Plant, Cell & Environment

Mildew Locus O (MLO) genes, initially identified as powdery mildew susceptibility factors, are increasingly recognized as multifunctional regulators implicated in diverse processes, including plant reproduction, root thigmotropism, and interactions with beneficial microbes. Recent evidence shows that MLO proteins can act as Ca2+-permeable channels in response to Rapid Alkalinization Factors (RALF) peptides in reproductive cells, pointing to broader roles in Ca2+-mediated signalling. In this study, we investigate the symbiotic clade IV member LjMLO4 in the model legume Lotus japonicus, focusing on its role in root development and responsiveness to LjRALF34 peptides. We show that LjMLO4 expression is strongly induced in root cells colonized by arbuscular mycorrhizal (AM) fungi, yet loss-of-function mutants exhibit only subtle AM-associated phenotypes. Instead, we uncover a previously uncharacterized function of LjMLO4 as a regulator of primary root growth and lateral root formation, acting even in the absence of AM fungal colonization and in a Ca2+-dependent manner. Heterologous expression in E. coli confirms that LjMLO4 facilitates Ca2+ transport, while genetic and physiological assays demonstrate its contribution to LjRALF34-triggered root growth responses and Ca2+ signalling. Together, these findings identify LjMLO4 as a molecular hub between peptide signalling, Ca2+ transport and root system architecture, highlighting how MLO proteins integrate developmental, nutritional and symbiotic cues.

Repairing damaged tissues is essential for the survival of all organisms. In plants, tissue injury rapidly triggers defense and repair programs. However, the molecular mechanisms linking early injury cue to the later stages of wound repair remain unclear. Here, we show that wounding of Arabidopsis leaves induces localized low temperature at the injury site, likely caused by evaporative cooling, which is accompanied by an activation of cold-responsive genes. Using thermal imaging combined with computer vision and deep learning, we developed a workflow to monitor the dynamics of wound healing in a quantitative, non-invasive and real-time manner. Mechanistically, we show that C-repeat Binding Factor (CBF) transcription factors are required for the activation of injury-associated cold response and downstream salicylic acid (SA) signaling. The CBF-SA module promotes lignin deposition and wound repair. Together, these findings reveal a link between a wound-induced biophysical cue and the tissue repair program.

Mechanical stimulation of the root triggers signal transduction involving Reactive Oxygen Species (ROS) and calcium, but their relationships are unclear. This study aims to clarify the temporal and spatial interrelations between calcium and ROS following a localized lateral compression of the root. We combined a microfluidic valve rootchip to apply controlled compression, with fluorescent probes and wide-field or confocal microscopy to monitor H2O2 and calcium dynamics in root tissues simultaneously. Pharmacological inhibitors were used to investigate the causal links between H2O2 and calcium responses. In response to compression, we observed transient H2O2 accumulation, with characteristics similar to the calcium response observed previously in the same microfluidic system. H2O2 and calcium response occurred in 3 kinetic phases: a fast calcium increase relying on mechanosensitive channels and external calcium entry, followed by a long-lasting H2O2 accumulation and a slow calcium increase depending on NADPH oxidase activity. H2O2 accumulated in all root tissues while calcium increases were confined to the root center. These results suggest that two mechanotransduction mechanisms are involved in root response to compression. One mechanism relies on plasma membrane mechanosensitive channels, triggering a fast calcium increase. Another independent mechanism, relying on FERONIA, induces H2O2 accumulation, which drives the slower secondary calcium increase.

