Plant, Cell & Environment

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is regulated by both intrinsic and environmental signals and is associated with the existence of a highly controlled cytoplasmic tip Ca{superscript 2} gradient, whose disruption impairs RH development. The molecular mechanisms underlying the Ca2+ homeostasis fine tuning and the Ca2+ organellar contributions to the cytoplasmic pool remain unclear. In the model plant Arabidopsis thaliana, many efflux routes are present, including those that employ Ca2+-pumps from the Autoinhibited Ca2+-ATPase (ACA) family. Here, we identified that the ER localized ACA2, and to a lower extent ACA7, are crucial ACAs required to control RH growth. By using genetically encoded Ca2+ biosensors we showed that Ca2+-dynamics are compromised in the aca2-2 mutant, having lower cytosolic Ca2+ concentration [Ca2+]cyt and growth rate, showing an altered homeostatic calcium setpoint compared to Col-0. Accordingly, the ACA2 mutation changed the dynamics of [Ca2+]cyt oscillations coupled to growth rate, inducing longer periods and more regular oscillations in the dominant high-frequency range (around 22 s), and slower oscillations (around 1 min) in the low-frequency range. Finally, expression of ACA2 with changes in four putative Ca2+ binding residues (ACA2{Delta}Ca2+) failed to rescue the RH growth phenotype in the aca2-2 mutant. Collectively, our findings indicate that ER-localized ACA2 and possibly ACA7 are crucial for modulating cytoplasmic Ca2+ signals, possibly composing a critical part of a negative feedback loop, and their absence leads to impairments in RH cell elongation.

Tropospheric ozone (O) is a major atmospheric pollutant that affects plant carbon metabolism, redox homeostasis, and secondary metabolism, including the biosynthesis and emission of biogenic volatile organic compounds (BVOCs). However, the contribution of BVOCs to O3 tolerance, particularly in tropical woody species, remains poorly understood. Here, we investigated whether acute O exposure (cumulative AOT40 of 3497.82 ppb h) induces alterations in photosynthetic performance, redox homeostasis, and BVOC partitioning in Eugenia uniflora. We evaluated gas exchange, photosynthetic pigments, ascorbate and glutathione pools, emitted BVOCs, modeled intercellular BVOC concentrations, and the relative carbon cost associated with BVOC emissions. O exposure significantly increased net CO2 assimilation without affecting stomatal conductance, transpiration, leaf water status, or chlorophyll concentrations, indicating maintenance of photosynthetic performance. Carotenoid concentrations and total glutathione decreased, whereas glutathione redox status was maintained. O induced marked compound-specific changes in BVOC composition and partitioning. Several monoterpenes appeared exclusively under O exposure, {gamma}-elemene emission increased significantly, and the relative distribution of individual BVOCs between the modeled intercellular and emitted pools was altered. These findings show that the response of E. uniflora to acute O exposure was characterized by interplay among carbon assimilation, glutathione redox regulation, and BVOC partitioning rather than by increased total volatile emission. Enhanced carbon assimilation occurred without additional carbon loss through BVOC release, while changes in the modeled intercellular pool indicate that part of the volatile response remained within the leaf. Our findings highlight BVOC partitioning as an important dimension of the plant response to oxidative stress and demonstrate that emission measurements alone may not fully capture the fate and potential physiological role of volatile carbon under O exposure. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/743946v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@653af1org.highwire.dtl.DTLVardef@ca5forg.highwire.dtl.DTLVardef@1e641bforg.highwire.dtl.DTLVardef@1e68fae_HPS_FORMAT_FIGEXP M_FIG C_FIG BVOC Partitioning Contributes to Oxidative Stress Defence Under Acute O Exposure

Photorespiration significantly influences terrestrial carbon fluxes, yet empirical measurements of its variability across tree species and temperature conditions remain limited, constraining predictions of vegetation and climate models. We quantified apparent photorespiratory CO2 loss (Lapp) and its temperature response for seven temperate broadleaf tree species in northern Europe, using in situ O2-shift measurements in Uppsala, Sweden during peak summer. Apparent loss was derived as the difference between net CO2 assimilation under ambient (Anet) and O2-free conditions at three leaf temperatures (25, 30, and 35 {degrees}C), spanning typical and heat-wave scenarios. Apparent photorespiratory CO2 loss showed pronounced interspecific variation and increased with temperature, while net photosynthesis remained relatively stable. The ratio of apparent loss to net photosynthesis ({phi} = Lapp/Anet) rose sharply with temperature, reaching species-mean values up to 0.94 at 35 {degrees}C, indicating that photorespiration can represent nearly the entirety of net carbon gain under heat stress even when leaves remain net CO2 sinks. Suppression of photorespiration under N2 and associated changes in leaf temperature systematically reallocated photosynthetic electron transport: the fraction of ambient electron transport rate (ETR) allocated to net CO2 assimilation declined with temperature, whereas the complementary fraction allocated to apparent photorespiratory loss and other O2-dependent sinks increased, with ETR-based apparent loss and its proportional expression rising steeply across the 25-35 {degrees}C range. Together, these in situ flux and partitioning measurements reveal high variability and strong temperature sensitivity in apparent photorespiration among temperate trees. Compared to crop-based parameterisations, the {phi} values we report for temperate trees are substantially higher and more temperature-dependent, providing species-specific constraints that can improve Farquhar-von Caemmerer-Berry-type vegetation model representations of photorespiration in forest ecosystems.

