Plant Biology

O_LIEuropean beech (Fagus sylvatica L.) is a widely distributed, ecologically and economically important deciduous tree species in European forests, but is increasingly threatened by drought stress. Volatile organic compounds (VOCs) are ubiquitous plant metabolites that may serve as non-invasive biomarkers of drought stress, yet they have rarely been studied in European beech. C_LIO_LIIn this study, we examined VOC responses of European beech to experimental drought across diverse genetic backgrounds in a common garden. The 72 four-year-old beech saplings represented three genetic clusters, seven provenances (geographic seed sources), and 12 maternal seed families. Half of the saplings were assigned to the drought treatment and received no water for 14 days, while the remaining saplings served as controls and were watered as required. VOC profiles, quantified as peak heights of mass spectral features, were measured for all individuals during pre-drought, drought, and rewatering periods. C_LIO_LIWe found that pre-drought VOC profiles, in particular monoterpenes, varied significantly among genetic backgrounds. Experimental drought significantly altered VOC profiles, characterized by increased green leaf volatiles and decreased monoterpenes, oxidized terpenoid derivatives, and other fatty acid derivatives. Reductions in monoterpenes persisted after rewatering, indicating a drought legacy effect. Drought responses were largely conserved across genetic backgrounds, with significant seed family-specific responses detected for only three VOC features. C_LIO_LIOur findings suggest that VOC profiles are genetically structured yet highly plastic under drought and highlight their potential as non-invasive biomarkers for monitoring drought stress in European beech under climate change. C_LI

Heat tolerance determines the vitality of tree species under climate change independently of drought tolerance, but has been much less studied than tree water relations. We studied species-specific differences and the capacity for seasonal heat acclimation in Central Europes naturally most important tree species, Fagus sylvatica, in comparison with two exotic tree species (Fagus orientalis, Pseudotsuga menziesii) that are considered for silvicultural climate change adaptation in managed forests. Foliage of mature trees was incubated at temperatures from 35-50 {degrees}C for up to 4 h to simulate daily heat maxima during heat waves. The maximum quantum yield (Fv/Fm) of photosystem II (PS II) of dark-adapted leaves was measured, because the PS II is particularly sensitive to heat and its functionality can decide on plant survival under heat. Fagus sylvatica was much more tolerant to heat than Pseudotsuga menziesii, but weakly (albeit significantly) less tolerant than Fagus orientalis. Within its limits, Pseudotsuga menziesii showed high seasonal heat acclimation with constantly increasing tolerance during the growing season. Fagus orientalis, but practically not Fagus sylvatica, also acclimated to heat. This makes Fagus orientalis slightly superior over Fagus sylvatica in terms of heat tolerance, whereas the suitability of Pseudotsuga menziesii for silvicultural climate change adaptation is questionable. Strong heat acclimation, but also overall low heat tolerance, in Pseudotsuga menziesii might be the result of evergreenness, which requires the generation of both cold and heat tolerance during the year.

1. Stomata and leaf traits are key regulators of plant water use efficiency and are expected to have changed in response to rising atmospheric CO2 concentrations and climate warming over the past centuries. However, long-term data documenting such changes are rare. 2. We leveraged herbarium collections to track changes in stomatal characteristics and leaf traits in 656 individuals of invasive Japanese knotweed and its hybrid Bohemian knotweed collected across their European range and spanning 160 years of invasive spread. 3. We found that several functional traits including stomatal density and maximum anatomical stomatal conductance did not show significant changes over time but that plants adjusted their stomatal size and shape over time, and that these changes were associated with increased atmospheric CO2 levels. Interestingly, Reynoutria japonica showed increases in stomatal size and stomatal elongation, while the hybrid R. x bohemica showed a reduction in stomatal size. Traits also varied systematically with climates of origin. Plants from warmer origins with higher evaporative demands during the growing season had thicker leaves, lower SLA, smaller stomata and higher stomatal density, indicating more conservative water-use strategies. Stomatal density and gas exchange capacity co-varied with leaf structural traits, and there was a trade-off between stomatal size and number. Overall, fast leaf economic traits were associated with slow physiological traits. 4. Our results suggest that stomatal anatomical plasticity may enhance climate resilience by maintaining a stable maximum gas exchange capacity across environmental gradients. Herbarium collections provide a unique resource for reconstructing plant responses to historical environmental changes and understanding intraspecific trait variation.

