AoB PLANTS

O_LIAquatic algae are key primary producers in the Arctic and Antarctic, yet how cold-water species respond to environmental change is poorly understood. The Polar Regions are increasingly exposed to frequent heat waves, leading to declining ice cover, increased light availability, and decreasing salinity in polar waters. We compared three phylogenetically related but geographically distant polar Chlamydomonas species to test how habitat history shapes algal responses to light, salinity, and temperature stress. C_LIO_LIWe assessed the growth, morphology, and photochemistry of psychrophilic Chlamydomonas acclimated to native-like (lower light, higher salinity) and climate-shifted conditions (higher light, lower salinity). Next, we exposed acclimated cultures to a lethal heat shock and observed how acclimation affects algal temperature stress resilience. C_LIO_LIAll three species acclimated to climate-shifted conditions grew rapidly but showed the greatest sensitivity to temperature stress, with rapid loss of viability and photosynthetic efficiency. In contrast, slow-growing cultures acclimated to native-like conditions exhibited significantly greater resilience to temperature stress. C_LIO_LIOur work is the first to directly link light and salinity acclimation with temperature resilience in psychrophilic algae, suggesting that fast-growing polar green algae may be particularly vulnerable to increasingly frequent heat waves, with major implications for primary productivity in polar environments. C_LI

O_LISubmerged macrophytes are central to freshwater ecosystems functioning but are declining globally under multiple anthropogenic stressors. We aimed to identify general patterns in physiological responses and interaction types, and to assess whether a mechanistic understanding of stressor interactions can be developed from published evidence. C_LIO_LIWe systematically reviewed 12,858 records, identified 172 relevant papers, and extracted effect sizes from 124 experiments included in the meta-analysis. C_LIO_LIMost studies examined combinations of nutrient enrichment, shading, toxic trace metals, warming, and emerging contaminants such as PFAS and microplastics, typically under simplified 2 x 2 factorial laboratory designs. Additive effects dominated (50%), while synergistic interactions were relatively infrequent (14%). Antagonistic interactions often reflected dominance of a single stressor or compensatory responses, whereas synergisms were most frequent with metals combined with co-stressors enhancing bioavailability. C_LIO_LIOur synthesis suggests that accumulated stressors cause negative, but not necessarily amplified, responses, although the limited number of experiments testing more than two stressors means synergistic effects may be underestimated. We propose Stuckenia pectinata as a model organism because of its cosmopolitan distribution, experimental tractability, and available genomic resources, and argue that expanding stressor complexity, duration, and taxonomic breadth will strengthen predictions of macrophyte responses and inform freshwater conservation under global change. C_LI

CoverCress is a new winter annual oilseed crop developed from field pennycress within the past 20 years. Field pennycress is commonly considered to be self-pollinated but little basic research has been published and there is some misalignment of conclusions. Our experience working with pennycress plant growth in greenhouse and field conditions over the past 13 years suggests that outcrossing is uncommon. We conducted lab, greenhouse, and field experiments to strengthen the body of work. Pollen viability kinetics analysis showed that longevity of pollen viability is negatively impacted by increasing temperatures and by direct exposure to light. Samples treated at 4C declined to 50% viability in 12 hours while it took just 2.5 hrs at 37C, and 1.6 hrs in full sunlight on a cool early April day. Cross-pollination was absent among greenhouse-grown plants flowering inside an agitated plastic pollen-containment covering. Across greenhouse tests, high rates of cross-pollination occurred only in an emasculation treatment that rendered flowers male sterile and opened the pistil to cross-fertilization. Field trials designed to measure pollen flow distance using a trackable fae1 knockout reporter gene failed to show detectable movement of pollen under field conditions in two locations. This data strongly suggests that domesticated field pennycress may be considered a self-pollinated crop and managed as such.