We tested whether seasonal plasticity in photosystem II (PSII) heat tolerance aligns with leaf phenology in tropical trees, comparing evergreen and deciduous species across the wettopostwet transition in a seasonally dry tropical forest of the central Western Ghats, India. This transition, preceding droughtinduced senescence, represents the key window to assess true unstressed thermal plasticity. PSII thermal traits: damage onset (T5), damage midpoint (T50), and temperature between damage onset to full loss (T95-T5), decline width (DW) were quantified in 27 cooccurring species during the wet (27.5{square}{degrees}C) and postwet (31.6{square}{degrees}C) periods. Contrary to phenologybased predictions, PSII plasticity was not structured by leaf habit or successional status. Both T5 (+1.7{square}{degrees}C) and T50 (+0.9{square}{degrees}C) increased significantly across seasons, but responses were speciesspecific, with evergreen and deciduous trees acclimating similarly. The preventionversusforbearance tradeoff (T5 - DW relationship) remained conserved, though leaf habits diverged under postwet conditions. Thermal safety margins based on T50 were large, but T5 revealed vulnerable latesuccessional evergreens (Saraca asoca, Ficus spp.) and Careya arborea. These results show that PSII thermotolerance regulation operates largely independently of droughtavoidance phenology, indicating species identity and not leaf habit drives plasticity in PSII thermal response in seasonally dry tropical forests.

Grasslands are vulnerable to increasing drought with global warming, but process-based models lack the mechanistic knowledge required to predict the magnitude of drought impacts. While a plant hydraulics framework has been successful in advancing process understanding of drought responses in trees, and how drought responses vary across rainfall gradients, similar approaches have rarely been applied to grasses. Here, we quantified the progression of key drought response processes in sixteen dominant perennial grasses (seven C3 and nine C4) with differing climatic origins across eastern Australia. We found that stomatal closure, hydraulic impairment and leaf browning occurred concurrently, in contrast to the progressive sequence typically observed in trees. We also found that drought response traits were not correlated with species climate of origin. The early impairment of leaf hydraulic conductance and leaf browning along with the lack of correlation with climate of origin suggest that grasses may employ fundamentally different strategies to adapt to low water availability than trees. These results highlight the need for grass-specific parameterization of drought responses in process-based models.

Winter warming is altering plant exposure to cold events, yet its effects on seasonal cold hardiness dynamics remain poorly understood. Here we quantified bud cold hardiness across four dormant seasons in a boreal peatland forest whole ecosystem warming experiment. Across a +0.00 to +9.00{degrees}C warming gradient, we semi-regularly measured cold hardiness in two overstory (Larix laricina and Picea mariana) and two understory species (Chamaedaphne calyculata and Rhododendron groenlandicum). Warming reduced cold hardiness in fall and spring by delaying acclimation and advancing deacclimation. However, risk was only increased in late winter and spring for three species. Warming reduced snow cover, increasing temperature variability and cold damage to understory shrubs. Together, our results show that cold damage risk depends on species traits, microclimate, and seasonal timing.

Pattern recognition receptors (PRRs) mediate plant immune responses by detecting extracellular immunogenic patterns, including microbe-associated molecular patterns (MAMPs). PRR signaling is commonly assessed using assays such as reactive oxygen species (ROS) bursts, cytosolic calcium influx, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition (SGI), which are performed in distinct experimental systems, including seedlings grown on artificial media and soil-grown rosettes. Here, we systematically compare receptor kinase immune outputs triggered by the bacterial MAMPs elf18 and flg22 in Arabidopsis thaliana seedlings and rosettes across a range of concentrations. Rosettes exhibited greater sensitivity than seedlings in ROS assays, whereas cytosolic calcium responses measured using the Aeqcyt/pMAQ2 reporter were stronger in seedlings, correlating with reduced reporter transcript accumulation in rosette tissue. MAPK activation was consistently stronger in rosettes, whereas SGI assays revealed higher sensitivity to elf18 than flg22 in seedlings despite flg22 inducing stronger early signaling outputs. Together, these results demonstrate that canonical PRR-mediated immune outputs are differentially sensitive to experimental context and should not be interpreted as interchangeable measures of immune activation. These findings highlight the importance of considering experimental conditions when comparing immune responses across assays and developmental stages.