Attracting specific pollinators can be favoured by natural selection to avoid reproductive interference between sympatric plant species. However, the ways in which fine differences in floral traits lead to the attraction of specific pollinators are diverse and unknown in many pollination interactions. We surveyed pollinators on three sympatric Aristolochia species (A. clematitis, A. pistolochia and A. rotunda) pollinated by Diptera to investigate if specific pollination occurs. To decipher if specific pollination may be mediated by different floral odours, we characterized the volatile organic compounds (VOCs) emitted by flowers and highlighted those VOCs electrophysiologically detected by pollinators in A. rotunda and A. pistolochia. Among the most abundant pollinators, Forcipomyia monilicornis was a specific pollinator of A. pistolochia while two Dasyhelea species were specific pollinators of A. clematitis. Forcipomyia aristolochiae and T. ruficeps were non-specific pollinators of A. rotunda, although they were more frequently found in A. rotunda flowers. The floral odours of A. rotunda and A. pistolochia differed significantly from each other and elicited specific electrophysiological responses in their respective pollinators. Although several pollinator species visit more than one Aristolochia species, those pollinators are preferentially found in one Aristolochia species. Selective attraction is likely mediated by specific VOCs.

Due to climate change, extreme weather events such as droughts are becoming more frequent and intense. This has a profound impact on plant performance and ecological interactions, including those involving herbivorous insects. The combined impact of drought stress and insect herbivory on plant metabolism has rarely been studied, particularly in woody plants. In this study, we investigated the influence of varying degrees of drought, both alone and in combination with herbivory by the leaf beetle Chrysomela tremulae, on the morphological and chemical characteristics of black poplar (Populus nigra) trees using a full factorial experimental design. We quantified morphological traits, volatile organic compound (VOC) emissions, phytohormone and amino acid concentrations, and phenolic profiles. Drought conditions increased the concentrations of salicylic acid (SA) and abscisic acid (ABA), while feeding induced ABA and SA. Amino acid profiles shifted significantly under drought conditions, particularly in beetle-infested plants. In contrast, salicinoids, which are the most important phenolic defense compounds in poplars, remained relatively stable. We also observed significant compound-specific effects on both constitutive and herbivore-induced VOC emissions. Our results demonstrate that drought and insect herbivory exert a joint influence on the chemical responses of P. nigra across multiple metabolic pathways. These findings highlight how the interaction between abiotic and biotic stresses can influence the defense chemistry of trees, which will consequently affect ecological interactions in forest ecosystems in the face of climate change.

Background and AimsPlants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. MethodsLarvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC- qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key ResultsLarvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. ConclusionsInsect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Issue SectionOriginal article

O_LIVolatile organic compounds (VOCs) mediate plant-plant signalling and may contribute to phenotypic differentiation among populations. However, the extent to which VOC-mediated signalling varies among locally adapted populations, and how VOC traits relate to major fitness-related traits, remain poorly understood. C_LIO_LIWe conducted a greenhouse experiment using two genetically and phenologically divergent Iberian populations of Arabidopsis thaliana. Plants were exposed to herbivory by Spodoptera exigua, after which we quantified herbivore-induced VOC emissions, VOC-mediated signalling effects on neighbouring conspecifics, and relationships between VOC traits, flowering time, and seed germination. C_LIO_LIHerbivory altered VOC composition, but overall VOC profiles remained broadly similar between populations despite strong divergence in life-history strategies, constitutive resistance to herbivory, and genetic structure. In contrast, correlations between VOC traits and fitness-related traits differed between populations and herbivory treatments. Nevertheless, receiver plants from both populations exhibited reduced herbivore damage after exposure to herbivore-induced emitters, indicating conserved VOC-mediated signalling. C_LIO_LIOur results suggest that herbivore-induced volatile signalling may represent a relatively conserved component of plant defence across locally adapted populations. In contrast, relationships between VOC traits and life-history variation may reflect population-specific integration of defence and fitness-related traits. C_LI

