Chirality fundamentally influences the bioactivity and ecological roles of plant natural products. Steroidal glycoalkaloids (SGAs) in tomato and wild relatives exhibit this stereochemical variation. Forward genetics identifies C26 hydroxylase GAME8 as the key enzyme controlling SGA chirality. Cultivated tomato harbors two identical GAME8 copies yielding S-configured intermediates, whereas Solanum pennellii homologs GAME8a and GAME8b dominantly produce R- and S-configured products, respectively. Site-saturation mutagenesis and molecular docking reveal four residues sufficient to switch this chirality. Bioassays demonstrate that R-type SGAs, predominant in wild tomato, offer superior defense against the specialist pest Phthorimaea absoluta, although chirality does not affect resistance to the generalist Spodoptera litura. Collectively, these results elucidate the biosynthetic mechanism and ecological significance of SGA chirality, establishing a basis for tailoring phytochemical stereochemistry to enhance crop protection.

BACKGROUND:Olfactory conditioning, a key manifestation of behavioral plasticity, is known to enhance insect fitness in nature. It remains unclear, however, whether predatory mites-which share a common arthropod ancestry with insects but belong to a distinct evolutionary lineage-have a comparable capacity for olfactory conditioning. To bridge this knowledge gap, we assessed associative olfactory learning in the predatory mite Amblyseius largoensis by exposing it to herbivore-induced plant volatiles (HIPVs) from citrus plants infested with its prey, Panonychus citri. RESULTS:Trained mites showed preference shifts toward these herbivore-associated chemical signals, requiring less time to locate prey, consuming more prey individuals and producing more eggs than naive counterparts. Individual HIPV compounds (α-farnesene, humulene, linalool, β-ocimene, caryophyllene) each demonstrated unique training effectiveness, whereas cross-reactivity bioassays revealed hierarchical chemical information processing with both compound-specific and categorical responses. CONCLUSION:The success of olfactory training suggests that the mite chemosensory system provides reliability for ecological applications where consistent, robust conditioning responses are essential for predation effectiveness. © 2026 Society of Chemical Industry.

Argonautes (AGOs) are the effectors for the action of microRNAs (miRNAs). Plant genomes harbor large numbers of AGO genes whose functions remain to be fully understood. Here, we elucidated a function of AGO5 in the ecological model plant Nicotiana attenuata during its interactions with the specialist herbivore Manduca sexta. Plants silenced in NaAGO5 expression using inverted-repeat technology (irAGO5) were indistinguishable from the wild type (WT) in growth and development but were highly susceptible to M. sexta herbivory. M. sexta caterpillars grew faster and accumulated significantly more biomass on irAGO5 than on WT plants. Herbivory-elicited irAGO5 plants accumulated significantly lower amounts of auxin-dependent defense metabolites such as phenolamides, flavonoids, and diterpenoid glycosides, but not nicotine and trypsin protease inhibitors (TPI). Nicotine and TPI levels, which require intact jasmonate signaling, were attenuated in plants silenced in NaAGO8 expression (irAGO8). irAGO5 plants showed compromised herbivore-induced auxin levels and YUCCA gene expression but accumulated more salicylic acid; however, jasmonate accumulations were at WT levels. Exogenous auxin treatments restored resistance against M. sexta and auxin-dependent defense metabolites. Substantial temporal changes in the miRNome were observed in irAGO5 and were largely different from those in irAGO8. An AGO5-dependent miRNA-mRNA regulatory interaction network was inferred for defense-signaling components. Furthermore, double knockdowns of NaAGO5 and NaAGO8 revealed cooperative functions of the two genes during herbivory. We infer that AGO5 is a central component of the herbivore-induced smRNA pathway that modulates multiple nodes in the auxin-dependent metabolic space of the defense signaling network when N. attenuata plants interact with the specialist herbivore M. sexta.

Argonaute2 (AGO2) largely participates in maintaining viral defenses. However, its function is not understood in species that are not commonly challenged by viruses in their native habitats. The ecological model species, Nicotiana attenuata, grows in arid/desert habitats. Natural virus infections are not commonly observed in this species even when the genes essential for viral defenses, like the RdRs, are silenced. The biological function of NaAGO2 has remained elusive. Silencing NaAGO2 with inverted-repeats (irAGO2) did not alter morphology, growth, or reproductive performance of unstressed plants compared to the wild-type (WT). irAGO2 was also able to defend against herbivores or pathogens and compete with con-species neighbors. However, irAGO2 had increased tolerance to water stress, exhibiting enhanced reproductive output during drought and recovery. Water-stressed irAGO2 accumulated significantly more abscisic acid (ABA) and proline, which are critical signaling and protective metabolites. Drought-responsive miRNA accumulation patterns were largely altered in irAGO2, potentially modulating ABA and proline gene expression during water stress and recovery. The function of three such Na-miRNAs (miR156, miR172, and miR398) was examined by transient overexpression in mitigating water stress and regulating ABA and proline pathways. We infer that AGO2 functions in fine-tuning ABA and proline homeostasis that optimizes N. attenuata's growth in complex stressful environments.

