Plant cell walls are dynamic composite structures whose biogenesis, remodelling, and integrity maintenance require coordinated regulation across biosynthetic, trafficking, sensing, and signalling pathways. Plasma membrane-localised receptor kinases and mechanosensitive channels monitor wall status and transduce perturbations into intracellular responses, whilst biomolecular condensates, membrane-less or membrane-associated assemblies formed through liquid-liquid phase separation and related processes, have emerged as candidate organisational features of several of these pathways. Direct experimental evidence linking condensates to cell wall function nonetheless remains sparse, and for many systems it is unclear whether observed puncta represent bona fide phase-separated assemblies. In this review we survey cell wall biogenesis and integrity pathways during development and under stress and critically evaluate where condensates plausibly participate. To keep claims proportionate to the evidence, we apply an explicit hierarchy, classifying each system as direct, indirect, contextual, or speculative. On this basis, the RALF-pectin system, in which extracellular phase separation generates signalling platforms that recruit the receptor kinase FERONIA and its co-receptor LLG1 as client proteins, remains the only directly validated example; most other associations, including P-bodies, stress granules, and nuclear transcriptional condensates, appear to respond to osmotic stress or molecular crowding arising as secondary consequences of wall perturbations. We further assess how computational and artificial intelligence approaches might complement experimental work, alongside their present limitations for plant systems.
The development of microbial-based agricultural amendments that work consistently in the field requires an understanding of the molecular mechanisms of plant-microbe interactions. Studying these underlying mechanisms of interaction demands the ability to grow plants under environmentally controlled and gnotobiotic conditions (i.e., all microorganisms interacting with the plant are known, whether that is germ-free, defined microbial communities, or natural communities). The currently available plant gnotobiotic systems are not suitable for studying large plants of agricultural relevance, such as cereals. Moreover, most of these systems lack the ability to manage irrigation. Here, we introduce GNOVA, a new gnotobiotic system designed to accommodate cereal plants with the ability to manage irrigation. This new system is an accessible platform composed of a 3D printed base and commercially available materials. This protocol provides a step-by-step guide to assembling the system and experiment set up. Furthermore, we present a performance comparison of GNOVA to a gnotobiotic bag system. GNOVA extended plant growth from two weeks in the bag system up to 17 weeks for wheat and 4 weeks for maize. The germination rate of both crops also increased within GNOVA from 66% to 100% for wheat and from 75% to 100% for maize. Wheat grown within GNOVA developed tillers, which were absent in plants of the same age within the bag system. The fresh weight of maize grown in the GNOVA was 594% higher than in the bag system. Additionally, the shoot height and root length of maize were 89% and 57% greater within the GNOVA system than in the bags, respectively. The GNOVA system extends the toolbox available to scientists for the exploration of plant-microbiota interactions beyond the seedling stage in cereals by providing increased growth space and irrigation management.
Global environmental change can alter ecological mechanisms that maintain biodiversity. Interactions between plants and soil microbes mediate plant species coexistence, which can vary with abiotic factors such as light and soil moisture, and such context dependence is only beginning to be explored. We assessed how variation in light and soil moisture alters soil microbial effects on the predicted coexistence of two common tree species (Litsea floribunda and Symplocos racemosa) native to the Western Ghats, India. We conducted a shade-house reciprocal transplant experiment with different soil origins in factorial combinations of high/low water and light, and predicted coexistence outcomes using metrics that decompose microbial effects into stabilization and fitness differences. For high-water, low-light conditions, we also evaluated whether soil microbes alter plant-plant interactions, using a structural framework that quantifies the feasibility domain of coexistence from species interaction coefficients. Soil microbes were predicted to promote plant coexistence in high-water, low-light conditions, where stabilization exceeded the fitness differences due to microbes. Under low-water, high-light conditions, larger fitness differences predicted exclusion of S. racemosa. Microbes had weak effects on plant-plant interactions, but predicted coexistence improved slightly in soils with background microbes not shaped by either species, due to weaker estimated fitness differences in those soils. Drier and brighter conditions may weaken the potential for microbially mediated plant coexistence in tropical forests. These findings hint at shifts in microbially mediated plant community dynamics in response to global change factors such as forest fragmentation and drought.