O_LIHerbivory is a major biotic stress for plants, triggering the induction and modulation of diverse specialized metabolites. Such induction responses are well studied for leaves and have been shown to depend on the herbivore feeding mode. Little is known about changes in flower metabolites and chemodiversity due to florivory type. Moreover, we lack an understanding of the intraspecific variation in such responses and whether these are spatially structured. C_LIO_LIThe aromatic plant Tanacetum vulgare, which shows high intraspecific chemodiversity in terpene profiles, was used to examine chemotype-specific metabolic responses of flower heads to infestation by the inflorescence-infesting aphid Macrosiphoniella tanacetaria or the flower-feeding beetle Olibrus spp. under field conditions. At peak flowering, each plant received both florivory treatments on separate stems, leaving one stem herbivore-free as a control. After four days, flower heads were harvested to analyze terpenes (GC-MS) and metabolic fingerprints (LC-MS). C_LIO_LIWe found stem-specific floral metabolic responses, with florivory altering specific chemical families and their chemodiversity. Levels of a few terpenes decreased following infestation, while none increased. Untargeted analyses revealed that aphid infestation had a lower effect on flower chemistry than beetle infestation, with aphid infestation mainly causing decreases and beetle infestation predominantly leading to increases in some metabolite intensities, but little overlap across treatments and chemotypes. C_LIO_LIOur results demonstrate that floral metabolic responses to florivory are spatially structured, florivore type-specific and shaped by plant chemotype. These findings highlight that the interplay between vascular organization, insect feeding mode, and intraspecific chemodiversity governs how flowers adjust their chemical defenses. C_LI One-sentence summaryTanacetum vulgare showed chemotype-specific responses to florivory by aphids (Macrosiphoniella tanacetaria) and beetles (Olibrus spp.), with aphids causing decreased and beetles increased levels of metabolic features within the same plant individuals, with little overlap in significant features across chemotypes.

Xylem and bark properties influence tree growth and drought resistance, yet their functional coordination and their environmental drivers remain unclear. We assessed xylem-bark coordination in branches of eight temperate woody Rosaceae species spanning different ecological preferences. We quantified xylem hydraulic efficiency and safety alongside bark traits governing permeability, hygroscopic water exchange, water storage, and anatomy, and evaluated phylogenetic signal and climatic associations. Bark water vapor conductance (Gbark) increased with maximum xylem hydraulic conductivity (Kh) and with xylem water potential at 50% loss of conductivity (P50), indicating species with more efficient but more embolism-vulnerable xylem developed more permeable bark. Species with higher Gbark showed reduced hygroscopic absorption time, consistent with faster rehydration from atmospheric water vapor. Both Gbark and P50 were phylogenetically conserved and covaried with climatic factors, namely air temperature, vapor pressure deficit (VPD), and isothermality. Species from warmer, high-VPD climates with greater diurnal temperature variability combined higher bark permeability with more vulnerable xylem, implying a shift from embolism avoidance to embolism tolerance strategies. Overall, xylem and bark hydraulics in Rosaceae evolved in concert along diurnal and annual gradients of evaporative demand, showing that drought resistance in woody angiosperms cannot be understood without considering bark traits alongside xylem function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/730605v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@14872c2org.highwire.dtl.DTLVardef@1662c8corg.highwire.dtl.DTLVardef@f6b4aaorg.highwire.dtl.DTLVardef@cf1aef_HPS_FORMAT_FIGEXP M_FIG C_FIG Caption: This study shows that xylem and bark in Rosaceae species form an integrated functional system in which xylem hydraulic safety, efficiency, and bark permeability are jointly tuned along diurnal and annual gradients of air temperature and evaporative demand.