Belowground, plants are exposed to a wide range of biotic stresses that vary in severity and nature, including tissue damage, disruption of vascular connectivity, and depletion of assimilates. How plants adapt their root systems to cope with different types of belowground biotic stresses is not well known. In this paper we compare above- and belowground plant adaptations to three nematode species with distinct tissue migration and feeding behaviours to study mechanisms underlying tolerance to different types of biotic stresses. We monitored both green canopy growth and changes in root system architecture of Arabidopsis inoculated with Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita. This revealed three distinct phases in aboveground plant responses: (i) initial growth inhibition associated with host invasion and tissue damage, (ii) persistent growth reduction associated with nematode sedentarism, and (iii) late growth stimulus in more advanced stages of infection. Specific adaptations in the root systems further revealed fundamentally different stress coping strategies. Tissue damage and intermittent feeding by P. penetrans in the root cortex did not induce significant changes in root system architecture. Tissue damage to the root cortex and prolonged feeding on host vascular cells by H. schachtii induced secondary root formation compensating for primary root growth inhibition. Prolonged feeding on host vascular cell by M. incognita alone did not induce secondary root formation, but was accompanied by typical local tissue swelling instead. Our data suggest that local secondary root formation and tissue swelling are two distinct compensatory mechanisms underlying tolerance to sedentarism by root-feeding nematodes. HighlightHow plants utilize root system plasticity to cope with different types of biotic stresses by root feeding nematodes remains largely unknown. Here, we report on specific adaptive growth responses in Arabidopsis roots to three nematode species, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita, with fundamentally different strategies for host invasion, subsequent migration through host tissue, and feeding on host cells.

Spinach (Spinacia oleraceae) is a principal vegetable crop commercially grown in Controlled Environment Agriculture (CEA). Recent research suggests that root morphological and architectural differences among crop species influence yield, resource use efficiency, and environmental stress tolerance. These root traits may be exploited to increase yield, promote efficient nutrient use, and mitigate environmental stressors. This study measured differences between various spinach cultivars in CEA systems to reveal morphological and anatomical variation. We grew three spinach cultivars with different reported growing rates ( Income, Darkside, and El-Majestic) under NFT hydroponic and substrate-based systems in a controlled greenhouse environment over 45 days with destructive harvests at days 15, 30, and 45. Supplemental light (250 {micro}mol/m2/s) with 12-hour photoperiod and periodic fertigation was used. Harvests included the collection of leaf and root biomass, and scanning of root systems in WinRhizo software, measuring ten variables. On day 45, root cross-sections from orders 1-5 were embedded in JB-4 resin, sectioned, stained, and analyzed for diameter, vasculature, and rhizodermis characteristics. Results indicate that in spinach, differences in root system morphology are linked to cultivation systems over cultivar identity. Vascular and root anatomical alterations are minor compared to morphological differences in response to the cultivation system. Hydroponic-style growth systems are associated with the proliferation of fine-root ideotypes compared with substrate-based conditions. Such findings affirm previous studies, which suggest plastic root morphology in response to growth systems, and may be used to help create more resilient, resource-efficient cultivars. HighlightsO_LIIn spinach, root system morphology differences are linked to cultivation systems. C_LIO_LIRoot vascular and anatomical alterations are minor in response to cultivation system. C_LIO_LIHydroponic growth systems are linked to fine-root ideotype proliferation in spinach. C_LIO_LIFine-root ideotype proliferation may be a breeding target for CEA spinach. C_LI

Successful establishment of a species in a new range is a useful way to understand the impact of demography and selection on the evolution of globally distributed species. In particular, introductions influence genetic diversity and population structure in the introduced range in unpredictable ways. Additionally, introgressive hybridization is often associated with successful establishment in new ranges. In this study, we explore the impact of introgressive hybridization on the polyploid Capsella bursa-pastoris in the New York City metropolitan area. We find Capsella bursa-pastoris in the New York City metropolitan area likely originated from multiple introductions from northern Eurasia, and that populations across the New York City metropolitan area are generally panmictic. As with Capsella bursa-pastoris in Eurasia, we discover evidence of introgression from the diploid Capsella rubella in this population. By evaluating ancestry in regions across the genome, we find introgressed regions are rich in gene content and contribute to genetic diversity in this population. These results suggest that introgressive hybridization before introductions may buffer species from the negative effects of population bottlenecks and allow for successful establishment.

