Plant Physiology

Changes in intracellular calcium ion (Ca{superscript 2}) concentrations generate characteristic signatures that are decoded by specialized Ca{superscript 2}-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca{superscript 2} at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca{superscript 2}-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca{superscript 2} sensor that couples Ca{superscript 2} perception to JA-mediated defense and abiotic stress responses in plants.

Light is essential for plant growth and development. Sustainably feeding a constantly-growing human population will likely involve adapting crop plants to intercropping and high planting density by rational manipulation of light signaling. Here, we edited the tomato (Solanum lycopersicum) genome to generate lines with a light-stable version of ELONGATED HYPOCOTYL 5 (HY5), a master transcription factor involved in the integration of light and hormone signaling. Removing the tomato HY5 N-terminal domain required for interaction with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) prevented light-dependent protein degradation and resulted in a gain-of-function phenotype of short seedlings. Elongation growth was also compromised under proximity shade conditions either simulated by enriching white light (W) with far-red light (W+FR) or achieved by growing plants at a higher density. Transcriptomic analysis of gene expression changes after exposure to W+FR for 24h revealed a reduced number of shade-responsive genes in edited lines compared to unedited, wild-type controls, many of which are related to growth and hormone (notably auxin) biosynthesis and signaling. The reduced elongation observed in edited lines correlated with enhanced resistance to infection by viral, bacterial and fungal pathogens, both under low and high density conditions. These results indicate that our editing approach allows the generation of gain-of-function tomato plants in which HY5 is camouflaged to avoid COP1 recognition and eventual degradation. Our findings therefore provide a biotechnological tool to create more compact and pathogen-resistant plants amenable to high planting densities.

Photosynthetic carbon assimilation is sensitive to heat stress, with a key component being the thermal sensitivity of Rubisco activase (RCA). One approach to increasing heat tolerance of photosynthesis is to increase the thermotolerance of RCA. Here, we have used a transgenic approach to express RCA{beta} from Agave tequilana (AtRCA) in Setaria viridis. A second line was created where the endogenous SvRCA{beta} was substituted for AtRCA{beta} through complementation of a null mutant, {triangleup}rcaB (AtRCA[bKO]). In vitro assays showed that Agave RCA readily activates Setaria Rubisco, and that its thermostability is higher than that of Setaria RCA. AtRCA[bKO] plants exhibited higher CO2 assimilation at 25{degrees}C compared to WT and AtRCA plants, although Rubisco content and activation did not change. After six hours of heat stress, AtRCA[bKO] plants retained higher CO2 assimilation rates than AtRCA, however, after 24 hours CO2 assimilation declined to similar levels in all lines, suggesting that RCA may not limit carbon assimilation at the conditions tested. Taken together, our results show that substitution of RCA from a CAM plant into a C4 model allows normal growth and supports slightly increased carbon assimilation under control and short-term heat conditions.

Salinity severely impairs plant growth and development. Increasing evidence suggests that the cell wall (CW) plays a central role in salt stress acclimation, not only as a structural barrier but also as a dynamic sensor that activates downstream stress signaling pathways. To identify extracellular factors involved in cell wall remodeling and integrity signaling during salt stress, we performed a comparative proteomic analysis of the Arabidopsis seedling apoplast. Among the proteins that accumulated under salt stress, we focused on three members of the Subtilisin-Like Protease family (SBT1.1, SBT1.6, and SBT5.3), which have previously been implicated in the processing of signaling peptides and CW-associated proteins. Functional analyses revealed that sbt1.1 and sbt5.3 mutants exhibit enhanced lignin deposition under salt stress, suggesting altered CW remodeling during stress application. Given the established role of SBT1.1 in processing PHYTOSULFOKINE (PSK) peptides, we investigated the involvement of PSK signaling in salt stress responses. Mutants lacking the two PSK RECEPTORS (pskr1 pskr2) displayed reduced growth and increased lignification under salt treatment. Exogenous application of PSK attenuated salt-induced responses, including MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) phosphorylation, salt stress marker gene expression, and lignin accumulation, ultimately promoting root elongation under salt stress. Furthermore, PSK treatment mitigated salt-induced changes in CW composition. In cell wall integrity (CWI) mutants, PSK treatment failed to restore wild-type root growth under salt stress and induced a pronounced root bending phenotype in fer-4, indicating that CWI signaling influences PSK-mediated root growth responses. Together, these results support a role for PSK signaling in modulating CW-associated responses to salinity stress in Arabidopsis.