The ability of seeds to survive until dormancy recedes and the germination requirements are met is an adaptive strategy. Proteomics improves our understanding of the mechanisms that control the adaptation to environmental heterogeneity. In this study, we investigated two European beech populations from different habitats that differed in dormancy and germination traits. We found that the populations exhibited different germination strategies, which were reflected in coordinated but quantitatively different proteomic reprogramming. The Miekinia population exhibited stronger accumulation of proteins involved in nucleotide sugar biosynthesis, S-adenosylmethionine metabolism, and flavonoid biosynthesis. Enhanced nucleotide sugar biosynthesis indicates more intensive cell wall remodelling and carbohydrate metabolism, which support embryo growth and faster germination. Increased S-adenosylmethionine metabolism suggests the epigenetic and hormonal regulation of germination differences between populations. Higher flavonoid biosynthesis indicates an enhanced antioxidant capacity associated with environmental protection. In contrast, the Wisa population showed stronger accumulation of proteins involved in RNA processing, suggesting tighter post-transcriptional regulation and proteome reorganization during germination. Consistent with its deeper dormancy and later germination, the Wisa population appears to rely more on RNA-level regulation, whereas the Miekinia population prioritizes metabolic activation. These contrasting proteomic profiles likely reflect population-specific physiological strategies associated with dormancy depth and adaptation to different climatic conditions. HighlightProteomic reprogramming reveals population-specific germination strategies in European beech, linking dormancy depth with contrasting metabolic activation and RNA-level regulation during the transition from dormancy to germination.

Temperate forests increasingly face extreme air temperature, but plant physiological responses, particularly alterations in carbon allocation or protection via volatile organic compound (VOC) emissions, remain poorly understood. We pulse-labelled well-watered saplings of Fagus sylvatica and Pseudotsuga menziesii in a controlled heat stress experiment with 13CO2 to quantify heat-induced shifts in CO2, VOC and C pool exchange, specifically analyzing compound-specific {delta}13C of terpenoids, water-soluble organic matter (WSOM) and dark respiration. Under heat stress, up to 50% of fresh assimilates were directed to maintenance respiration and 1-2% to VOC emissions, while net assimilation and water use efficiency decreased by 50-75% in both species. Heat directly affected metabolic processes and reduced turnover rates of fresh assimilates in F. sylvatica, but accelerated them in P. menziesii. Strong 13C labelling of some compounds, particularly acyclic ones, suggested increased de novo synthesis of specific terpenoids for heat stress protection. By tracing the fate of recently assimilated 13CO2 we demonstrate that heat stress reduces net carbon uptake and water use efficiency, disrupts turnover of C pools and increases carbon loss via respiration and de novo synthesis of specific VOCs, potentially diminishing net carbon uptake of forests under future heat extremes.

The root meristem is essential for stem cell maintenance and root development in plants. In Arabidopsis, Root meristem Growth Factor (RGF) peptides and their receptors regulate root meristem size through reactive oxygen species (ROS)-dependent signalling. RGF1-mediated ROS redistribution post-translationally stabilises the root meristem master regulator PLETHORA2 (PLT2). Although genomic studies suggest that RGF-receptor modules are evolutionarily conserved across land plants, their functional characterisation has remained largely limited to Arabidopsis. Here, we show that Oryza sativa RGF1-1 (OsRGF1-1) functions as a rice homologue of Arabidopsis RGF1 (AtRGF1). CRISPR/Cas9-generated Osrgf1-1 mutants exhibited shorter seminal roots, reduced root meristem size, and decreased superoxide (O2*-) accumulation. EdU staining further confirmed that cell proliferation activity was reduced in the Osrgf1-1 mutants. The Osrgf1-1 mutants were sensitive to low concentrations of chemically synthesised mature OsRGF1-1 peptide. This low dose of OsRGF1-1 peptide restored seminal root growth and O2*- accumulation in the Osrgf1-1 mutants but had no detectable effect on the wild type. Functional analyses using Arabidopsis rgfr receptor mutants further demonstrated that OsRGF1-1 is perceived through conserved RGF receptor machinery. Together, our findings provide the first functional evidence that the RGF1-receptor-ROS signalling module is evolutionarily conserved between dicots and monocots in the regulation of root meristem development.