Oaks (Quercus L.) are among the most ecologically important tree genera in the northern hemisphere, with an intricate evolutionary history reflected in a reticulated phylogeny. Oak diversity has been profoundly shaped by introgression and diversification, yet the Iberian Peninsula remains an understudied natural laboratory for understanding these evolutionary processes. We used RAD-seq to characterize 38 taxa (including nothotaxa) and investigate the evolutionary history of the Iberian white oaks, with an emphasis on hybrid swarms. Results led to a readdressing of Iberian white oak species, expanding our current understanding of the phylogeography of the European Section Quercus. Furthermore, molecular evidence led to the circumscription of two new subsections, reflecting the contrast between typical temperate and Atlantic distributed species (Group A), and the submediterranean marcescent oaks (Group B). The former unveiled the recovery of Q. estremadurensis and a Northwestern Iberian lineage represented by Q. broteroana and Q. orocantabrica as southwestern representatives of the broad European pedunculate oaks (Q. robur s.l.). The latter led to the validation of hybrid swarms, emphasizing the Iberian oak syngameon and the importance of gene flow to oak evolution. Ultimately, our approach advances the understanding of European white oak evolution across different evolutionary scales, establishing the Iberian Peninsula as an important reservoir of oak diversity.

Plants produce a vast diversity of specialized metabolites that function as chemical defenses against herbivores, pathogens, and competing plants. Many of these compounds also act as powerful tools for biological discovery, revealing fundamental cellular mechanisms through their effects on living systems. Among these metabolites, the harmala alkaloids from Peganum harmala (Syrian rue) possess cross-kingdom biological effects, including medicinal and neuroactive activity in humans, and allelopathic, growth-inhibiting effects on other plant species. However, the cellular processes in plants that are targeted by the harmala alkaloids are unknown. Here, we investigated the effects of the harmala alkaloids on plant growth and cell division using Arabidopsis thaliana as a model system. Of the harmala alkaloids, harmaline was identified as the most potent compound for root growth inhibition. Quantitative live cell imaging demonstrated that harmaline exposure causes progressive defects in cell division orientation and root cell morphology in a temporal manner. Furthermore, we identified harmaline-mediated phragmoplast orientation and morphology defects, pointing to a potential target related to phragmoplast guidance proteins. These findings position harmaline as a promising chemical probe for investigating the mechanisms that govern division plane positioning in plant cells and highlight a putative pathway by which harmala alkaloids exert allelopathic effects in competing plants. One sentence summaryHarmala alkaloids regulate cell division plane as an allelopathic mechanism

O_LIBackground and Aims: Chemodiversity is a fitness-relevant trait shaped by genetics, environment, and their interaction. Populus trichocarpa naturally inhabits broad climatic gradients and shows extensive variation in specialised metabolism. We investigated whether provenance and climate of origin imprint leaf chemodiversity and class-level relationships under common-garden conditions, and how these patterns relate to gene expression. C_LIO_LIMethods: Leaves from 87 P. trichocarpa genotypes representing 22 provenances from the west coast of North America growing in a common garden were profiled by untargeted FT-ICR-MS (1030 features) and targeted LC-MS/MS. A subset of 41 genotypes was subject to RNA-seq analyses. We tested whether provenance influenced multivariate patterns and whether metabolomic differences were related to geographic and climatic distance, where chemodiversity was quantified as Functional Hill Diversity. C_LIO_LIKey Results: P. trichocarpa metabolomes differed among origins despite shared growth conditions and showed distance-decay with both geography and climate. North-south extremes were well separated, and within-drainage samples shared high similarity. Flavonoid and isoprenoid pools strongly co-varied across individuals, whereas isoprene synthase activity did not predict total isoprenoids. Transcriptomes showed within-pathway coherence but limited overall provenance separation. C_LIO_LIConclusions: Leaf chemistry in P. trichocarpa retains signatures of geographic origin even under common-garden conditions. Coordinated investment in flavonoids and isoprenoids, together with among-origin differences in functional chemodiversity, reveals provenance-linked chemical fingerprints that complement genomic and metabolic trait data for climate-informed deployment. C_LI