Most transgene applications require high and sustained expression, particularly in stably transformed plants. Achieving optimal transgene performance, however, depends on the combined influence of multiple genetic and regulatory factors. In previous work, we systematically evaluated the contribution of different genetic elements to transient transgene expression and demonstrated that terminators are key determinants of transgene performance by reducing transcriptional read-through and preventing transgene silencing. Here, we extend these findings by investigating the roles of terminators and introns in the expression of transgenes in stably transformed plants. Our results show that optimal transgene performance arises from the complementary actions of these two elements. Terminator choice was a major determinant of transgene expression levels, whereas introns played a critical role in maintaining expression stability. We further demonstrate a strong relationship between transgene expression levels and small RNA accumulation and show that intron-containing endogenous genes are enriched among highly expressed and stress-responsive genes, suggesting that intron-mediated protection from silencing may facilitate higher levels of gene expression and have contributed to the emergence and evolutionary retention of intron-containing genes.

In nature, plants experience complex combinations of environmental stresses. Prior studies using Arabidopsis thaliana show that transcriptional responses to combinatorial stress are unique and cannot be predicted from responses to individual treatments, making it imperative to study combinatorial stress in the monocotyledonous cereals that comprise the majority of our food supply. However, crop species are not easily amenable for such studies because of their large size, long generation time, and organismal complexity. In contrast, the monocotyledonous aquatic duckweeds are small, grow rapidly, and show reduced organismal and genomic complexity. Here, we explore the physiological responses to combinatorial stress in three duckweed species, S. polyrhiza, L. minor, and W. australiana and examine their potential to serve as multiplexable models. Focusing on S. polyrhiza, the most complex and best annotated species, we investigated transcriptional response to individual and combinatorial stress treatments. While most individual treatments elicit few transcriptional changes, combinatorial stress elicited a non-additive transcriptional response unique to each combination. Exploring the duckweed regulatory landscape, we found that regulatory regions near genes that were differentially expressed in combinatorial stress in S. polyrhiza resemble environmentally responsive regulatory elements found in terrestrial plants. Examined duckweed species show numbers and genomic distributions of regulatory elements similar to those of A. thaliana and maize, with subtle effects of genome size but none of organismal complexity. Taken together, our results establish that duckweeds respond to combinatorial stress in a manner similar to terrestrial plants and can therefore serve as high-throughput, multiplexable models for studying its molecular underpinnings.

Water availability in drying soils is most often spatially heterogeneous, requiring plants to adjust their root system architecture to sustain water uptake. This acclimation response to water deficit (WD) relies on the integration of local and systemic signals. The later and their mode of action remain poorly understood. Using hydroponic split-root systems in maize and Arabidopsis, we show that, with respect to control well-watered plants, a local WD does not affect shoot biomass but suppresses lateral root (LR) growth in the water-deprived compartment while promoting LR elongation in the well-watered compartment. Spatiotemporal analyses in maize revealed an early and transient (<2h) transcriptomic response of LRs triggered by both local and systemic WD signals. This response involved activation of jasmonate (JA) biosynthesis and signaling components, including MYC2 like regulators and JAZ repressors. It occurred in both the water-deprived and well-watered root halves of split roots, with no evidence of long-distance transport of JA. Furthermore, exogenous methyl jasmonate locally inhibited LR elongation, whereas the Arabidopsis JA insensitive jar1-1 mutant showed an enhanced systemic LR growth response to a local WD. Together, these findings demonstrate that JA acts as an early negative regulator that counterbalances the systemic enhancement of LR growth induced by a local WD, thereby fine-tuning root system architecture and water uptake during local soil drying.