Vector-borne plant viruses depend on insect vectors for transmission and often suppress host defenses that limit vector survival and spread. However, their impact on volatile-mediated indirect defenses remains unclear. Here, we show that rice viruses inhibit methyl salicylate (MeSA) emission, impairing parasitoid recruitment and promoting vector persistence. Field experiments demonstrate that MeSA, a key herbivore-induced volatile, suppresses vector populations by attracting egg parasitoids. Viruses counter this by targeting basic-helix-loop-helix transcription factor OsMYC2, a jasmonic acid signaling hub, thereby down-regulating OsBSMT1 and MeSA biosynthesis, responses conserved across diverse rice viruses and vector species. MeSA applications in the field restore parasitoid-mediated vector suppression, highlighting its potential for sustainable disease control. MeSA is a central ecological signal in a previously unidentified viral strategy that enhances transmission.

Plants emit volatile compounds that orchestrate complex ecological interactions, with methylated catabolites of interaction-induced phytohormones being common examples. Salicylic acid (SA) mediates plant antipathogen responses, while its methylated derivative, MeSA, broadly mediates plant-insect interactions without specificity. Here, we identified dimethyl salicylate (DMSA), an unappreciated dimethylated SA catabolite, emitted by rice when attacked by the major destructive pest, the brown planthopper. DMSA biosynthesis requires an O-methyltransferase cascade, BSMT1 (benzoic acid/salicylic acid carboxyl methyltransferase 1)-MSOMT (methyl salicylate O-methyltransferase), which is directly activated by a jasmonate (JA)-responsive MYC2-JAMYB transcriptional cascade. Natural variation in the MSOMT promoter confers its herbivory-induced expression in indica but not japonica cultivars. Functionally, DMSA acts as a specific volatile signal attracting the egg-parasitoid wasps of brown planthoppers (BPHs) without mediating direct resistance, which demonstrably suppresses BPH populations in paddy fields. DMSA is an optimized advance in SA signaling derived plant "alarm calls" with great potential in sustainable rice pest management.

Summary Trichomes are epidermal projections, crucial components of plant defense systems. Whether these epidermal defenses are effective against leafminers, which infest the internal mesophyll layers, remains unclear. Using the devasting invasive tomato pest, Phthorimaea absoluta (tomato leafminer), and the trichome-rich wild tomato, Solanum pennellii , we demonstrate that trichome produced acylsugars effectively inhibit the growth of endophagous P. absoluta larvae feeding inside leaves. High spatial resolution leaf- and frass-metabolite analysis by MALDI-MSI and Stimulated Raman Scattering microscopy revealed that trichome-synthesized acylsugars are translocated into the mesophyll tissues when larvae infest leaves. We identified an ATP-binding cassette (ABC) transporter, SpABCB5, as the mediator of this translocation and as an essential component of defense against leafminers. These data reveal that epidermal trichomes not only defend against free-living (ectophagous) herbivores through direct contact or rapid deposition of chemical defenses onto leaf surfaces, but also defend against endophagous leafminers by rapidly depositing chemical defenses into internal tissues. By delivering mines against leaf miners, trichomes offer new avenues for breeding crops with resistance to leafminers.