Flower color has primarily been studied in the context of pollinator attraction, although effects on thermal energy balance are also important, especially in the context of global climate change. We used infrared imaging to compare petal temperatures of white versus pigmented cultivars of ten angiosperm taxa under controlled environmental conditions. Excised sets of flowers (n= 6 sets per species) exhibiting white, light, and/or dark anthocyanin (red to purple) coloration were mounted perpendicularly to the sun at mid-day, under clear sky, low wind (<1 m s-1) conditions. Sunlight was filtered through either UV-transparent or UV-opaque film, and petal temperatures were measured using an infrared camera after one minute equilibration. In all species, pigmented flowers were significantly warmer than lighter-colored conspecifics. Mean differences averaged +5.3{degrees}C for darker-colored versus white morphs, +2.9{degrees}C for lighter-colored versus white. Most warming was associated with visible wavelengths, but additional warming under UV-inclusion was also observed in some species. In situ observations of intact landscape plants under low-wind, high-light conditions corroborated experimental results, with differences exceeding 10{degrees}C observed in some taxa. Temperature differences >7{degrees}C were also recorded for purple versus white sections of the same flower in multicolored Viola and Petunia cultivars. Follow-up experiments using dark-pink and white varieties of Impatiens x hybrida corroborated well-known effects of sunlight intensity and wind speed on floral temperatures, helping to explain inconsistent reports in the literature. Our results clearly demonstrate that anthocyanin pigments can have significant and dramatic impacts on floral temperatures, which could be an important factor driving evolution of flower color. In the context of climate change, floral pigments could amplify the effects of rising global temperatures, negatively impacting plant reproduction and crop yields, especially on the warmer end of species ranges. Changes in flower color could also potentially induce shifts in pollinator communities, which could have community-scale effects.

Plant root cellular architecture and cell wall composition influence plant productivity, stress resilience, biotic interactions, and potentially soil carbon accumulation. This study establishes comprehensive compositional parameters for roots of a lowland switchgrass genotype, DVR3. Root traits were analyzed in 12.5 cm depth segments, from Zone 1 near the surface to Zone 4 down to 50 cm. Mean abundance ({micro}g/mg) for major cell wall components included cellulose 470 {+/-} 20, xylose 250 {+/-} 20, lignin 170 {+/-} 15, and total suberin 35 {+/-} 5. Composition and cellular anatomy varied with depth, in a partially coordinated manner. Cross sections showed extensive aerenchyma in mature root regions despite greater root mass density, corresponding to abundant lignin and cellulose. Deep roots were enriched for pectin-associated traits, including arabinogalactan II, homogalacturonan, and arabinose-associated linkages. Suberin content did not vary significantly, though Casparian strip formation, endoderm and exoderm thickening, and suberin surface staining progressed with development. Similar trends in root lignin and specific root length were observed for another lowland switchgrass genotype, AP13. These results suggest that it may be possible to genetically enhance native switchgrass root chemistry to promote soil penetration and below-ground carbon accumulation by reducing variability with development, potentially via cell-type specific adjustments. HighlightOlder, shallower switchgrass crown roots are enriched in lignin and cellulose, and deeper, younger roots are pectin-rich with juvenile cellular anatomy. A more uniform compositional distribution might enhance below-ground traits. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/744798v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@159de98org.highwire.dtl.DTLVardef@124d714org.highwire.dtl.DTLVardef@1a49c14org.highwire.dtl.DTLVardef@2fa67_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic summary of switchgrass root anatomy and composition across four 12.5-cm depth zones of a 50-cm root system. Zone 1 represents older, shallow roots and Zone 4 includes younger roots and root tips. Representative cross-sections show greater aerenchyma development in older roots than in young root tips. The compositional heatmap shows higher cellulose, lignin, and xylose in Zone 1, higher pectin and nitrogen in Zone 4, and relatively little variation in suberin across zones.