Fluctuating light is common in field environments. Yet, the mechanisms by which C4 crops coordinate carbon and nitrogen metabolism during short-term carbon deprivation remain poorly understood. Here, we imposed transient darkness at different phases of the diel cycle to assess how the timing of light loss affects photosynthesis, carbohydrate turnover, amino acid dynamics, and sugar-sensing pathways in commercial sugarcane leaves. Early-day darkness significantly impaired photosynthetic induction and revealed a temporal disconnect between stomatal and metabolic limitations, whereas midday and late-day treatments caused temporary, time-specific disruptions in carbon assimilation. These shifts altered the balance between sucrose preservation and catabolic mobilization, leading to treatment-dependent changes in starch reserves and free amino acids. Core circadian components largely maintained their phase relationships, but their amplitudes varied across treatments, consistent with partial decoupling from carbon status. Darkness also reorganized energy signaling, with SnRK1 and DIN6 responses associated with greater declines in sucrose. Notably, trehalose-pathway transcripts showed marked changes in network connectivity, with ScTPSIIG consistently emerging as a highly connected candidate associated with photosynthetic performance, water-use traits, sugar sensing, and amino acid metabolism. Overall, these results indicate that the timing of carbon limitation and residual sucrose availability shape distinct metabolic responses, while trehalose metabolism provides a candidate regulatory layer coordinating carbon-nitrogen adjustment during the diel cycle, highlighting class II TPS proteins as targets for functional investigation of metabolic resilience in sugarcane.

Sporadically, all plastids produce dynamic tubular extensions in response to myriad stresses. Although the use of fluorescent proteins revealed these transient tubules to be stroma-filled and led to their being called stromules, the mechanism underlying tubule extension from a spheroidal plastid remained unknown. As plastids in viridiplantae have two bounding membranes the fluid stroma remains confined within the inner envelope membrane (IEM) while the outer envelope membrane (OEM) around the IEM forms the interface with the rest of the cytoplasm. Here, using a sucrose treatment for inducing tubules in the dark in transgenic Arabidopsis plants expressing different stroma and OEM-targeted fluorescent proteins we carried out comparative molecular-genetic and lipidomics analyses. Our data strongly suggest that plastid tubule formation occurs due to increase in bilayer-forming galactolipids such as digalactosyldiacylglycerol (DGDG) on the OEM. While the dynamic behaviour of stromules occurs within an external encasing OEM, stresses such as phosphate starvation and high light intensity cause the galactolipid-rich OEM-tubule projecting beyond the stromule to be shed into the cytoplasm. OEM shedding from long tubules with extended plastid outreach into the cell suggests their important role in dispersing plastid-derived, membrane-stabilizing, galactolipids to the rest of the cell to augment its resilience to stress.

Salinity tolerance is commonly associated with whole leaf Na exclusion and maintenance of K homeostasis, but whether spatial ion partitioning among functional leaf compartments contributes to stress adaptation remains unclear. Here, we investigated the relationship between bulk leaf and stomatal complex ionomes and gas exchange performance under salinity using two contrasting genotypes in both maize and faba bean crops. Maize generally maintained higher photosynthesis and stomatal conductance than faba bean under salt stress, which was associated with lower Na accumulation, stronger K retention and distinct ion partitioning patterns between bulk leaf tissue and the stomatal complex. Enrichment analysis revealed that stomatal complex ion composition provided information beyond bulk leaf ion concentrations, with Na and Cl- showing distinct distribution patterns associated with photosynthetic performance. Integrating physiological and ionomic traits further demonstrated that stomatal-complex ion traits captured additional variation in salinity responses. These findings identify the stomatal complex as a functionally distinct ionomic compartment and reveal compartment-specific ion partitioning as an important mechanism underlying species-specific salinity tolerance.