The escalating threat of plant diseases to global agriculture and food security necessitates innovative and sustainable control strategies. Conventional biological control agents (BCAs), while environmentally friendly, often suffer environmental challenges and secretion of limited/poor antimicrobial compounds. Advances in CRISPR/Cas genome editing, protease engineering, and synthetic biology have enabled precise modifications that improve pathogen targeting and secretion efficiency. Interest should now be shifted on development of "Super Bioagents (SBs)" with enhanced secretion systems (SSs) for plant disease suppression against changing environmental factors. This will create sustainable ecofriendly alternative to chemical pesticides. This review explores a detailed overview of molecular mechanisms of microbial SSs and the potentials of SBs as a frontier in plant disease management. While there are still challenges in mass deployment of BCAs in sustainable agriculture, this review is guided by the hypothesis that rational, quantitative engineering of microbial SSs can transform conventional BCAs into integrated SBs. It synthesizes current advances within a systems‑level bioengineering framework linking secretion efficiency, regulation, and field performance. It further explores possible integration of SBs in plant-microbiome interactions to further enhance their adaptability and effectiveness. Finally, the review dives into recent breakthroughs, current challenges, and future directions for SBs development and application as next-generation plant disease control agents.
Protein-protein interactions underpin virtually all biological processes in plants, from signal transduction and immune responses to development and stress adaptation. Despite their fundamental importance, the plant interactome remains far from complete, and existing maps are systematically biased by the technical limitations inherent to conventional detection platforms. This review critically traces the evolution of protein-protein interaction methodologies, from foundational approaches to advance in vivo and quantitative platforms. Classical techniques such as the yeast two-hybrid system and in vitro pull-down assays operate outside physiological cellular environments and are poorly suited to capturing transient or condition-dependent interactions. Affinity purification coupled with mass spectrometry improves throughput but remains vulnerable to artifacts introduced during cell lysis and to the preferential loss of weak interactors. To address these shortcomings, proximity labeling with engineered biotin ligases, most notably the fast-acting variant TurboID, has emerged as a powerful strategy, enabling covalent biotinylation of protein neighborhoods within living cells prior to lysis and thereby preserving associations that conventional methods routinely miss. Because TurboID reports proximity rather than direct binding, its output requires downstream binary validation. Complementary in planta validation tools are equally critical for moving beyond discovery. Split-luciferase complementation assays based on the NanoLuciferase reporter provide exceptional sensitivity for binary interaction detection under native expression conditions, while Förster Resonance Energy Transfer measured through fluorescence lifetime imaging microscopy offers quantitative biophysical evidence of molecular proximity at endogenous expression levels, serving as a high-confidence validation approach. Emerging technologies, including high-throughput protein microarrays and optogenetically controlled dimerization systems, further expand the methodological repertoire available to the plant biology community. We propose a practical, integrative three-tier framework, combining proximity labeling for broad in vivo discovery, split-luciferase complementation for sensitive binary validation, and fluorescence lifetime imaging microscopy for quantitative confirmation, that systematically funnels candidate interactions from initial identification to physiologically rigorous verification. This framework synthesizes established best practices into a structured workflow applicable to mapping dynamic plant interactomes, though its optimal implementation will depend on the biological question, target protein class, and available resources.
Colorectal cancer (CRC) is one of the leading contributors to cancer related mortality worldwide highlighting the need for novel therapeutic agents. This study investigated the potential anti-colorectal cancer activity of phytochemicals from Artemisia annua L. plant using an integrated in silico approaches. Gene expression analysis, ADMET screening, network pharmacology, molecular docking, density functional theory (DFT), molecular dynamics (MD) simulation, and post-simulation trajectory analyses were employed to identify potential therapeutic compounds and molecular targets. Among the identified phytochemicals, toxicity screening identified 13 predicted non-toxic compounds and molecular docking results revealed that cirsilineol (-8.3 kcal/mol), 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone (-7.9 kcal/mol), and isobonducellin (-7.9 kcal/mol) exhibited strong binding affinity toward AKT1 protein than control drug 5fu (-5 kcal/mol) and capivasertib (-7.6 kcal/mol). Additionally, ADME analysis confirmed favorable drug likeness profiles of these active compounds. The 200ns molecular dynamics simulation analysis revealed that the isobonducellin-AKT1 complex possessed stable conformation with good RMSD (2.253 ± 0.243 Å), RMSF (1.184 ± 0.852 Å), Rg (4.0 ± 0.064 Å), SASA (45.93 ± 36.75 Å2), and hydrogen bond (84.649 ± 5.306), compared to other ligands and control capivasertib. DFT, PCA, DCCM and MM-GBSA binding free energy analysis further supported isobonducellin (CID: 10423880) as a strong AKT1-targeting drug candidate. Although MM-GBSA suggested slightly better binding for another ligand, isobonducellin was selected based on its overall superior dynamic stability and consistent interaction profile across simulations. Our results proposed that isobonducellin from Artemisia annua L. shows potential as a colorectal cancer therapeutic by modulating multiple signaling pathways and targeting AKT1 protein. However, additional experimental studies such as cancer cell-line assays and animal-model testing are required to validate this study.