Chloroplasts comprise photosynthesis and other important processes. Plants protect chloroplasts from stresses including Cd accumulation. Common terrestrial plants, excluders apply a set of mechanisms to restrict Cd penetration to chloroplasts. Removal of accumulated Cd from chloroplasts should also be a beneficial strategy. However, we do not know whether excluder plant species have ability to remove Cd from chloroplasts. We used barley as a common excluder plant species. To barley plants, we applied a model with two stable isotopes 111Cd and 114Cd to distinguish Cd accumulated earlier and later. A portion of Cd absorbed by roots continued translocation to shoot for some days after the external source of Cd was changed from one isotope to another. Chloroplasts acquired new portions of Cd and lost part of Cd accumulated earlier; a total Cd content remained rather unchanged. Cd loss from thylakoids was detected in vivo and in vitro. Cd loss from stroma and envelope was observed in vivo but not in vitro. Therefore, barley has at least two distinct mechanisms for Cd removal from chloroplasts: one from thylakoids and another from stroma. We hypothesized diverse chlorophagy pathways as a potential mechanism for Cd removal from chloroplasts. Cd accumulation by chloroplasts was mainly light-independent. In chloroplasts, Cd accumulated in vivo was tightly bound and mainly located in thylakoids. In vitro, chloroplasts from Cd-treated plants accumulated much less Cd than chloroplasts from untreated plants in a previous study. This implies reorganization of transport across chloroplast envelope membranes. HighlightsO_LICd was removed from thylakoids both in vivo and in vitro C_LIO_LICd was removed from stroma and envelope in vivo but not in vitro C_LIO_LIIn chloroplasts, Cd accumulated in vivo was tightly bound C_LIO_LICd accumulation by chloroplasts was mainly light-independent C_LIO_LIRoot barrier slowed down Cd translocation to shoot but not halted it C_LI

Plant cold acclimation emerges from coordinated adjustments in photosynthesis, primary metabolism, and intracellular carbon allocation. Yet, the regulatory role of subcellular metabolite compartmentation in natural variation of cold acclimation remains insufficiently understood. Here, we investigated four Arabidopsis thaliana accessions grown either individually or in bulk to determine how growth configuration and genotype shape the metabolism of sugars and organic acids during cold exposure. Using non-aqueous fractionation, we quantified plastidial, cytosolic, and vacuolar sugar pools alongside whole-cell carbohydrates, organic acids, enzyme activities, photosynthetic parameters, and stress markers. A neural-network classifier revealed that subcellular sugar distribution together with sugar amounts and organic acids provided the strongest discriminatory power among accessions, surpassing photosynthetic traits and enzyme activities. Our findings demonstrate that natural variation in cold acclimation is strongly determined by genotype-specific subcellular metabolite architectures, and that the cultivation strategy modulates these intracellular signatures. We conclude that subcellular compartmentation of metabolites represents a cellular control layer for natural variation of cold acclimation and resilience in Arabidopsis thaliana.

Forest soils are an important source of volatile organic compounds (VOCs), yet little is known about how different tree species influence soil VOC emissions and the role of rhizosphere processes in mediating VOC release form roots. We analysed soil VOC emissions from the soil surface and bulk soil as well as from roots with intact rhizosphere and washed roots of Picea abies and Fagus sylvatica. Tree saplings were grown on natural forest soil, and VOC emissions and gas exchange of soils and roots were measured under controlled conditions using online gas analysers integrated into an automated system. To assess the contribution of rhizosphere soil and microbial communities to root VOC emissions, roots were analysed (a) without washing, preserving the rhizosphere, (b) water-washed, and (c) ethanol-washed (70 vol%) to minimize microbial contributions. Species-specific VOC emission patterns were observed in both soils and roots. P. abies showed higher total emission rates and a more diverse, terpenoid-rich VOC profile dominated by -pinene, {beta}-pinene, {beta}-myrcene, and -phellandrene than F. sylvatica. Notably, these differences were evident not only at the soil surface but also in root and litter free bulk soil. Root washing further revealed that the rhizosphere is a hotspot of terpenoid production in P. abies, with significantly higher monoterpenoid emissions from unwashed roots than from water or ethanol-washed roots. This study demonstrates how tree species shape net soil VOC emissions, potentially leading to cascading effects on atmospheric VOC concentrations, and highlights the importance of the rhizosphere in regulating belowground VOC production.

Concurrent exposure to heat and drought stress compromises legume productivity, yet their combined effects are rarely quantified systematically. We compiled a database of 18 studies covering seven legume species. From these, we extracted 929 physiological, biochemical, and yield-related traits and calculated actual-to-additive ratios to classify heat-drought interactions as antagonistic (ratio < 1), additive (ratio = 1), or synergistic (ratio > 1). Additive heat-drought relationships accounted for 59 % of all classifiable observations, 37% relationships were antagonistic, and 4% synergistic. The relationship varied with species, genotype, trait, and experimental conditions highlighting the complexity of combined abiotic stress effects. The results challenge the common assumption that concurrent stresses invariably exacerbate damage and underscore the need for more realistic, quantitatively defined stress treatments as well as frameworks that integrate trait-level responses into predictive models of crop growth and development. Our synthesis provides a quantitative foundation to understand legume phenotypes under the increasingly frequent co-occurrence of heat and drought stress and identifies research areas where further work is needed to improve insight into combined stress responses. HighlightsO_LICombined heat and drought responses were mainly additive or antagonistic. C_LIO_LIEvidence is biased toward few legumes and controlled environments. C_LIO_LIField-based, multi-species studies are needed to identify adaptive traits. C_LI