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

Salinity stress alters cellular ion homeostasis and photosynthetic activity, yet how guard cell chloroplast architecture contributes to salt adaptation remains poorly understood. Here, we investigated salt-induced chloroplast remodeling in guard cells of two faba bean genotypes, Fuego and Scoop, by integrating 3D chloroplast imaging, ion enrichment analysis and photosynthetic measurements. Salt stress induced distinct genotype-dependent changes in chloroplast morphology, with Fuego exhibiting pronounced chloroplast enlargement and reduced surface area-to-volume ratios under Na2SO and high NaCl, whereas Scoop showed treatment-dependent remodeling with larger chloroplasts under low NaCl and higher surface area-to-volume ratios under Na2SO and CaCl2. These structural responses were associated with differential Na and Cl partitioning at the stomatal complex surface. In Fuego, chloroplast size was negatively associated with photosynthetic rate, whereas Scoop showed positive relationships between chloroplast size and photosynthetic performance. Multivariate analysis further revealed coordinated associations among chloroplast architecture, ion enrichment and photosynthesis that distinguished the two genotypes under salinity. Our findings demonstrate that guard cell chloroplast remodeling is closely associated with genotype-specific salt responses and local ion partitioning. Integrating organelle structural plasticity with local ion homeostasis provides a spatially resolved perspective on the cellular basis of genotype-dependent salinity adaptation.

Plants produce diverse small interfering RNA (siRNA) molecules that modulate development, environmental responses, and immunity. Although transgene-derived siRNAs are traditionally viewed as mediators of gene silencing, whether they can actively regulate endogenous host pathways remains largely unexplored. Previously obtained Arabidopsis, wheat, and soybean plants expressing the sunflower gene encoding the transcription factor HaHB4 exhibited water deficit tolerance. Here, we show that expressing inverted-repeat constructs that generate HaHB4-derived siRNAs without producing the HaHB4 protein bypasses transgenic growth penalties and instead enhances vegetative vigor and reproductive performance. In Arabidopsis, these DCL-dependent siRNA-producing lines exhibited enhanced root growth, increased stem and pith areas, increased cauline branching, and higher seed yield under both optimal and water-limiting conditions. Transcriptomic analysis revealed convergent repression of biotic stress-related genes, accompanied by increased bacterial susceptibility and reduced sensitivity to salicylic acid-mediated growth inhibition, suggesting an altered balance between immunity and growth. Functional characterization of candidate endogenous HD-Zip I targets further showed that athb20 and athb53 mutants recapitulated the increased stem expansion and cauline branching of the RNAi lines, respectively, pointing to endogenous HD-Zip I genes as candidate mediators of these traits. Remarkably, these effects were observed in newly obtained transgenic soybean plants, where expression of HaHB4-derived siRNAs enhanced vegetative vigor under controlled growth conditions. Overall, these findings show that transgene-derived siRNAs act independently of protein function to rewire endogenous regulatory networks, providing a potential strategy to optimize crop architecture and yield.

Tomato (Solanum lycopersicum) is classified as a day-neutral plant, whereas its wild relatives exhibit delayed flowering under long day (LD) conditions due to higher activity of the SELF-PRUNING 5G (SP5G). In Arabidopsis thaliana, CONSTANS (CO) activates FLOWERING LOCUS T (FT), a homolog of SP5G, but whether and how CO integrates with SP5G in tomato flowering was unclear. Here, we demonstrate that SlCOL1 (the tomato CO homolog) delays flowering by directly activating SP5G in a PHYTOCHROME B1 (PHYB1)-dependent manner. Importantly, genetic and molecular analyses combining a photoperiod-responsive tomato line carrying the wild SP5G allele from S. pennellii, together with SlCOL1 and flowering-pathway mutants, revealed synergistic crosstalk among the photoperiodic SlCOL1-SP5G module, age-dependent pathway (mediated mainly by the microRNA156-SlSBP module), gibberellin (GA) pathway, and SINGLE FLOWER TRUSS (SFT) pathway. Mechanistically, we show that SP5G forms a complex with miR156-targeted SlSBP13 to directly regulate SFT expression, and that GA may interfere with SP5G activity. Together, these findings revealed a coordinated network that integrates multiple flowering signals to modulate both shared and pathway-specific targets. Our findings provide a significant advance in understanding the molecular regulation of tomato flowering and offer promising avenues for breeding strategies optimized for diverse environmental conditions and latitudes.