PremiseHerbarium specimens are increasingly used to extract morphological traits for ecological and evolutionary studies, yet the effects of tissue desiccation on trait measurements remain poorly understood. Here, we tested whether higher tissue water content leads to greater measurement changes after herborization (H1) and whether fresh trait values can be reliably predicted from herbarium measurements (H2). MethodsWe evaluated the reliability of herbarium-based measurements by comparing fresh and dried traits of leaves, flowers, fleshy fruits, and seeds across 262 individuals representing 133 Neotropical Myrtaceae species. Phylogenetic least square models and machine-learning regressions were used to test H1 and H2. ResultsLeaves and flowers generally shrank after herborization, fruits size metrics tended to increase, and seeds were largely unaffected. Water content was significantly associated with the magnitude of herborization effects in flowers and some leaf and seed traits. Fresh trait values were accurately predicted from herbarium measurements. Prediction errors were lowest for leaf traits, followed by fruits, flowers, and seeds. DiscussionThese results partially support H1 and support H2, indicating that herbarium specimens can be reliably used for trait analyses when organ-specific responses are considered, providing a practical framework to account for potential desiccation bias in functional trait research.

In cereal-legume intercrops, weed suppression is primarily driven by cereals, whose competitiveness is shaped by trait plasticity--morphological adjustments in response to the intercrop environment. However, how individual cereal traits respond plastically and contribute to system performance remains unclear, hampering improvements through breeding or system design. We combined field experiments with functional-structural plant modelling to quantify plastic responses of four cereal traits (tiller number, tiller angle, specific leaf area (SLA), and specific internode length (SIL)) and their effects on weed suppression and crop productivity. Field measurements revealed plasticity in tiller number, tiller angle, and SIL between sole crops and intercrops, while SLA showed minimal differences. Simulations showed that intermediate tiller numbers resulted in the strongest weed suppression and highest productivity, indicating an optimum, while more horizontal tillers suppressed weeds slightly better than vertical ones. Weed suppression increased with higher SLA values, while SIL showed a saturating response, increasing to intermediate SIL values and plateauing thereafter. In simulations with short-statured cereal phenotypes (low SIL), the reduction in cereal weed suppression was compensated by the legume component. This study demonstrates how FSP modelling can be used to investigate trait plasticity mechanisms and generate testable hypotheses about trait effects in complex intercrop systems. HighlightCereal trait plasticity shapes weed suppression in cereal-legume intercrops, with distinct response patterns per trait, while legumes can compensate for weakly competitive cereals, suggesting balanced competition over cereal dominance.

Timing of flowering is shifting with climate change. Although climate-driven shifts in phenology sometimes affect seed production, whether changing phenology will scale up to affect population dynamics of long-lived plants remains largely unknown, particularly under changing precipitation. Understanding how phenology affects persistence and extinction risk is a pressing need given contemporary biodiversity loss. We combined nearly a decade of demographic censuses and a four-year phenological survey in a rainfall manipulation experiment to examine the effects of experimental drought and irrigation on flowering phenology, vital rates (e.g., survival and individual growth), and population growth in the perennial herb Lomatium utriculatum. We found that drought advanced flowering by 3.3 days on average, and that earlier-flowering plants produced more seeds regardless of treatment. However, both rainfall treatments reduced seed production compared to controls. We quantified the phenology-mediated and direct, non-phenological effects of rainfall manipulation on population growth rates using integral projection models and a life table response experiment. Drought and irrigation increased {lambda} through increased individual growth, but these effects were partially negated by treatment-driven declines in seed output. In contrast, changes to seed production resulting from shifting flowering times had negligible effects on population growth. Our results suggest that climate-driven phenological shifts may only marginally impact population dynamics in perennial plants and highlight that assessing phenologys consequences for persistence under climate change must also account for direct demographic effects of the climate driver(s) themselves. SignificanceWill changing flowering times under climate change increase extinction risk in plant populations? Despite well-documented earlier flowering and its influence on the number of offspring produced, how changing flowering times will affect population growth or decline is still mostly unknown. We study this in a perennial wildflower subject to changes in rainfall. While we found that drought meant earlier flowering and that, all else equal, early flowering meant more seeds, these effects only marginally affected population growth. Instead, population growth was influenced mostly by rainfall-driven changes to individual plant growth. While shifting flowering times remain an important indicator of climate change, assessing extirpation in plants requires considering flowering times as only one of many life cycle processes changing with climate.