Accurately modeling carbon (C) allocation is essential for predicting crop yield and the performance of new cultivars in various environments. Most crop models allocate C empirically, using fixed partitioning tables or harvest indices that prescribe allocation without representing the underlying physiology, limiting their predictive power under novel conditions. A mechanistic alternative, in which C allocation emerges from local utilization and transport, could instead respond dynamically to environmental changes, source-sink perturbations, and organ-level trait modifications. To achieve this design, we integrated a utilization-transport-resistance (UTR) allocation model into the Soybean-BioCro crop growth modeling framework. We calibrated and validated the model using organ biomass data from two soybean cultivars grown at two CO2 levels over eight seasons, achieving accuracy comparable to partitioning-based models while predicting more reasonable carbon allocation fractions. Further, the UTR-BioCro model predicted leaf and stem total nonstructural carbohydrate concentrations with reasonable accuracy compared to experimental measurements across the 2022 growing season. A local sensitivity analysis of the model parameters indicated that the onset of reproductive growth influenced yield more strongly than utilization or transport parameters suggesting the timing of this transition as a potential target for crop improvement. Finally, the UTR-BioCro model reproduced yield responses to source-sink perturbations including shading and pod removal, and captured the qualitative response to defoliation without requiring scenario-specific tuning as most partitioning approaches require. By grounding C allocation in physiological mechanisms, this work provides a foundation for predicting crop responses across diverse environments and engineered traits, supporting crop improvement for a changing environment.

Ultraviolet-C (UV-C) radiation can be highly damaging to plants, yet its effects on gibberellin (GA) homeostasis are not well understood. In this study, we show that short daily UV-C pulse treatments (12 s, 1,200 J m-2) applied for seven days reduce plant height and delay flowering in Arabidopsis thaliana. Endogenous levels of the GA biosynthesis precursors GA12, GA53, GA15, and GA24, the bioactive GA4, and the GA catabolites GA34 and GA110 are all lower in UV-C treated plants than in untreated controls. These changes were accompanied by lower transcript levels of the GA biosynthesis genes KS, GA13ox1, GA20ox1, and GA3ox1, together with opposing changes in the expression of GA2ox genes. Exogenous GA4 restores growth in UV-C-treated plants, suggesting that reduced GA availability contributes to UV-C-induced growth inhibition. Consistent with this finding, the GA-signalling mutant gdella and the GA-biosynthesis mutants kao1 and kao2 show strongly reduced UV-C responses. Together, these findings highlight the importance of GA metabolism and signalling in the developmental response to repeated UV-C exposure, and suggest that exposure regimen influences the dynamics of UV-C-induced hormonal responses.

Plant roots exhibit remarkable developmental plasticity, resulting in the adaptation of growth direction and architecture upon environmental changes. Previously, we demonstrated that inorganic phosphate (Pi) triggers Arabidopsis roots to skew to the left when grown on tilted agar plates. This so-called 'phosphate-dependent skewing' (PDS) is caused by a right-handed (clockwise, CW) circumnutation of the root tip, which is driven by a left-handed (counterclockwise, CCW) cell file rotation (CFR) of epidermal cells in the root elongation zone, and involves the cortical microtubule cytoskeleton (Sheng et al., 2024). In the present study, we demonstrate that NaCl triggers a skewing response in the opposite direction and that all other helical movements are also reversed. Thus, 'Salt-Induced Rightward Skewing' (SIRS) is accompanied by a right-handed (CW) epidermal CFR, a left-handed (CCW) circumnutation of the root tip, and hence, a left-handed (CCW) helical root growth. Comparing different Na+- and Cl- salts revealed that SIRS is predominantly caused by cations, and can be induced by K+ and osmotic stress as well, although Na+ is most efficient. To get further insight into the mechanism underlying this response, we tested candidate genes from an earlier GWAS on root responses to salt stress (Deolu-Ajayi et al., 2019) for their potential involvement. This identified GLT1 and DOB1 as being involved in the root skewing response to Pi and NaCl, respectively. Our findings reveal that Pi and salinity elicit opposing effects on root circumnutation, and hence root skewing and growth direction. Understanding the molecular machinery driving this helical behaviour may help explain adaptive mechanisms, including changes in the spatial architecture of roots, and may facilitate the optimization of crop yield under abiotic stress conditions through breeding or crop management strategies. Our results also shed new light on halotropism, which is typically measured as a change in root growth direction to the right, which in the present study has been identified to represent SIRS.