Peptide hormone signaling coordinates plant growth and osmotic stress responses, yet how the transition between these responses is regulated remains poorly understood. Here, we investigated the function of the rice PLANT PEPTIDES CONTAINING SULFATED TYROSINE 8 (OsPSY8) peptide in osmotic stress responses. OsPSY8 was predominantly expressed in root tissues under non-stress conditions, with preferential expression in lateral roots where it promoted root growth. Osmotic stress rapidly reduced OsPSY8 expression in roots through the OsWRKY24 transcription factor. Loss-of-function ospsy8 mutants exhibited enhanced osmotic stress tolerance, whereas OsPSY8 overexpression increased osmotic stress susceptibility. Transcriptomic analyses revealed that disruption of OsPSY8 activated stress-responsive pathways, including those associated with lignin biosynthesis, compatible solute production, cell wall remodeling, and reactive oxygen species (ROS) scavenging, and was accompanied by increased lignin accumulation in roots. In contrast, overexpression of OsPSY8 resulted in maintenance of growth-associated transcriptional programs while suppressing stress-responsive pathways under osmotic stress. Together, these findings identify OsPSY8 as an important regulator of the transition from growth to stress adaptation in rice and suggest that stress-induced repression of PSY signaling is required to disengage growth programs and activate adaptive responses during osmotic stress. Significance StatementCrop survival during drought depends on the ability to transition from growth to stress adaptation. Plant peptide hormones have emerged as important regulators of this critical transition, highlighting the importance of investigating their roles and potential for improving crop resilience. We show that a rice peptide hormone regulates this transition. Under non-stress conditions, this peptide hormone, predominantly expressed in rice roots, promotes root growth while suppressing stress responses. During osmotic stress, expression of the peptide hormone decreases, resulting in activation of stress-responsive pathways, such as lignin biosynthesis and reactive oxygen species scavenging. These findings demonstrate that a peptide hormone coordinates the balance between growth and stress adaptation in rice, with broader implications for understanding and improving crop resilience.

Rhamnogalacturonan I (RG-I) is a major pectin domain and an important wall component for cell-cell adhesion. Arabidopsis seed mucilage serves as a powerful model to identify and characterize pectin-related enzymes, since this gelatinous capsule is composed predominantly of RG-I. Although multiple glycosyltransferase (GT) families participate in pectin biosynthesis, only a limited number of their members have been functionally characterized in vivo or biochemically characterized in vitro. In this study, we characterized the biological functions of PEELING WALLS1 (PEEL1), a Golgi-localized GT106 protein that is related to known RG-I rhamnosyltransferases (RRTs). Knocking out PEEL1, but not its close paralog PEEL1-LIKE (PEEL1L), led to patchy mucilage release from seeds upon hydration. However, RG-I content was not compromised in mucilage extracted from peel1 or peel1 peel1L mutant seeds. Treatment of seeds with a cation chelator restored mucilage expansion but failed to rescue the underlying defect in epidermal cell adhesion. Histological experiments and transgene complementation demonstrated that optimal expression of PEEL1 is required for the adhesion of primary walls to the seed surface. Collectively, these findings identify PEEL1 as a previously unrecognized contributor to primary cell wall adhesion and suggest that different RRT-related proteins contribute to unique pectic structures or localizations in vivo, rather than total mucilage RG-I abundance.

Rising atmospheric CO2 concentrations consistently reduce mineral concentrations in C crops, yet the mechanisms driving this decline - dilution, reduced transpiration-driven mass flow, and altered root nutrient acquisition capacity - have rarely been tested simultaneously under field conditions. Here, we grew two soybean (Glycine max Merr.) varieties with contrasting yield responses to elevated [CO2] (Loda and HS93-4118) at the Soybean Free Air CO2 Enrichment (SoyFACE) facility and quantified the independent contributions of dilution, transpiration, and root uptake to observed changes in tissue mineral concentration and nutrient yield. Elevated [CO2] increased grain yield by 21% and 6% in Loda and HS, respectively, and reduced transpiration by 13-14% across both varieties. A decomposition analysis revealed that while dilution and reduced transpiration each exerted negative effects on tissue nutrient concentrations, with their combined effect representing a 31-40% potential reduction in whole-plant concentration, active root uptake responses were positive for nearly all nutrients, largely offsetting these losses and resulting in observed concentration declines substantially smaller than either mechanism alone would predict. Consequently, whole-plant nutrient yield increased under elevated CO2 for all elements measured, and grain nutrient yield increased for most. The relationship between seasonal transpiration and nutrient yield was steeper under elevated CO2 for most macronutrients, indicating that plants acquired more nutrients per unit water transpired, not less, under elevated CO2. These results demonstrate that root uptake is the dominant compensatory response to elevated [CO2] driven dilution and reduced mass flow in soybean, that nutrient yield is maintained or increased for most elements under elevated [CO2], and that Fe represents a physiologically and nutritionally significant vulnerability in future CO2 environments.