Premise of studyUnder increasingly frequent pollinator-limited environments, plants need to rely on modes of reproductive assurance such as selfing and cloning. However, few studies investigate the interplay between selfing and cloning in plants that can do both. Here, we explore mechanisms determining the relative expression of selfing and cloning throughout the European distribution of the wild woodland strawberry (Fragaria vesca) under a pollinator-free environment. MethodsWe established an outdoor common garden with 121 woodland strawberry genotypes from across Europe and excluded them from pollinators. For each genotype, we recorded reproductive traits and performed hand-pollination treatments. Key ResultsWe found a weak trade-off between cloning and selfing, driven by increased seed and fruit provisioning rather than flower production. The capacity to autonomously self-fertilize was determined by the lateral proximity of the anthers to the pistils (lateral herkogamy), but not by early inbreeding depression. Genotypes sampled at lower latitudes and altitudes were better at self-fertilizing and had smaller petals. The propensity to clone increased towards the east, where genotypes also had smaller petals, particularly at higher latitudes. ConclusionAt the species level, we detected a trade-off between the propensity for clonal reproduction and the capacity for self-fertilization. At a continental scale, the capacity to self-fertilize varied along a north-south gradient, whereas clonal propensity varied along an east-west gradient. Our results suggest that these two modes of reproductive assurance may compensate for reduced pollinator attractiveness (smaller petals) in regions where each mode is most strongly expressed.

Enset (Ensete ventricosum), a multipurpose crop domesticated exclusively in Ethiopia, serves as a staple food for millions of smallholder farmers. It is primarily cultivated as a monocrop in homegardens, leaving it vulnerable to climate change risks. One potential nature-based solution involves agroforestry systems; however, ensets response to canopy cover remains unclear. This study examined how scattered trees in enset farms affected microclimate and enset morpho-physiology in South Ethiopia. Trees significantly modified the microclimate conditions in enset homegardens. The average daily reductions in air, soil surface, and soil temperatures ranged from -0.5 to -1.9 {degrees}C, -0.4 to -2.1 {degrees}C, and +0.4 to -1.0 {degrees}C, respectively. The minimum soil moisture offset ranged from +0.8% to +5.7%. Although the tree identity effect on enset growth was negligible, planting position relative to the overstory trees significantly influenced enset responses. Most morphophysiological traits were higher under tree canopies, with progressively lower values at the edge and outside the tree canopy. In contrast, leaf dry matter content exhibited an inverse trend, aligning with the leaf economics spectrum. These results demonstrate ensets adaptability to canopy shade, suggesting potential for agroforestry expansion. Cultivar-specific shade tolerance and ideal shade levels to maintain enset productivity should be investigated further.

Improving photosynthesis is a promising approach to enhance sugar beet productivity. However, genetic variation in leaf photosynthesis and its relationship with disease resistance remain underexplored. We evaluated 98 sugar beet genotypes representing different breeding categories, including commercial F1 hybrids, seed-parent lines, and pollinator lines, in Hokkaido, northern Japan. Leaf gas exchange was measured during early growth under field conditions around the infection period of Cercospora leaf spot (CLS). To account for fluctuating irradiance during large-scale phenotyping, we applied a multilevel mixed-effects light-response model to estimate genotype-specific photosynthetic characteristics. Substantial genotypic variations in photosynthetic characteristics were detected. F1 hybrids exhibited higher photosynthetic capacity than breeding lines, whereas differences among breeding categories were unclear due to large within-category variation. Some breeding lines exhibited photosynthetic rates higher than those of hybrids, indicating exploitable genetic resources within the present genetic panel. We did not detect statistically significant trade-off between leaf photosynthesis and CLS resistance among 98 genotypes; in a subset of 19 genotypes analysed in detail, the relationship was even synergistic. Our results highlight the genetic diversity of leaf photosynthesis and its category-dependent structure, and suggest that selection for enhanced photosynthesis can proceed without substantial trade-off with CLS resistance. HighlightLeaf photosynthesis of 98 sugar beet genotypes showed significant genetic variation and dependence on breeding category. Active photosynthesis incurred minimal trade-off with Cercospora leaf spot resistance.