Background and aims Plant volatile organic compounds (VOCs) change dynamically with plant development and in response to environmental conditions. However, their potential as non-invasive indicators of phenological progression remains poorly explored. In this study, we developed a framework integrating automated VOC sampling, time-resolved VOC profiling, and machine-learning analysis for the non-invasive assessment of plant phenology. Using soybean (Glycine max (L.) Merr.), we investigated whether development-associated temporal variation in VOC emissions could delineate and predict developmental phases. Methods We collected VOCs daily under controlled environmental conditions from 16 to 43 days after sowing, spanning the transition from vegetative to reproductive stages, using an automated sampling system coupled with thermal desorption-gas chromatograph-mass spectrometer (TD-GC-MS). To characterise temporal changes in VOC profiles associated with phenological progression, we analysed the daily VOC data using a multi-step pipeline combining statistical filtering and similarity-based network analysis. We defined VOC-derived developmental phases from similarity patterns in the VOC profiles, then developed and evaluated machine-learning models to predict these phases. Key results Seven VOCs exhibited distinct phase-dependent dynamics, including green leaf volatiles and monoterpenes showing characteristic temporal changes during phenological progression. Network-based clustering of VOC profiles resolved five developmental phases closely aligned with conventional developmental stages. A machine-learning model predicted these phases from the VOC profiles with high predictive accuracy on independent test data, demonstrating that phenological progression could be quantitatively inferred from VOC emission patterns. Conclusions Our findings support VOC profiling as a reliable and non-invasive approach for assessing phenological progression in soybean. By extracting temporally structured VOC signals, this framework captures developmental information that may be difficult to obtain through visual observation alone, particularly after canopy closure. VOC profiling offers a practical tool for monitoring crop developmental dynamics and has broader potential for plant phenotyping and precision crop management.