Rhizosphere oxidation is a key adaptive mechanism in reductive soil environments, in which oxygen released from roots alters rhizosphere redox conditions and regulates biogeochemical processes. Rice plants possess an internal oxygen transport system, and radial oxygen loss (ROL) from roots is closely associated with root development. However, the spatial patterns of ROL in soil and their relationships with root traits remain poorly characterized. In this study, we developed a multimodal imaging system that integrates planar oxygen optodes with X-ray computed tomography to simultaneously visualize rhizosphere oxidation and root development in rice. Daily time-course tracking of individual crown roots revealed dynamic changes in the spatial distribution and magnitude of rhizosphere oxygen in relation to root elongation and aging. Root thickness was positively correlated with dissolved oxygen levels near root tips. Genotypic comparisons further identified a cultivar with reduced rhizosphere oxidation despite possessing thicker roots among the tested genotypes, thereby indicating the involvement of additional physiological processes. Overall, these findings demonstrate that rhizosphere oxidation is regulated by root growth stage and thickness and dynamically modulated during root development.

Compound droughts, i.e. the co-occurrences of heat and drought, represent a serious challenge for temperate forest trees leading to significant losses in forest biomass. We studied the physiological response of Norway spruce (Picea abies) saplings to heat and drought individually, and in combination. Continuous measurements of leaf gas exchange and VOC emission allowed us to identify fast-response reactions, while discrete VOC and root exudate samplings added qualitative information on compositional changes. Additionally, we used 13CO2 and 2H2O label pulses to investigate C-allocation and root water uptake in response to stress. Heat as well as drought reduced assimilation rates in the saplings, whereas transpiration, leaf VOC emission and root exudation rates increased in response to heat. Drought alone increased VOC emission but decreased exudation rates. Combined heat and drought triggered an amplified response in both processes despite negative net CO2 assimilation rates. Label incorporation showed compromised water uptake capacity of drought-stressed plants and illustrated de novo C-allocation to VOC emission and root exudates. The results point at the high susceptibility of Norway spruce saplings to drought and heat. Combined stress resulted in synergistic responses in VOC emissions and root exudates, showing the detrimental effect of compound droughts on Norway spruce. HighlightIn this study, we found synergistic effects of heat and drought on carbon losses from leaf VOC emission and root exudates despite negative assimilation rates in Norway spruce saplings.

The article introduces a new Forest Stress Index (ISF) based on a plant hydraulic modelling approach rather than classical climatic drought indices. Unlike other index like scPDSI or SPEI, ISF is grounded in xylem embolism dynamics simulated with the mechanistic SurEau model. The goal is to better link climatic anomalies to tree physiological functioning and mortality risk. ISF is defined using a locally adapted ideotype characterized by an optimal P50 value under a reference hydraulic functioning threshold. Simulations are performed across Europe and France using multiple climate datasets. The index is robust to model parameterization choices and assumptions about plant functional traits. Results show strong spatial and temporal consistency and significant correlations with SPEI and scPDSI. However, ISF more strongly highlights extreme drought years and exhibits a more skewed distribution. Future projections under SSP5-8.5 indicate a widespread increase in hydraulic stress with strong regional contrasts. Overall, ISF provides a mechanistic and complementary drought indicator more directly linked to forest mortality processes.

The composition and quantity of root exudates are strongly influenced by physiological and environmental conditions, reflecting dynamic changes in plant metabolism. Although many studies report that root exudate metabolite profiles vary with plant phenology, few have disentangled the effects of phenological stage from those of nutrient availability. We collected root exudates from white lupin (Lupinus albus) grown in a fine phosphorus (P) gradient (5, 10, 20, 30 and 50 {micro}M P) in hydroponics at three developmental stages, performed untargeted metabolomics using GC-MS, and measured 10 above and belowground traits. Our results show that plant phenological stage exerts a stronger influence on exudate metabolomic profiles than variation in P supply. During leaf development, exudates were dominated by metabolites associated with carbon metabolism, whereas flowering was characterized by compounds related to secondary metabolism and cell wall turnover. Phosphorus influenced exudate profiles only at the flowering stage, with distinct profiles observed at 5 and 10 {micro}M P compared with 20-50 {micro}M P. These findings provide new insights into the temporal regulation of root exudation and demonstrate that plant developmental stage is a primary determinant of metabolic responses to phosphorus availability and, potentially, rhizosphere functioning under nutrient limitation. HighlightRoot exudate quantity and composition in white lupin is shaped more by plant phenological stage than phosphorus supply, with phosphorus effects emerging only during flowering.