Phenotypic plasticity is a key mechanism by which plants adjust their traits to environmental changes. These phenotypic adjustments are driven by plastic changes in gene expression regulated by gene regulatory networks. Drought, a major selective force in Mediterranean ecosystems, provides a powerful context to examine how genomic plasticity translates into phenotypic responses. Here, we used Dianthus inoxianus, a drought-tolerant Mediterranean carnation, in order to characterize the phenotypic and transcriptomic plasticity in response to drought stress combining ecophysiological measurements with RNA-seq, gene co-expression and gene regulatory network analyses. Most of the phenotypic traits exhibited low plasticity in response to drought, except water and osmotic potential. At transcriptome level, we identified 57 plastic genes, suggesting that drought tolerance in D. inoxianus relies predominantly on constitutive gene expression. These plastic genes were enriched in processes typically related to drought response, such as cell wall components and abscisic acid (ABA) signaling. Some plastic genes belonged to drought-responsive modules, while others were hubs in different modules acting as inter-modular connectors. Furthermore, the regulatory network revealed that these plastic genes were strongly regulated by multiple stress-responsive transcription factors, and that drought-associated modules were regulated through both ABA-dependent and ABA-independent pathways. In addition, we identified contrasting patterns of canalization and decanalization, with immune and post-transcriptional regulation remaining canalized under drought, whereas photosynthesis and amino acid metabolism became decanalized, potentially releasing cryptic genetic variation. Overall, our results emphasise that drought tolerance in D. inoxianus emerges from a strategy combining preadaptation with targeted plasticity in key molecular pathways.

The ecological and evolutionary processes determining species range limits remain poorly understood. Ultimately, range limits depend on the species abilities to persist under heterogeneous conditions, by adaptive differentiation and phenotypic plasticity, including transgenerational effects. To investigate ecological differentiation and transgenerational effects in the clonal invasive knotweed, Reynoutria japonica, in Europe, we conducted a two-phase transplant experiment: plants sampled along the entire latitudinal gradient were planted in three sites located at the northern range margin, mid-range and near the southern range margin, and then re-transplanted among all three sites after two years. Biomass production and allocation were generally not associated with latitude of origin and previous growth at the same site did not promote performance. We therefore find no evidence that adaptive differentiation or transgenerational effects contribute to the wide distribution of R. japonica in Europe. However, at the northern site, with a 25% shorter season, knotweed plants invested much less biomass below-ground, and the pattern was further strengthened in plants that had grown in the northern site in the previous generation. Overwintering below-ground rhizomes are essential for survival and spread. We further explored limiting climate conditions in a species distribution model for the European range and found that mean annual temperature and temperature annual range are the main predictors of the European distribution of R. japonica. Taken together, our study suggests that low temperatures and associated short seasons may pose a limit to the broad environmental tolerance of R. japonica and restrict its northward spread by reducing below-ground biomass accumulation.