Vegetative desiccation tolerance requires specialised cell wall (CW) adaptations to withstand severe mechanical stress during dehydration and rehydration. While intracellular protective strategies in resurrection plants, including Ramonda serbica, are well documented, the CW response remains poorly understood. Here, we integrated immunocytochemical profiling, FTIR spectroscopy, quantification of CW-bound phenolics, transcriptomics, and ionically bound CW proteomics across hydrated (HL), desiccated (DL), and rehydrated states (R1-1h, R2-24h, R3-48h). Reversible CW folding was facilitated by condensed arabinogalactan-proteins (AGPs) and extensins, alongside a site-specific balance between pectin methylesterification and demethylesterification. Structural compaction was further reinforced by the accumulation of CW-bound hydroxycinnamates, which persisted through R1 phase. Moreover, basic 7S globulin, miraculin, -galactosidase, and two LEA4 protein family members were strongly accumulated during DL and R1, providing the first evidence of ionically CW-bound LEA proteins. Initial rewatering (R1) triggered a rapid transcriptomic reactivation of pectin-degrading/modifying enzymes, carbohydrate-active enzymes, and subtilases, accompanied by unesterified pectin enrichment. By 24-48 h (R2-R3), CW-bound hydroxycinnamic acids declined, and CW architecture, gene expression, and proteome profiles returned to baseline levels. Overall, our findings reveal a coordinated spatiotemporal apoplastic network - driven by glycoproteins, pectin modulation, CW-bound hydroxycinnamates, and LEA4 proteins - essential for rapid desiccation recovery.

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.

The Arabidopsis thaliana nonhost resistance proteins 2A (AtNHR2A) and 2B (AtNHR2B) play crucial roles in plant immunity as the single mutants Atnhr2a and Atnhr2b and the double mutant Atnhr2bAtnhr2a are susceptible to the non-adapted pathogen Pseudomonas syringae pv. tabaci that is unable to infect wild-type Col-0 plants. The localization of fluorescent versions of AtNHR2A and AtNHR2B to compartments of the endomembrane system together with their interaction with secreted proteins suggested a function in endomembrane-mediated secretory processes participating in plant immunity. Comparative apoplastic proteomics analysis between wild type Col-0 and the double mutant Atnhr2bAtnhr2a after treatment P. syringae pv. tabaci, revealed that AtNHR2A and AtNHR2B are indeed required for the secretion of proteins containing N-terminal signal peptides that occurs through the conventional protein secretion pathway. In this work, we leveraged these apoplastic proteomics datasets to identify proteins lacking N-terminal signal peptide and expected to be secreted through unconventional secretion pathway(s). We discovered that AtNHR2A and AtNHR2B are also required for the secretion of proteins through an unconventional secretion pathway that, intriguingly, included proteins previously associated with abiotic stress. These findings led us to define the subcellular dynamics of AtNHR2A and AtNHR2B, and through co-localization analyses and the use of vesicle trafficking inhibitors, we uncovered their trafficking pathways transitioning through Golgi-dependent and Golgi-independent pathways to ultimately reach the central vacuole. Our findings suggest that AtNHR2A and AtNHR2B participate in a multivesicular bodies-vacuole-mediated unconventional secretion pathway that results in the release of proteins involved in plant responses to environmental stresses.