Background and AimsThe development of unisexual flowers relies on the tight coordination of flower organ identity and sex determination. The genus Quercus is typically considered strictly monoecious, bearing fully segregated male and female flowers within the same individual tree. However, several reports of atypical flowering across the genus challenge this canonical view, suggesting that flowering in oaks may be more flexible than traditionally assumed. In this work, the dynamics of flower development in Quercus orocantabrica were examined to correlate contrasting floral morphologies with divergent molecular profiles. MethodsThe flowering phenology of Q. orocantabrica trees was closely monitored over several individuals and years, together with a detailed floral morphological analysis of male, female and atypical flowers. Key floral homeotic gene homologues were identified, and their expression assayed in the development of different flowers. Key ResultsRecurrent and widespread hermaphroditic flowering was detected in several Q. orocantabrica trees, frequently associated with unseasonal flowering events. Gene expression analysis of male, female and hermaphroditic flowers revealed a sex-biased expression of Q. orocantabrica B- and C-class genes, with the B-class gene QoPI in particular being tightly associated with the presence of fully-developed stamens. In addition, the expression of the C-class gene QoSHP contrasted with reports in other Fagaceae, highlighting a potential functional divergence of the C/D-class lineage within the family. ConclusionsThe results here depicted indicate that the dynamics of floral sex identity in oaks are more plastic than traditionally assumed, supporting a reinterpretation of oak reproductive biology based on a versatile and resilient framework responsive to different developmental contexts.

O_LIThere is growing interest in developing perennial cereal crops for sustainable production, yet key differences in trait syndromes between annual and perennial grasses and their influence on environmental adaptation remain poorly understood. C_LIO_LIWe measured 25 traits in 16 annual and perennial Hordeum species (45 accessions), including barley, grown over three seasons in a common garden. Using a phylogenetic framework and repeated transitions between annual and perennial forms, we (i) identified traits distinguishing these life strategies and (ii) tested how they relate to climate at the accessions origins. C_LIO_LIWild and cultivated barley are distinguished within the Hordeum clade by high growth rates and large organs, which may have predisposed wild barley to domestication. Annual and perennial accessions differed in resource allocation: annuals had higher harvest index and leaf and grain nitrogen, while perennials produced carbon-rich tissues and sustained vegetative growth. Seasonal temperature variation shaped trait syndromes: annual traits aligned with temperature in the driest quarter, reflecting selection under terminal stress, while perennial traits correlated with temperature in the wettest quarter, the main growth phase shaping long-term performance and survival. C_LIO_LIWe provide important information on traits and climate adaptations underlying perennial persistence and annual resource strategies, relevant for developing perennial cereal crops. C_LI

O_LIPhotoperiod is a stable seasonal signal. Although the photoperiodic flowering is well understood in short-day (SD) and long-day (LD) annual plants, regulatory mechanisms in perennials remain elusive. In a perennial woodland strawberry (Fragaria vesca L.), flowering is induced in SDs in autumn and plants flower following spring, while in plants with mutated FvTERMINAL FLOWER1 (FvTFL1), LDs induce flowering. C_LIO_LIWe investigated photoperiodic flowering of F. vesca through phenotypic and molecular characterization of transgenic lines and their crosses. We studied natural variation in flowering time and gene expression in European accessions, and explored their correlations with climatic, geographical and genetic origins. C_LIO_LIWe showed that FvGIGANTEA (FvGI) and FvCONSTANS (FvCO) activate FvFLOWERING LOCUS T1 (FvFT1) in LDs resulting in early flowering in fvtfl1 mutant, while in SD F. vesca, activation of FvTFL1 by FvFT1 reverses the photoperiodic requirement of flowering. In natural accessions, decreasing expression of FvFT1 and FvTFL1 towards colder climates in the east and north correlated with earlier flowering. C_LIO_LIWe define a photoperiodic flowering mechanism controlling floral transition of perennial F. vesca in autumn that differs from known mechanisms in annual and perennial plants. Our findings open new avenues to understand how perennial plants cope with changing seasons across climatic and geographical ranges. C_LI