Heat-tolerant rice cultivars are essential for mitigating global warming impacts. Basal anther dehiscence length (BDL) is a promising visible morphological marker for heat tolerance through stable pollination. We investigated the effects of sowing date on anther morphology, pollination, and fertility under controlled high-temperature conditions (35, 37, or 39 {degrees}C at flowering). Three japonica cultivars-- Akitakomachi (early heading), Koshihikari (medium), and Hatsushimo (late)--were sown monthly over 3 months and grown in pots. At heading, the plants were exposed to the temperature treatments for 3 days, and the proportion of florets with [≥]10 germinated pollen grains on the stigma (GP10) and seed set were assessed. Among anther traits, BDL showed the greatest variation, with all cultivars from the second sowing exhibiting the shortest BDL. Analysis of variance revealed significant effects of genotype, sowing date, and their interaction on anther traits and fertility. Regression analysis indicated that fertility was associated with GP10, with BDL contributing significantly to GP10 in the late-heading Hatsushimo, together with maximum temperature at flowering. Thus, both genotype and environment shape anther morphology, pollination, and fertility, indicating that BDL plasticity and genotype-specific environmental responses must be carefully considered when using BDL as a breeding marker for heat tolerance. HighlightVariation in sowing date significantly affects anther morphology and heat tolerance in rice. Genotype-specific responses to the growing environment require careful consideration for reliable breeding assessments.

Black spruce (Picea mariana [Mill.] B.S.P.) is an emblematic and ubiquitous species of the North Americas boreal forest. While conifer breeding programs have traditionally focused on growth and wood property traits, the study of climate adaptation traits is becoming increasingly prevalent, given the predicted impact of climate change on North Americas boreal zone. Through this study, we aimed to identify genes associated with climate adaptation in black spruce across Canada. A total of 254 black spruce trees from 30 populations, covering most of the species distribution range, were sampled and genotyped for SNPs located in [~]5000 gene loci. Uni- and multivariate Genotype-Environment Association (GEA) approaches, namely LFMM and RDA, as well as an outlier method based on population differentiation (FST) were used to identify genes significantly associated with climatic factors. As such, a total of 77 genes carrying significant candidate SNPs were identified, among which 14 candidates were corroborated by at least two methods. Many of these gene SNPs were also confirmed at a smaller geographic scale, across west - east partitions corresponding to the two main black spruce historical lineages. Notably, significant gene SNPs were more frequently associated to moisture/aridity factors in the western part of the range, and more to temperature factors in the eastern part. The genes carrying these SNPs were also frequently associated to abiotic and biotic stress response. In the context of rapid climate change in the Canadian boreal forest, the results obtained within the framework of this study should support implementing gene conservation efforts while assisting prediction in black spruce breeding programs, which are instrumental to producing adapted planting stock for the large-scale reforestation efforts conducted annually across the Canadian boreal forest.

The growth of cultures and formation of mucilage blooms in reaction to salt stress of cyanobacterial cultures are investigated with a focus on the influence of pH. In non-buffered medium, cultures show their pH increasing from 6.5 just after inoculation, up to 11 during the exponential phase. We record the time-evolution of concentration and pH, with different initial OD0. In a second set of experiments, we extract the doubling time of the unbuffered cultures in comparison with those inoculated in pH-buffered BG11 media at four different pH from 6.3 to 10.5 : in the most acid media, all cultures die or grow very slowly. At pH = 10.5, we obtain the fastest growth for all four strains, allowing to qualify these cyanobacteria as being alkaliphiles, though for all strains with comparable initial OD0, the doubling time is shorter for unbuffered cultures. Following a previous study [31]), we finally investigate the influence of pH on mucilage formation and biomass uplift induced by salt stress, involving EPS floculation by cations. Our results show that operating in buffered media significantly influences the mucilage formation, though the observed regimes cannot be simply correlated to the pH value.