Soybean, a widely cultivated leguminous crop valued for its protein, amino acids, and oil, faces the challenge of maintaining protein levels, which have an inverse correlation with yield. Reducing leaf chlorophyll levels could increase seed protein levels without compromising yield; however, this is yet to be tested. Therefore, to understand the impacts of low chlorophyll mutations on soybean yield and seed composition, we screened and compared 25 low chlorophyll soybean mutants to their 11 dark green parents. PI548210 (Lincoln mutant) demonstrates a higher concentration of protein without affecting yield compared to its dark green parent PI548362 (Lincoln), suggesting it as a good candidate for further large-scale field trials. PI547555 (Y11/y11, Clark mutant) demonstrates a lower concentration of oil without impacting yield, alongside lower gross photosynthesis, but with chlorophyll levels in the pod and seed tissues that are comparable to its dark green parent PI548533 (Clark). These findings are consistent with the oil concentration of the soybean being influenced by pod and seed photosynthesis, which is correlated with pod height and row spacing. Chlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed. SIGNIFICANCEO_LIPI548210 (Lincoln mutant), one of twenty-five low chlorophyll soybean mutants, demonstrates a higher concentration of soybean protein without affecting yield compared to its dark green parent (Figure 1 and Table 1). C_LIO_LIPI547555 (Y11/y11, Clark mutant), a low chlorophyll soybean mutant, demonstrates a reduced concentration of soybean oil without impacting yield, alongside lower gross photosynthesis in pod and seed tissues compared to its dark green parent (Figures 3 and Table 2). These findings suggest that the oil concentration of the soybean is influenced by pod and seed photosynthesis, which is in turn influenced by pod height and row spacing (Figure 2). C_LIO_LIChlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed (Figure 5-6). C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/744892v1_fig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@4282dcorg.highwire.dtl.DTLVardef@9d565forg.highwire.dtl.DTLVardef@1918292org.highwire.dtl.DTLVardef@1359b1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Two low chlorophyll mutants are as healthy as their dark green parents. Lincoln and its low chlorophyll mutant, left; Clark and its low chlorophyll mutant, known as Y11/y11, right. It can be seen by eye that the plants have low chlorophyll (light green/yellow leaves) but a similar growth habit to their dark green parents. See Supplemental Figures 1-4 for contrast, where low chlorophyll mutants are stunted in growth compared to their dark green parents. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@657ec9org.highwire.dtl.DTLVardef@166e75borg.highwire.dtl.DTLVardef@df23c7org.highwire.dtl.DTLVardef@1a60124org.highwire.dtl.DTLVardef@194ed96_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONComparison of seed yield, weight, seed composition between low chlorophyll mutants and their dark green parents. ANOVA is used with linear mixed model (random effect = block, fixed effect = variety). Least squares mean is used to compare. For yield and seed composition, N=4 blocks. For leaf chlorophyll (SPAD), N=40. Yield is average yield per plant (g). n.s. = not significant. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/744892v1_fig3.gif" ALT="Figure 3"> View larger version (26K): org.highwire.dtl.DTLVardef@7a368aorg.highwire.dtl.DTLVardef@192b8f0org.highwire.dtl.DTLVardef@1abb738org.highwire.dtl.DTLVardef@89e978_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO Light response curve of low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Rates of net and gross photosynthesis of low chlorophyll (white) and dark green parents (black) pods under field conditions. Each dot represents a value (n=4) {+/-}SE. We assumed that the seeds greatly inhibited the transmittance of light through the pod and used photosynthetic photon flux density for a single-side. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@3f0528org.highwire.dtl.DTLVardef@16ba712org.highwire.dtl.DTLVardef@a5ab2aorg.highwire.dtl.DTLVardef@889254org.highwire.dtl.DTLVardef@3efa4f_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONPod photosynthetic parameters for low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Photosynthesis was measured 1 September through 15 September 2021 at the University of Illinois Energy Farm in Urbana, IL, USA. The statistical analysis was done using ANOVA with linear mixed model (alpha=0.05). N=4 {+/-} SEM for Clark and N=3 {+/-} SEM for Y11. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/744892v1_fig2.gif" ALT="Figure 2"> View larger version (23K): org.highwire.dtl.DTLVardef@a36c26org.highwire.dtl.DTLVardef@1116c8forg.highwire.dtl.DTLVardef@ee5e61org.highwire.dtl.DTLVardef@1766712_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533) differ in concentration of seed oil, which interacts with height of pod and row spacing. The box plots show the median (central line), the lower and upper quartiles (box) and the minimum and maximum values (whiskers). The statistical analysis was done using ANOVA with linear mixed model (n=3 blocks, alpha=0.05). Least squares mean is used to compare. N.s., non- significant in the analysis. A. Concentration of oil in low chlorophyll mutant seeds from the upper canopy decreased by 4% compared to the dark green parent (18.2% vs 19%) while there was no difference between them in the seeds from the lower canopy (20.2% vs 20.6%). B. Schematic layout of 2013 field setting showing two different row spacings. C. Concentration of oil in low chlorophyll mutant decreased by 2% in 38cm spacing (21.4% vs 22%) while there was no difference in 19cm spacing (21.3% vs 21.7%) in 2013 field. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/744892v1_fig5.gif" ALT="Figure 5"> View larger version (22K): org.highwire.dtl.DTLVardef@68e508org.highwire.dtl.DTLVardef@94a6ccorg.highwire.dtl.DTLVardef@152a187org.highwire.dtl.DTLVardef@1eae137_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5C_FLOATNO (greenhouse). Correlation between the level of leaf chlorophyll (x-axis: SPAD reading) and the level of immature pod or seed chlorophyll (y-axis, mg/g DW). Line represents the linear regression model. R-squared is a coefficient of determination, the percentage of the response variable variation that is explained by the linear model. Pod is labeled by the fresh weight of seeds it contained. A. Level of chlorophyll of 25-100mg pod (n=18). B. Level of chlorophyll of 100-200mg pod (n=17) . C. Level of chlorophyll of 25-100mg seed (n=17). D. Level of chlorophyll of 100-200mg seed (n=20). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/744892v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@167fd88org.highwire.dtl.DTLVardef@361472org.highwire.dtl.DTLVardef@786325org.highwire.dtl.DTLVardef@1b53855_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Levels of gene expression in chlorophyll synthesis pathway. A. CHL common pathway genes; Glutamyl-tRNA reductase (GluTR). Glutamate 1- semialdehyde aminotransferase (GSA-AT). ALA dehydratase (ALAD). Uroporphyrinogen III synthase (UROS). Uroporphyrinogen III decarboxylase (UROD). Protoporphyrinogen IX oxidase (PPO). B. Mg branch; Mg-chelatase (Mgch). Magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC). Protochlorophyllide reductase (POR). 3,8-divinyl protochlorophyllide a 8-vinyl-reductase (4VCR). Heme pathway; Ferrochelatase (FECH). Heme oxygenase (HO). Phytochromobilin synthase (HY). Data come from Severin et al (2010). RPKM, reads per kilobase per million mapped reads. DAF, days after flowering. The source seed is experimental line A81-356022 which was generated by introgressing G. soja (PI468916) into G. max (A81-356022). C_FIG