AO_SCPLOWBSTRACTC_SCPLOWO_LIAirborne imaging spectroscopy enables species-level classification in hyperdiverse tropical forests, but accuracy varies enormously among species. We asked which ecological and evolutionary attributes make a tropical tree species spectrally separable. C_LIO_LIUsing 3,256 field-verified crowns spanning 169 species in a hyperdiverse moist forest in French Guiana, we tested seven hypothesised determinants of classification accuracy at species, pairwise, and individual-crown scales using random forest, beta regression, elastic net, and binomial GLMM analyses. C_LIO_LIPhenological regularity - the strength and consistency of seasonal leaf-cycling - was the single strongest predictor of separability, emerging as the top-ranked variable across all analyses. The presence of congeneric species in the classification pool also reduced accuracy, while broader phylogenetic isolation contributed in multivariate models. At the crown level, crown area was the strongest predictor of correct classification, while liana infestation reduced odds of correct identification by 38%. Leaf chemical traits did not predict separability. C_LIO_LIIt is the consistency of a species ecological signal - its phenological rhythm, spatial sampling, and freedom from canopy contamination - rather than any single functional trait, that determines whether it can be reliably mapped from imaging spectroscopy. C_LI

The Eastern skunk cabbage (Symplocarpus foetidus) is a thermogenic plant with remarkably precise thermoregulatory control. Its unique sexual dimorphism in thermogenic strategy, homeothermy in female-phase flowers versus diurnal heterothermy in male-phase flowers, offers a powerful natural framework to identify thermoregulatory signaling pathways. We used untargeted metabolomics and LC-MS/MS to compare metabolite profiles across four groups: homeothermic females sampled at midday and midnight, and diurnally heterothermic males sampled at midday and midnight. Pairwise comparisons and cross-filtering against circadian- and sex-specific metabolite changes yielded 190 candidate thermoregulatory metabolites. Principal component analysis revealed that thermogenic state (warm vs. cold) was the primary axis of metabolic variation, accounting for nearly 55% of variance. Metabolite set enrichment analysis identified significant enrichment for octadecanoid formation from linoleic acid, linoleic acid oxylipin metabolism, and eicosanoid metabolism via cyclooxygenases. Oxylipins including 9-oxoODE, 9(S)-HOT, 13(S)-HOT, and 15-OxoETE were among the compounds most strongly associated with low thermogenic activity. We propose that lipoxygenase-derived cyclopentenone oxylipins may suppress thermogenesis by inhibiting thioredoxin o (TRXo), thereby inactivating the alternative oxidase (AOX) pathway that drives heat production. These findings suggest convergence between plant and mammalian thermoregulatory pathways, with linoleic acid-derived oxylipins and prostaglandins emerging as conserved regulators of metabolic heat production across deeply divergent lineages.

Maple syrup production is strongly influenced by spring weather conditions, particularly the frequency and intensity of freeze-thaw cycles. However, marked individual differences in sap and sugar yields persist among trees growing under similar stand conditions, indicating additional tree-level sources of variation. This study aimed to explain inter-individual variability in maple yields in commercial high-vacuum syrup production using structural, morphological, and growth characteristics. We used terrestrial light detection and ranging to derive variables describing crown, stem, and whole-tree size, biomass, and structure in 38 mature sugar maples. We related these variables to individual yields, including sap volume, sugar content, and syrup production. Our models explained substantial inter-individual variability: 52% in sap sugar content, 44% in sap volume, and 47% in syrup production. Laterally expanded crowns were associated with higher sap sugar content, as were lower growth rates in the first 25 mm of wood. Sap volume was highest in large, heavily branched trees with crowns extending vertically along the stem. Projected crown surface area was the strongest predictor of syrup yield, with an estimated increase of 250 mL per 5.6 m2. These findings highlight the importance of multidimensional crown development in maximizing individual yields in maple syrup production.