The construction and performance of plants capable of detoxifying the organochlorine pesticide (lindane, γ-hexachlorocyclohexane or γ-HCH) is reported. To this end, the bacterial linA gene from Sphingobium japonicum strain UT26, which encodes a dehydrochlorinase initiating γ-HCH degradation, was engineered into Arabidopsis thaliana. The resulting lines expressing linA exhibited markedly enhanced tolerance to lindane compared to wild-type controls, both in synthetic media and in contaminated soils. Furthermore, thereby engineered plants also removed more than 90% of the pollutant from the medium within 4 weeks. Chemical analyses revealed not only the formation of 1,2,4-trichlorobenzene (TCB), the expected γ-HCH degradation intermediates, but also detected 1,4-dichlorobenzene (DCB), suggesting that native plant activities could further push the degradation process beyond the canonical microbial pathway. This points to a potentially synergistic interaction between the introduced bacterial enzyme and the endogenous plant detoxification systems. By combining bacterial catabolic activity with plant resilience and root-mediated soil interactions, our data advocate a new strategy for the remediation of γ-HCH contaminated environments and demonstrate the feasibility of designing plants for enhanced degradation of persistent organic pollutants.
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A significant proportion of global photosynthetic carbon fixation relies on the pyrenoid, a biomolecular condensate found in the chloroplast of most unicellular algae, where the CO2-fixing enzyme Rubisco is exposed to saturating concentrations of the gas. In this review, we highlight recent advances in our understanding of the molecular basis of diverse pyrenoids. Phase separation of phylogenetically distant Rubiscos is mediated by convergently evolved linker proteins, with an emerging theme of pyrenoid condensation being organized via Rubisco-binding motifs. To minimize CO2 leakage out of the pyrenoid, starch sheaths and protein shells have evolved to surround the pyrenoid in various algal lineages. Crucially, the pyrenoid is a biomolecular condensate with an increasingly well-defined function that has evolved multiple times over the past billions of years. The emerging similarities and differences of these various pyrenoids will inform our appreciation of phase separation in biology and empower engineering efforts aimed at enhancing photosynthetic CO2 assimilation.
Sulfonylurea herbicides, such as bensulfuron-methyl (BM), are widely used around the world, but they pose a severe risk of phytotoxicity to crop plants that are not the herbicide's target, disrupting their physiological and metabolic homeostasis. Although biostimulants (BS) are increasingly recognised for alleviating abiotic stress, the mechanisms by which they mitigate herbicide toxicity across multiple pathways remain poorly investigated. This study comprehensively elucidates the morphological, physiological, biochemical, cytogenetic and molecular responses of cucumber (Cucumis sativus L.) seedlings to BM toxicity, as well as the restorative capacity of an amino acid-based BS. Our findings demonstrate that exposure to BM significantly suppresses plant biomass and triggers severe oxidative stress, as evidenced by the excessive accumulation of reactive oxygen species (ROS; H₂O₂ and malondialdehyde (MDA)). This systemic toxicity severely disrupted nutritional homeostasis and phytohormone profiles, notably inhibiting the biosynthesis of indole-3-acetic acid (IAA), gibberellic acid (GA) and salicylic acid (SA) while increasing abscisic acid (ABA). Conversely, the application of exogenous BSs effectively reversed these phytotoxic damages by upregulating antioxidant defence enzymes (SOD, CAT and POD) and restoring mineral uptake and hormonal networks. Furthermore, anatomical and cytogenetic assessments in Allium cepa revealed that BM induced structural deformations and chromosomal aberrations, which were significantly mitigated by BS pretreatment. Molecular docking simulations confirmed that BM exerts its toxicity by directly binding to essential proteins (e.g. ICL, CAT and POD) and DNA structures, thereby blocking their normal functions. In conclusion, this study provides profound mechanistic insights into the multiple stress responses induced by BM and reveals the ability of amino acid-based BSs to mitigate the effects of herbicide contamination, offering a sustainable strategy to protect agricultural productivity.