The application of in-field and aerial spectroscopy to assess functional and phylogenetic variation in plants has led to novel ecological insights and supports global assessments of plant biodiversity. Understanding how plant genetic variation influences reflectance spectra will help harness this potential for biodiversity monitoring and improve understanding of why plants differ in functional responses to environmental change. Here, we use a well-resolved genetic mapping population derived from Multiparent Advanced Generation Inter-cross (MAGIC) lines of Nicotiana attenuata to associate genetic differences with differences in leaf spectra between plants in a field experiment in their natural environment. We analyzed the leaf reflectance spectra using a hand-held spectroradiometer (350-2500 nm) on 616 fully genotyped plants of N. attenuata grown in a randomized block design. We tested three approaches to conducting genome-wide association studies on spectral variants. We introduce a new hierarchical spectral clustering with parallel analysis (HSC-PA) method. This method efficiently captured the variation in our high-dimensional dataset and allowed us to discover a novel association, between a locus on chromosome 1 and the 734-1143 nm spectral range, spanning the red-edge and near-infrared regions that are sensitive to leaf structure and photosynthetic activity. This locus contains a candidate gene annotated as carbonic anhydrase, an enzyme involved in CO₂ hydration and regulation of photosynthetic efficiency, suggesting a physiological link between variation in leaf optical properties and carbon assimilation. In contrast, an approach treating single wavelengths as phenotypes identified genetic signals highly consistent with HSC-PA, but suffered from massive statistical redundancy without pinpointing significant, interpretable features. An index-based approach, which reduces complex spectra to a few dimensionless variables, detected two significant associations for ARDSI _ C w $$ \mathrm{ARDSI}\_{C}_w $$ (a water-content-related index) with loci on chromosome 1 near genes annotated as a Zeta toxin domain-containing protein, and an Exocyst subunit Exo70 family protein. While these findings are biologically plausible, they represent a very narrow subset of the spectral variation captured by HSC-PA. The HSC-PA approach supports a comprehensive understanding of the genetic determinants of leaf spectral variation that is data-driven but human-interpretable and is thus a tool to discover genetic differences underlying intraspecific variation, a foundation of biodiversity.

Nicotine, tobacco’s addictive and potent insecticidal alkaloid, has shaped human history, agriculture, and the plants that produce it. However, the enzymatic steps and reaction mechanisms involved in nicotine biosynthesis remain elusive. Here, we reveal that the final coupling reaction is stabilized by glycosylation via a uridine diphosphate (UDP)-glycosyltransferase, reduced and activated by an A622, condensed through a stereoselective intermolecular Mannich-like reaction, sequentially oxidized by a berberine bridge enzyme-like (BBL), and finally deglycosylated by a β-glucosidase to yield nicotine. A 5-component metabolon assembles at vacuolar membranes to channel both nicotine biosynthesis and its transport. We reconstituted this metabolon both in vitro and heterologously in vivo. Abrogating any of these components depletes nicotine accumulations. A multidrug and toxic compound extrusion (MATE) transporter is essential for efficiently engineering nicotine production in heterologous plant species, which confers pest resistance. This work completes the nicotine biosynthesis pathway and provides critical insights into the intermolecular Mannich-like reaction, a fundamental mechanism for scaffold formation in many plant alkaloids.

Summary The effects of drought stress on stomatal opening dynamics, plant volatile organic compound (VOC) emissions and plant–insect interactions have been well‐documented individually, but how they interact mechanistically remains poorly studied. Here, we studied how drought‐triggered stomatal closure affects VOC emission and plant–trophic interactions by combining RNAi silencing, molecular biological and chemical analyses (GC‐MS) of a potato‐tuber moth‐egg parasitoid tritrophic system. Drought stress attenuated stomatal apertures and VOC emissions, which made the potato (Solanum tuberosum L.) plants more attractive to the herbivore but less attractive to the parasitoid. Stomatal aperture manipulations through StSLAC1 gene knockdown and chemical treatments (ABA and 5‐aminolevulinic acid) consistently affected drought‐triggered VOC emissions and plant–herbivore–parasitoid interactions, supporting aperture‐dependent VOC emission. RNA‐Seq analysis revealed that drought stress did not transcriptionally inhibit VOC biosynthesis. Collectively, our findings are consistent with the stomatal regulation of plant–insect interactions through the modulation of VOC emissions under drought stress. This highlights the intricate interplay between stomatal dynamics, VOC emission and plant–insect interactions.

Herbivore attack elicits jasmonate (JA) signaling which in turn elicits both anti-herbivore plant defenses and growth inhibitions. The resulting growth-defense trade-offs constrain the utility of JA-based plant defense inducers to enhance endogenous pest resistance. Here, we designed and screened selective JA receptor agonists by synthesizing 6-substituted 1-oxoindanoyl isoleucine (In-Ile) conjugates and their free-acid forms, structural mimics of the bioactive hormone (+)-7-iso-jasmonoyl-L-Ile. These compounds differentially activate JA responses through selective binding of specific COI-JAZ coreceptor complexes. Notably, In-Ile treatments enhanced rice resistance to brown planthopper attack, a destructive rice pest, under both laboratory and field conditions, without compromising rice's growth or yield. Mechanistically, this agonist activates the OsMYB55-mediated lignin biosynthesis defense receptor module [OsCOI1a/2-OsJAZs (3,4,6,7,12)] without activating the growth-suppression receptor module (OsCOI1b-OsJAZs). These findings demonstrate that synthetic JA agonists can provide nuanced manipulations of endogenous plant defenses without yield penalties-a promising biorational strategy for pest control in rice.