Norway spruce (Picea abies) responds to attacks by the spruce bark beetle (Ips typographus) through the rapid activation of local defense mechanisms, but field studies can be difficult to standardize due to variable attack pressure and environmental heterogeneity. Here, we developed a phytotron-based assay that mimics early beetle-associated stress using insect-derived protein extracts, enabling reproducible molecular analyses under controlled conditions. Ten-week-old spruce seedlings were stem-treated with mock buffer or beetle protein extracts, followed by transcriptomic analyses of stem tissues and targeted metabolomic profiling of needles at 2 and 48 h post-inoculation. RT-qPCR analysis revealed rapid transcriptional activation of signaling and defense genes in Norway spruce, with NP-40-based protein extracts producing the most consistent early response. RNA-seq analysis revealed transcriptional dynamics, with 488 differentially expressed genes detected at 2 h and 84 at 48 h post-inoculation relative to mock-treated controls. Early responses at 2 h were characterized by activation of genes associated with immune perception and signal transduction. By 48 h, the response shifted toward accumulation of transcripts encoding defense proteins such as chitinases, defensins, proteinase inhibitors, and pathogenesis-related (PR) proteins. Importantly, a substantial proportion of differentially expressed genes overlapped with those previously identified in mature Norway spruce trees during pioneer bark beetle attack under field conditions, supporting the biological relevance of the assay. In contrast, targeted analyses of secondary metabolites performed in needle tissue revealed limited systemic changes across time points, suggesting that early induced defenses may remain largely localized to the stem. Together, these results demonstrate that beetle-derived proteins trigger a rapid and temporally structured defense response in Norway spruce seedlings and establish a reproducible elicitor-based platform for dissecting conifer immune responses and screening spruce genotypes for bark beetle resistance. HighlightBark beetle protein elicitors trigger temporally structured immune responses in Norway spruce seedlings that overlap with responses observed in mature trees, with rapid immune signaling at 2 h followed by defense protein accumulation at 48 h.

The current understanding of Crassulacean Acid Metabolism (CAM), including semi-controlled studies in pineapple, does not fully explain outcomes observed under commercial field conditions. Although empirical agronomy confirms a strong climatic influence on growth and development, mechanistic explanations at the metabolic level--particularly for photosynthate allocation--remain scarce. This study evaluated how environmental variation affects diel CAM outputs and how such effects can be agronomically interpreted. MD-2 pineapple plants were cultivated in contrasting natural environments across Costa Rica. Leaf samples were collected at defined phenological stages and at the end of CAM Phases I and IV. Field data revealed distinct metabolic balances between soluble sugar accumulation and nocturnal malic acid content. Under high radiation and temperature, sucrose concentrations increased markedly, reflecting shifts toward leaf growth over stem reserve storage. These shifts were associated with differences in harvest index, highlighting the role of sucrose dynamics in phenotypic plasticity. From a seed selection perspective, integrating CAM diel profiling into research protocols--together with physiological age (thermal units)--could provide a stronger basis for classifying planting material beyond current fresh-weight standards. Such integration would improve the prediction of photosynthetic performance, early establishment success, and ultimately, crop uniformity at harvest. Approximately 38% of diel metabolic patterns deviated from the classical CAM model, indicating dynamic regulatory mechanisms under field conditions. Understanding these patterns could improve the interpretation of yield variability, natural flowering incidence, and harvest index across agroecosystems. Recognizing these CAM particularities offers a path to bridge the gap between fundamental CAM biochemistry and agronomic application, enabling the development of precise management strategies that respond to metabolic plasticity under real-world conditions. Closing this gap is essential to enhance productivity, uniformity, and sustainability in pineapple agroecosystems.