames. A singular value decomposition-based heme-plane filtering strategy objectively identified distal active-site residues for flexible treatment, eliminating user-dependent subjectivity. Cross-family validation against four phylogenetically distinct P450s (CYP73, CYP711, CYP706, and CYP701) generated catalytically competent binding poses with substrate-to-heme-iron distances of 2.8-4.4 [A] without enzyme-specific parameterization. Released as an open-source tool, this pipeline furnishes the plant science community with a standardized, reproducible computational framework to accelerate functional annotation of the largely unexplored plant P450 families.