During drought, numerous compounds accumulate in plant tissues, but their physiological roles remain unclear - they may function as osmolytes, osmoprotectants, or merely arise as by-products of stress-induced metabolic shifts. We developed an experimental approach to link accumulation patterns with specific functions, using Scots pine (Pinus sylvestris L.) saplings subjected to water deprivation and subsequent rewatering as a model system. We monitored changes in relative water content (RWC) and osmotic adjustment dynamics, employed untargeted primary metabolite profiling for preliminary screening of compounds correlated with water status, and performed quantitative GC-MS and LC-MS analyses of selected metabolites. Major inorganic cations (K, Ca{superscript 2}, Mg{superscript 2}) were also quantified to assess their potential roles. Our results revealed that tryptophan, valine, and lysine - though generally present in low abundance - exhibited selective accumulation under severely reduced RWC ([≤] 70%), suggesting their involvement as osmoprotectants. Major organic acids, particularly shikimic acid, showed trends consistent with osmotic adjustment. Notably, neither sucrose nor inorganic cations appeared to function as primary osmolytes in this context. The proposed approach offers a viable strategy for identifying compounds involved in plant adaptation to water deficit, with potential applications in breeding programs aimed at improving drought tolerance. HighlightsAn approach to identify osmolytes and osmoprotectants was implemented Accumulation of Trp, Val and Lys was consistent with their role in osmoprotection Osmotic adjustment relied predominantly on organic acids than on inorganic ions Monosaccharides but not sucrose correlates with changes in needle water status

BackgroundPlants are exposed to various environmental challenges. With ongoing climate change, droughts and insect outbreaks are expected to become more frequent. Thus, a better understanding is needed of how different plant species respond to such single and combined challenges. This study investigated common versus species-specific responses to environmental challenges in three perennial plant species of different growth forms and whether responses differ intraspecifically among accessions. Clones of different accessions of the herbaceous species Tanacetum vulgare, the woody vine Solanum dulcamara, and the tree Populus nigra were subjected to similar control, herbivory, drought, and combined (drought and herbivory) treatments for the same periods. After the exposure, concentrations of foliar phytohormones and various morphological traits were measured. ResultsAcross all species, several foliar phytohormones and one of ten morphological traits responded consistently to the environmental challenges. Jasmonoyl-isoleucine was induced by herbivory and the combined treatment, abscisic acid (ABA) by drought and the combined treatment, and indole acetic acid by the combined treatment in all species. Root mass remained unchanged in all species. However, structural equation models (SEMs) revealed a shared regulatory pathway across species in which ABA connected treatment and root mass, indicating a common hormonal response potentially linking challenges to growth responses. Despite these common patterns, species-specific responses were pronounced. In P. nigra, a unique induction of salicylic acid was found under the combined treatment, while aboveground mass and root-shoot ratio remained unaffected by any treatment, in contrast to the other two species. Species-specific SEMs further indicated distinct phytohormone-mediated pathways underlying morphological variation. Phenotypic plasticity reflected these species-specific patterns, with none of the phytohormones or morphological traits exhibiting uniform plasticity across species. Intraspecific variation further shaped responses, as phytohormone and morphological trait plasticity depended on accession, indicating substantial accession-specific plant responses. ConclusionsOur results indicate that some responses to comparable challenges may be conserved across species, while others are species-specific. The combined treatment elicited the most pronounced responses, and such complex responses may become more frequent under current global change. Our study highlights that comprehensive understanding of plant responses requires systematic comparisons at both interspecific and intraspecific scales.

In C4 photosynthesis, incoming CO2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phosphoenolpyruvate carboxykinase (PEPCK). NADP-ME generates NADPH in the BSC chloroplast and species that use it as the major route for decarboxylation typically have dimorphic BSC chloroplasts with little or no photosystem II. They operate an energy shuttle: much of the 3-phosphoglycerate formed in the Calvin-Benson cycle diffuses to the MC, enters the chloroplasts and is reduced to triose phosphates that return to the BSC. In species where carboxylation occurs mainly via NAD-ME or PEPCK, BSC chloroplasts possess photosystem II. Indirect evidence indicates they nevertheless have the capacity to operate an energy shuttle. We show here that NAD-ME and PEPCK species possess large pools of 3PGA and triose phosphates and, for two examples of each subtype, opposed concentration gradients of 3-phosphoglycerate and triose phosphates to drive rapid exchange between the BSC and MC. Reasons for and consequences of the widespread operation of the intercellular energy shuttle in C4 plants are discussed. Highlight StatementAn intercellular energy shuttle in which 3-phosphoglycerate moves from the bundle sheath to the mesophyll and triose phosphates return to the bundle sheath is a general feature of C4 photosynthesis.