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.

Increasing global temperatures and the rising frequency of heat waves pose a significant threat to plant reproduction. The reproductive phase is particularly sensitive to heat stress, yet the underlying mechanisms regulating thermotolerance during this stage remain insufficiently understood, despite significant advcances in its understanding during vegetative growth. Heat stress responses are largely controlled by heat shock factors (HSFs) and their downstream targets, including heat shock proteins (HSPs). Among these, HSP101 is essential for acquired thermotolerance and recovery from stress, while HEAT SHOCK BINDING PROTEIN (HSBP) acts as a negative regulator of HSF activity, modulating the heat shock response. Here, we investigated the impact of elevated temperature regimes on the reproductive development of Arabidopsis thaliana, with a particular focus on pollen development and fertility. Our results show that heat stress negatively affects pollen development in a dose-dependent manner, leading to reduced reproductive success. We confirmed the critical role of HSP101 in reproductive thermotolerance using the hot1-3 mutant, deficient in HSP101. Furthermore, we provide evidence that the hot1-3 mutant is tetraploid. The origin of this event is unknown, but it is tempting to speculate that disruption of heat stress responses and interference with meiotic processes may lead to whole genome duplication. Overall, this study provides new insights into the regulation of plant reproductive development under heat stress and highlights the importance of HSP101 in maintaining fertility. These findings contribute to a better understanding of plant responses to rising temperatures and may inform strategies to enhance crop resilience under climate change. Main ConclusionFlowering Arabidopsis plants adapt to long-term high temperature by shortening the flowering period and reducing their fertility. The study also demonstrated that the commonly used hot1-3 mutant is tetraploid.

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.

O_LIStored seeds are crucial repositories of plant genetic diversity. However, long-term storage inevitably causes seed deterioration and loss of viability, and chemical processes within the seeds during storage can influence germination and seedling establishment. Emerging evidence suggests that seed ageing can also affect traits of adult plants, yet the extent to which this phenomenon is relevant across species, particularly for wild plant species with high genetic variation, remains unclear. C_LIO_LITo address this, we focused on 14 grassland species and subjected their seeds to simulated long-term storage by exposing them to artificial ageing conditions (60% rH, 45{degrees}C). We then compared plants grown from the aged seeds with plants from fresh seeds in a common garden experiment. C_LIO_LIArtificially aged seeds germinated later, the developing seedlings had lower survival rates and reduced growth. Adult plants grown from aged seeds flowered later, produced fewer flowers, and had less biomass by the end of the first vegetation period than those from fresh seeds. The effect of the ageing treatment varied between species, but the trend was overall significant across species, with minor differences between perennials and annuals. Interestingly, in perennial plants, the effects vanished or were inverted in the second growing season, with plants growing from aged seeds flowering earlier and producing more biomass. C_LIO_LISynthesis. Our results show that seed storage affects seedling performance, plant growth, and flowering phenology. These direct storage effects should be considered when using stored seeds for species conservation, ecosystem restoration, or evolutionary research relying on stored seeds. C_LI

Cytokinin (CK) N-glucosides are the most abundant CK metabolites in Arabidopsis and most angiosperms, yet their role in cytokinin activity and response is unclear. Here, we examined metabolomic, transcriptomic, and proteomic profiles of seven CK N-glucoside conjugates in detached Arabidopsis leaves across a 144-hour dark-induced senescence (DIS) timecourse. All tested N-glucosides were found to undergo a slow conversion to their corresponding base forms at position-dependent rates, with N9-glucosides releasing base faster than their corresponding N7-glucosides. Conversion during DIS was strictly isoform-specific and not accompanied by coordinated induction of CK biosynthesis genes, arguing against de novo synthesis as the source of accumulated base. Despite progressive base accumulation, N-glucoside-treated leaves produced substantially fewer Differentially Expressed Genes than direct base application at comparable base concentrations, revealing a disconnect between hormone presence and transcriptional output. Unbiased model comparison identified the base:glucoside ratio as a stronger predictor of CK-Two Component Signaling (TCS) gene expression than absolute base concentration, though modulated by base-type-specific receptor affinities. Early proteomic profiling further revealed a coordinated response shared across N-glucosides but largely absent from base treatments. Together, these findings support that CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms. HighlightsPhysiology, metabolomic, transcriptomic, and proteomic findings here support CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms.