Chromophobe renal cell carcinoma (ChRCC) is a renal malignancy typically associated with indolent behavior, with nodal involvement being rare. We report a 78-year-old African American male with incidentally discovered node-positive (pN+) ChRCC with sarcomatoid differentiation who developed spinal metastases despite adjuvant pembrolizumab. Histopathologic evaluation demonstrated distinct plant-like cell membranes, while genomic profiling revealed CDK4 amplification and MTAP and CDKN2A alterations associated with aggressive tumor biology. Notably, the tumor also exhibited additional unique molecular alterations that have not been previously reported in the literature.
Foxtail millet (Setaria italica) is a drought-tolerant C4 cereal that grows on marginal lands and serves as a nutrient-rich food for millions in Asia and Africa. Despite its resilience and nutritional value, the genetic basis underlying plant height variation in foxtail millet remains incompletely understood, thereby constraining the effective implementation of semi-dwarfing strategies analogous to those that drove the Green Revolution in major cereals. This study identified sidt1, a semi-dwarf, high-tillering mutant exhibiting a compact architecture and enhanced lodging resistance. It is demonstrated that SiDT1 encodes a GA3-oxidase orthologous to rice D18/XIAOWEI. A single A-to-T mutation disrupted its catalytic function, reducing bioactive gibberellin biosynthesis. CRISPR-Cas9 knockout lines recapitulated the sidt1 phenotype, confirming SiDT1's functional role. Combined transcriptomic profiling and SiD53 immunoblot analysis indicated that strigolactone-related signaling is perturbed in sidt1, in agreement with its enhanced tillering phenotype. Notably, under high-density planting conditions, sidt1 maintained grain yield and quality while exhibiting superior lodging resistance. These findings identify SiDT1 as a key regulator of plant architecture and establish a semi-dwarf ideotype reminiscent of the rice Green Revolution, providing a valuable genetic resource for high-density and mechanized foxtail millet production.
Nickel (Ni) contamination is an increasing environmental concern that negatively affects plant growth, physiological performance, and the biosynthesis of medicinally important secondary metabolites. The use of natural biostimulants such as chitin and nitric oxide (NO) has emerged as a promising strategy to enhance plant tolerance against heavy metal stress. Therefore, this study investigated the potential of chitin and NO to enhance the physiological and phytochemical responses of Andrographis paniculata under Ni stress. The study was designed to assess the effects of varying concentrations of chitin (0, 15, and 30 µM) and NO (0, 0.5, and 1 g/L) on several growth parameters, including photosynthetic pigments, total phenolic content, total flavonoid content, protein accumulation, key secondary metabolites (andrographolide, neoandrographolide, and 14-deoxy-11,12-didehydroandrographolide), and the expression of isoprenoid biosynthesis-related genes (HMGR, HMGS, DXR, and DXS) in A. paniculata under different levels of Ni stress (0, 1.5, and 3 mM). The results showed that Ni stress significantly reduced chlorophyll, carotenoid, phenolic, and protein contents, whereas it altered secondary metabolite profiles and gene expression patterns. Application of NO and chitin significantly improved chlorophyll a, chlorophyll b, carotenoids, total phenols, and protein content under Ni stress conditions. In addition, NO and chitin treatments enhanced the accumulation of key bioactive compounds and positively regulated the expression of genes involved in terpenoid biosynthesis pathways. Overall, the findings indicate that NO and chitin alleviate Ni-induced stress in A. paniculata primarily through improving physiological performance and enhancing the accumulation of non-enzymatic antioxidant compounds such as phenolics and flavonoids, thereby contributing to improved metabolic stability and secondary metabolite production under heavy metal stress. Andrographis paniculata emerges as a valuable medicinal-industrial species with diverse pharmaceutical applicationsChitin-nitric oxide synergy significantly boosts nickel stress tolerance and phytochemical production in A. paniculataNickel stress upregulates terpenoid biosynthesis genes (HMGS, HMGR, DXS, DXR), amplified by chitin-NO elicitation.