Summary In Solanum pennellii LA0716, three stylar UI (sui) factors and one pollen UI (pui) factor were shown to be involved in S‐RNase‐independent unilateral incompatibility (UI). However, additional pui factor(s) and the antagonistic relationships among pui and sui factors remain to be investigated. Quantitative trait loci (QTL) mapping, functional and genetic analysis of LA0716‐based crosses, and integrated multi‐omics data are used to identify pui QTLs and functionally dissect pui QTLs from various types of stylar UI. In addition to the reported pui10.1 (SpFPS2), two pui QTLs (pui6.2 and pui12.1) were identified. In LA0716 styles, the three pui loci additively attenuate stylar UI, among which pui6.2 and pui12.1 appear to antagonize the sui factor, SpHT, via independent mechanisms. Furthermore, pui12.1′s function was found to be conserved in the SC styles of Solanum habrochaites LA0407 and Solanum chmielewskii LA1028. Candidate genes linked to pui6.2 and pui12.1 are identified for further analysis. This study reveals several mechanisms for three newly described types of stylar UI and the corresponding pui QTLs in LA0716, which advance our understanding of the complex genetic mechanisms underlying UI in the tomato clade.

Lignin is a key structural polymer that also serves as a potent defense against biotic stress. Herbivore-induced, jasmonate-dependent pith lignification in Nicotiana attenuata plays a crucial role in defense against the stem-borer Trichobaris mucorea. However, the regulatory mechanisms underlying herbivore-induced lignification remain largely unknown. We demonstrate that NaMYC2 and NaMYC3 orchestrate pith-specific lignification in response to T. mucorea attack. RNA-seq analysis reveals that monolignol biosynthetic genes and polymerization-associated genes fail to be induced in Namyc2/3 double mutants upon T. mucorea attack. Among NaMYC2/3-dependent genes in the attacked pith, we identify NaTHT1, responsible for synthesizing the noncanonical monolignol N-FT. Using Natht1 mutants, we further show that N-FT plays a key role in stem defense. Additionally, we identify NaNEC1a and NaNEC1c, NaMYC2/3-dependent superoxide dismutases in the pith. Nanec1a/1c double mutants exhibit reduced lignification and enhanced larval performance, supporting a direct link between superoxide metabolism and induced lignification. Our findings reveal a NaMYC2/3-mediated regulatory network in the pith that integrates monolignol biosynthesis, lignin polymerization, and noncanonical monolignol biosynthesis, thereby enhancing stem defense against T. mucorea.

Survival in desert ecosystems poses significant challenges for plants due to harsh conditions. Plant microbiomes are thought to promote resilience; however, whether plant hormones, specifically strigolactones (SLs) and karrikins (KARs), shape plant microbiomes remains unknown. The recruitment of root-associated microbiomes in Nicotiana attenuata, a model desert plant, silenced in specific genes associated with SL biosynthesis (CCD7) and perception (D14), karrikin perception (KAI2), and in the shared receptor (MAX2), required for both pathways, was studied. SL and KAR signaling, with MAX2 as a co-regulator, fine-tuned the assembly of root-associated microbiomes, with unique and shared regulatory functions on bacterial microbiome recruitment, particularly in taproot. Significant variation among the different plant genotypes in bacterial diversity and composition in taproot and lateral roots provides a foundation for future research to explore how microbiomes function in plant resilience in these harsh environments.

Plants use two major defense strategies, resistance and tolerance, to counter herbivore attack; how these are coordinated remains poorly understood. Here, we demonstrate that damage by a specialist leafminer (Phthorimaea operculella) triggers tissue-specific responses in potato plants: compensatory growth in immature leaves, enabling tolerance, while resistance is induced in the mined leaf. The tissue-specific regulation of Sugars Will Eventually Be Exported Transporter 11 (SWEET11) mediates both responses. In unattacked sink leaves, SWEET11 is up-regulated, enhancing sugar import, promoting compensatory growth and herbivore tolerance. In contrast, SWEET11 is rapidly down-regulated in mined source leaves, inhibiting sugar export and increasing cellular sugar levels. The resulting sugar accumulations suppress SNF1-related protein kinase 1 (SnRK1) and thereby enhance herbivore resistance. SWEET11-mediated sugar signaling enhances jasmonic acid (JA)-mediated resistance by degrading MCPI1a and MCPI1b (metallocarboxypeptidase inhibitor 1), two JA-induced herbivory susceptibility factors. These findings reveal a dual role for sugar transport in regulating tissue-specific defense responses to herbivory.