Transgenerational plasticity can shape plant phenotype and influence plant response to environmental changes in interaction with the current conditions. While how past stress interact with either current optimal or stress conditions is increasingly documented within a single plant, transgenerational plasticity remains particularly poorly understood especially at the metabolome level. In our study, we investigated whether heat stress induces transgenerational metabolic and phenotypic modifications along two successive clonal ramet generations of the sub-Antarctic aquatic plant Limosella australis. We performed untargeted metabolomic approaches and measured morphologic and performance traits, to assess both transgenerational plasticity of the metabolome and the phenotype. We found that heat stress remodelled the metabolic profile and influenced the foraging strategy of our clonal plant, and that some of these metabolic changes persisted into the first clonal generation. This one therefore adopted an intermediate growth strategy, even though culture conditions were optimal. By comparing differentially accumulated features between daughter ramets from heat stressed mother ramets and from unstressed mother ramets, we identified common and specific metabolites accumulation to heat stress response, belonging to diverse compound families. However, we did not observe any adaptative advantage and any metabolic imprint during another heat stress applied on the second clonal generation. This work provides especially new clues into how plant metabolome integrates and transfers previous stressed clonal generation's information.

Bacterial canker remains a major constraint affecting apricot production in South-East of France. It is primarily caused by Pseudomonas syringae, a Gram-negative bacterium, many strains of which exhibit ice nucleation activity. By promoting ice formation at relatively high subzero temperatures, ice nucleation-active bacteria may facilitate tissue disruption and pathogen entry. Concurrently, climate-driven shifts toward warmer winter-spring periods have advanced flowering phenology, increasing exposure to late frost events. Despite breeders having developed less susceptible varieties to bacterial canker and early flowering varieties, the link between these traits and frost sensitivity, an emerging risk in this location, remains unresolved. Here, we have evaluated the links between canker susceptibility and frost sensitivity using three cultivar pairs contrasting in disease response and flowering time. Ice nucleation temperature was measured in excised buds under controlled conditions throughout the frost-risk period, alongside field-based diameter variation monitoring over two years. Disease susceptibility (P < 0.001), phenology (P = 0.003), varieties (P < 0.001), locations (P < 0.001), and sampling date (P < 0.001) significantly affected nucleation temperature, whereas epiphytic bacterial abundance and xylem vessel diameter did not. Trees froze at higher temperatures in situ than in laboratory assays (1 to -2{degrees}C versus -3 to 4{degrees}C, respectively), indicating strong environmental modulation of freezing processes beyond Psy-like bacterial activity, which is reflected in contrasting disease susceptibilities (P < 0.001) and precocities (P < 0.001). These results shed light on the complexity of the freezing process in trees under natural conditions. We discuss the potential roles of microclimatic conditions and alternative ice nucleation sources beyond Psy-like bacteria in driving these physiological processes.

Crop models are essential for predicting climate change impacts on agriculture, yet their validation under multi-stress conditions remains limited. This study evaluated two widely-used wheat models, APSIM and STICS, using data from three Free-Air CO2 Enrichment (FACE) experiments (USA, Germany, Australia) combining elevated CO2 (eCO2), water deficit, and warming. Environmental characterisation using simulation-based stress indices revealed that intended "controls" frequently experienced hidden heat and water stress, meaning models were calibrated on crops already undergoing physiological adjustments. Evaluation of simulated yield and components revealed a clear hierarchy in prediction errors (RRMSE): unlimited conditions (3-9%) < single stress (4-27%, with a need to improve response to heat stress) < combined stress (17-123%). Elevated CO2 generally increased prediction uncertainty for crops experiencing water stress. Our results suggest that current stress functions from the models fail to capture the synergistic coupling between drought and heat stress. This highlights the urgent need for more mechanistic modelling to improve the reliability of climate change impact assessments.