Ultraviolet (UV) radiation generates DNA lesions, primarily cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts ([6-4] PPs), that can block DNA replication. Although nuclear UV-induced lesions are repaired or bypassed by specialized pathways, how plant organellar DNA polymerases replicate UV-damaged templates remains unclear. Here, we show that the two Arabidopsis thaliana organellar DNA replicases, AtPolIs, efficiently synthesize across CPDs with 80%-90% bypass efficiency, exceeding that reported for individual specialized translesion synthesis (TLS) polymerases. Furthermore, although [6-4] PPs impose a major barrier to most TLS polymerases, wild-type AtPolIs exhibit measurable lesion-bypass activity (∼10%), and reduction of exonuclease activity enhances bypass by ~8-fold, reaching levels comparable to synthesis on undamaged templates. We further demonstrate that TLS across UV photoproducts depends on three unique amino acid insertions within the polymerase domain, as disruption of these insertions severely compromises lesion bypass. These findings reveal that AtPolIs are replicative polymerases with an intrinsic and unusually robust capacity for UV-lesion bypass, suggesting a specialized adaptation that helps maintain plant organellar genome stability under UV stress.
Colletotrichum viniferum, the causal agent of grape ripe rot and leaf spot, poses a serious threat to grape yield and fruit quality. Like many phytopathogens, C. viniferum secretes effector proteins; however, the molecular mechanisms by which these effectors manipulate host immune responses remain poorly understood. In this study, we functionally characterized a candidate effector, CvA10999. CvA10999 suppressed INF1 (infestans 1, P. infestans PAMP elicitor) triggered cell death in Nicotiana benthamiana and was significantly upregulated during C. viniferum infection of susceptible grape V. vinifera cv. Thompson Seedless (TS) leaves. Targeted deletion of CvA10999 resulted in reduced sporulation, abnormal appressorium formation, and attenuated virulence on TS leaves. Further analysis revealed that CvA10999 interacts with the grape protein β-subunit of sucrose non-fermenting 1-related protein kinase (VvSnRKb1). Transient overexpression of VvSnRKb1 in TS leaves, as well as stable transgenic grapevines overexpressing VvSnRKb1, conferred enhanced resistance to C. viniferum. Mechanistically, CvA10999 bound to VvSnRKb1, disrupting its interaction with nonexpressor of pathogenesis-related genes 1 (VvNPR1) and interfering with VvNPR1 phosphorylation. This likely impaired the transcriptional activator function of VvNPR1 and downregulated salicylic acid (SA)-responsive pathogenesis-related (PR) genes. Collectively, these findings demonstrate that CvA10999 targets VvSnRKb1 to subvert host immunity and promote C. viniferum infection.
Extracellular freezing challenges insects in several ways, including mechanical damage from ice crystals, exposure to stressful levels of cold, and cellular dehydration. While stress-response mechanisms activated during and immediately after freezing are well-studied, less is known about how freeze-tolerant organisms recover from freezing in the longer-term and whether this recovery carries energetic costs. Here, we tracked changes in gene expression and energy stores over the course of 15 days following extracellular freezing using the world's southernmost insect, Belgica antarctica Jacobs (Diptera: Chironomidae), as a study system. We found that B. antarctica employed a coordinated "emergency" stress-response system during early-recovery (0-1 d), which includes the upregulation of genes involved in inhibiting cell death (e.g., Bcl-2, IAP-1) alongside mechanisms involved in damage repair and clearance of cell debris (e.g., heat shock proteins, autophagy, proteasome). Concomitantly, genes involved in ecdysone biosynthesis and juvenile hormone degradation were downregulated through the 1st day of recovery, and several genes involved in cuticle development were downregulated between the 3rd and 15th day of recovery, suggesting that homeostasis may not have been completely restored for two weeks after B. antarctica had thawed. These physiological changes did not lead to a detectable depletion of energy stores, indicating that this emergency response system does not incur significant energy drain. These results suggest that evoking a coordinated stress-response with minimal energy usage may be crucial for B. antarctica to persist in cold environments, and that blocking apoptosis and pausing development could provide sufficient time for freezing injury to be fully addressed.
Ficus hirta Vahl (F. hirta) is a traditional medicinal plant used for over 300 years in Chinese ethnic medical systems, including the Yao, Zhuang, and Dai. To provide a rigorous and balanced synthesis, this review conducted a systematic literature search across PubMed, Scopus, CNKI, and Web of Science, focusing on original research regarding the plant's phytochemistry and pharmacology. The extract contains diverse bioactive compounds, including flavonoids, phenolic acids, terpenoids, polysaccharides, and phenylpropanoids, which exhibit significant antibacterial and anti-inflammatory activities. This review summarizes their mechanisms of action: antibacterial effects are achieved through cell wall disruption, metabolic inhibition, and biofilm suppression; while anti-inflammatory effects involve modulating inflammatory mediators, enhancing antioxidant capacity, and regulating immune cells. Furthermore, the synergistic network between these properties and clinical effects-such as digestion promotion and cough relief-is explored. These findings provide a theoretical foundation for the medicinal development of F. hirta and highlight its potential in novel drug discovery.