Successful establishment is a critical determinant of seagrass restoration, yet the molecular mechanisms underlying seedling acclimatisation to natural environments remain poorly understood. Here, we combined seasonal physiological observations, transcriptome profiling, and gene co-expression network analysis to investigate the mechanisms underlying the early post-transplantation phase of Posidonia oceanica, a dominant foundation seagrass species, following transplantation. Transplanted seedlings were compared with plants from adjacent natural meadows over the first six months after transplantation using leaf and root samples collected in spring, summer, and autumn. Tissue identity was the primary driver of transcriptomic variation, but transplanted seedlings remained transcriptionally distinct from plants in natural meadows throughout the study, with roots showing greater divergence than leaves, suggesting tissue-specific trajectories of post-transplantation acclimatisation. The early post-transplantation phase was characterised by the activation of genes associated with RNA processing, transcriptional regulation, and abscisic acid signalling. During a summer marine heatwave (28 {degrees}C), both plant groups induced conserved heat-response pathways, including heat-shock proteins and protein-folding mechanisms. Furthermore, transplanted seedlings maintained higher expression of genes involved in photosystem II repair and photoprotection and exhibited reduced leaf growth and extensive leaf necrosis, consistent with a greater requirement for photosynthetic maintenace under prolonged thermal stress. Gene co-expression network analysis revealed that regulatory networks governing structural integrity, hormone signalling, and defence were more stable in natural meadow plants, while transplanted seedlings progressively reorganized their gene co-expression patterns to resemble those of natural meadow plants, particularly in leaves. Our findings reveal tissue-specific molecular trajectories of acclimatisation during early seedling establishment and identify candidate molecular indicators of field acclimatisation and thermal stress responses, providing new mechanistic insights relevant to seedling-based seagrass restoration under climate change.

{middle dot} Globally, forest ecosystems face widespread mortality events. However, the independent impacts of distinct stressors, such as heat stress vs edaphic drought, remain poorly understood and physiological early warning indicators for tree mortality are urgently required. {middle dot} We exposed well-watered saplings of Fagus sylvatica, Pseudotsuga menziesii and Picea abies to summer heat waves and subsequent natural winter-desiccation. Physiological parameters (e.g. gas exchange, water uptake velocity via 2H labelling, and volatile organic compound emissions) were monitored throughout the growing season and survival was assessed regularly until subsequent spring to capture immediate and delayed mortality as a consequence of legacy effects. {middle dot} Heat exposure without soil water deficit, followed by winter desiccation, triggered species-specific mortality rates (51.8% F. sylvatica, 48.2% P. abies, 16.9% P. menziesii), with P. abies exhibiting significantly faster mortality response than the other species. Reduced water uptake, lower stomatal conductance, impaired photosynthetic efficiency, and altered VOC emissions distinguished non-surviving from surviving saplings months before visible damage in all three species. {middle dot} Heat stress drives mortality independent of edaphic drought, with sub-lethal physiological indicators detectable up to 10 months before visual signs. These early warning indicators could enable damage detection before lethal thresholds are crossed, offering new strategies for mitigating climate change-driven forest decline.

Accelerating photoprotective regulation to improve carbon assimilation is a promising strategy to increase crop productivity. Although rapid non-photochemical quenching (NPQ) relaxation has been validated as a target through metabolic engineering, it remains unclear whether conventional breeding has improved this trait. Here, we investigated whether more than a century of soybean breeding enhanced NPQ relaxation alongside light-saturated carbon assimilation and seed traits. We evaluated a historical panel of 24 soybean genotypes across vegetative and reproductive developmental stages by integrating NPQ relaxation, gas exchange parameters, xanthophyll-cycle pigment profiles, expression of key photoprotective genes (VDE, PsbS, and ZEP), seed number and seed weight. NPQ relaxation parameters were not consistently associated with genotype release year, seed number, or seed weight at either developmental stage. The only exception was the amplitude of the rapidly relaxing NPQ component (AqE), which was negatively correlated with all three variables during the reproductive stage. In contrast, genotype release year was positively associated with maximum net CO2 assimilation rate (Amax), maximum carboxylation rate of Rubisco (Vcmax), maximum electron transport rate (Jmax), seed number, and seed weight, while Amax and Vcmax were positively correlated with seed number and seed weight. These findings indicate that the greater photosynthetic capacity of modern genotypes was not accompanied by faster photoprotective response. Thus, photoprotective regulation has not kept pace with gains in photosynthetic capacity under field conditions. We conclude that rapid NPQ relaxation remains an important target for synchronizing photoprotection with the high photosynthetic capacity of modern soybean lines.