Feeding and oviposition by phytophagous insects are both known to trigger defenses in plants. Whether these two defenses functionally interact remains poorly studied, although these interactions are likely important for pests with overlapping generations. Here we investigated the differences and interaction between feeding- and oviposition-induced plant defenses triggered by the brown planthopper (BPH, Nilaparvata lugens), which gregariously feeds and oviposits on rice. Analyses of host-plant transcriptomes, phytohormones, and direct and indirect defense compounds all show that BPH gravid females (GFs), but not nymphs and non-gravid females (NFs), strongly induce rice defenses. BPH nymphs and GFs prefer to feed on plants previously infested by nymphs over un-attacked plants, but are repelled by plants previously infested by GFs. Moreover, nymph feeding is found to reduce the attractiveness of rice plants to natural enemies and decrease egg parasitism by suppressing GF-induced volatiles that mediate indirect defenses in both growth chambers and paddies. Intergenerational interactions between oviposition- and feeding-induced plant defenses not only promote the development of the population of pest insects but may also contribute to the aggregation behavior of pest insects by suppressing oviposition-induced indirect plant defenses.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant, Nicotiana attenuata . Despite their ecological significance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Three multifunctional cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, NaCYP716E107) from N. attenuata were shown to oxidize the pentacyclic triterpene skeleton as evidenced by heterologous expression in Nicotiana benthamiana . NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of β-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of β-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6β position of β-amyrin/oleanolic acid. Three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata , suggesting the ecological significance of these specialized metabolite biosynthetic steps.One-sentence summary Three CYP716 enzymes diversify N. attenuata’s triterpenoid sector with potential roles in growth and development.

The feeding of piercing-sucking insect herbivores often elicits changes in their host plants that benefit the insect.1 In addition to thwarting a host’s defense responses, these phloem-feeding insects may manipulate source-sink signaling so as to increase resources consumed.2,3 To date, the molecular mechanisms underlying herbivore-induced resource reallocation remain less investigated. Brown planthopper (BPH), an important rice pest, feeds on the phloem and oviposits into leaf sheaths. BPH herbivory increases sugar accumulations 5-fold in the phloem sap of leaf sheaths and concurrently induces the expression of two clade III SWEET genes, SWEET13 and SWEET14, in leaf tissues, but not in leaf sheaths of attacked rice plants. Mutations of both genes by genome editing attenuate resistance to BPH without alterations of known chemical and physical defense responses. Moreover, BPH-elicited sugar levels in the phloem sap were significantly reduced in sweet13/14 mutants, which is likely to attenuate BPH feeding behavior on sweet13/14 mutants. In one of the two field seasons tested, the sweet13/14 mutants showed comparable yield to wild types, and in the other season, the mutants demonstrated stronger BPH resistance. These preliminary results suggested that the mutations in these SWEET transporters could enhance BPH resistance without yield penalties. Given that sweet13/14 mutants also exhibit resistance to bacterial blight pathogen, Xanthomonas oryzae pv. oryzae, these SWEET genes could serve as excellent molecular targets for the breeding of resistant rice cultivars.

Plant specialized metabolites (PSMs) are variably distributed across taxa, tissues, and ecological contexts; this variability has inspired many theories about PSM function, which to-date remain poorly tested because predictions have outpaced the available data. Advances in mass spectrometry–based metabolomics have enabled unbiased PSM profiling, and molecular biology techniques have produced PSM-free plants; the combination of these methods has accelerated our understanding of the complex ecological roles that PSMs play in plants. Synthetic biology techniques and workflows are producing high-value, structurally complex PSMs in quantities and purities sufficient for both medicinal and functional studies. These workflows enable the reengineering of PSM transport, externalization, structural diversity, and production in novel taxa, facilitating rigorous tests of long-standing theoretical predictions about why plants produce so many different PSMs in particular tissues and ecological contexts. Plants use their chemical prowess to solve ecological challenges, and synthetic biology workflows are accelerating our understanding of these evolved functions. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that 3 cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive 3-step oxidation reaction at the C28 position of beta-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2 alpha position of beta-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6 beta position of beta-amyrin/oleanolic acid. The genes encoding these 3 CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps. Three cytochrome P450 monooxygenases from Nicotiana attenuata participate in triterpenoid biosynthesis and may influence its growth and development.