Salvia plebeia R. Br. exerts favorable therapeutic effects on multiple diseases such as bronchitis, urticaria and colorectal cancer. Endophytes play vital roles in the growth and development of medicinal plants. They can not only promote plant growth, but also synthesize active ingredients identical to those of host plants, as well as induce hosts to produce active components. For the first time, this study systematically investigated the endophytes of S. plebeia. We evaluated the antagonistic activity and plant growth-promoting traits of endophytes, explored the bacteriostatic effects of strain fermentation broths and volatile organic compounds, and screened strains with excellent functional properties. Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was adopted to analyze the differences in metabolic components between S. plebeia and its dominant endophytes. Combined with network pharmacology, we further elucidated the potential molecular mechanisms by which their active ingredients intervene in diseases. Network pharmacology analysis revealed that S. plebeia and its dominant endophytes share a variety of active metabolites, which exert synergistic regulatory effects through common core targets and key signaling pathways. Meanwhile, endophytes possess unique regulatory pathways, a more complex protein-protein interaction network and stronger target binding capacity. They perform medicinal functions via a synergistic mode of multiple components, multiple targets and multiple pathways. This is the first study to apply network pharmacology to predict the correlation of metabolic components between endophytes and their host plants.
Sesame (Sesamum indicum L.), is one of the earliest domesticated oilseed crops. It is valued for its exceptional oil content, bioactive compounds, and adaptability to diverse agroclimatic conditions. However, its production remains highly susceptible to water scarcity particularly terminal drought and intermittent moisture deficits that in turn severely compromise the yield and oil quality. Recent advances in genomics, transcriptomics, proteomics, metabolomics, epigenomics, and phenomics have placed sesame as an emerging model for multi-omics-driven stress research. These approaches have uncovered key regulators of drought (NAC, MYB, WRKY), protective proteins (late embryogenesis abundant proteins, heat shock proteins, antioxidant enzymes), osmolyte- and redox-related metabolites, and hormonal signalling modules such as PYL-SnRK2-ABF (ABA), LOX/AOS/OPR (jasmonate), and EIN/ERF (ethylene). Addressing these gaps will require investments in precision phenotyping, robust pan-genomic databases, functional validation using CRISPR/Cas9 tools, and global data-sharing networks. This review highlights the current advances in sesame drought research across omics platforms, critically evaluates their relevance to breeding programs, and offers the first comprehensive multi-omics perspective on moisture-stress adaptation in sesame. Additionally, KEGG pathway-guided multi-omics integration connects ABA signaling, phenylpropanoid metabolism, and antioxidant pathways to drought adaptation in sesame. By bridging mechanistic insights with applied strategies, it highlights pathways to accelerate the development of climate-resilient sesame cultivars.
Cannabis sativa L. roots have been less studied than aboveground organs, despite their key role in plant physiology, metabolism, and interactions with biotic and abiotic factors. Metabolomic and phytochemical analyses reveal that roots synthesize a diverse array of bioactive compounds with antimicrobial, anti-inflammatory, antioxidant, and cytotoxic properties, highlighting their biotechnological potential. Root exudation patterns and interactions with endophytic microorganisms modulate rhizosphere microbial networks that support nutrient uptake, stress tolerance, pathogen resistance, and whole-plant physiology. Root-derived phytohormones and other signalling molecules may participate in coordinating biochemical pathways between belowground and aboveground tissues, with potential effects on secondary metabolism in aerial tissues. Recent advances in metabolomics, transcriptomics, microfluidic rhizosphere systems, and root-specific genetic engineering now enable detailed investigation of root metabolism in Cannabis sativa L. This review synthesises current knowledge on the metabolic roles of Cannabis sativa L. roots, their interactions with the rhizosphere microbiome, and root-derived systemic signalling. It emphasises aspects of root biology that are central to fundamental plant processes and to the development of sustainable strategies for optimising phytochemical yields. By placing roots at the forefront, this synthesis underscores the need to expand research beyond aerial tissues to fully understand and harness the biotechnological potential of Cannabis species. KEY POINTS: • Root metabolism and signalling regulate whole-plant-metabolic pathways • Root-associated microbiomes influence nutrient dynamics and phytochemical profiles • Root culture systems provide a scalable platform for biotechnological manipulation aimed at the production of bioactive compounds.