Plant Physiology

Aldoximes are amino acid-derived metabolites that serve as precursors of auxins and modulate phenylpropanoid production in Arabidopsis. However, the enzymes responsible for aldoxime production in Solanaceae remain unknown. Here, we report the identification of aldoxime-producing enzymes in tomato (Solanum lycopersicum) and examine how altered aldoxime production affects auxin production and phenylpropanoid metabolism. Through homology-based analysis, we identified five putative CYP79 homologs in tomato, among which SlCYP79DB32 and SlCYP79DB52 exhibited aldoxime-producing activity toward multiple amino acids, including phenylalanine and tryptophan. SlCYP79DB32 and SlCYP79DB52 converted phenylalanine into phenylacetaldoxime (PAOx), whereas only SlCYP79DB52 converted tryptophan into indole-3-acetaldoxime (IAOx). Stable isotope-labeled feeding experiments revealed that IAOx and PAOx can be converted to the auxins indole-3-acetic acid (IAA) and phenylacetic acid (PAA), respectively. Consistently, tomato plants engineered to overproduce IAOx and PAOx accumulated elevated levels of IAA and PAA. These plants also accumulated lower levels of phenylpropanoids. In Brassicaceae plants such as Arabidopsis and Camelina, aldoxime accumulation represses phenylpropanoid production by promoting degradation of phenylalanine ammonia-lyase (PAL). However, aldoxime accumulation did not reduce PAL activity in tomato, suggesting an alternative mechanism in this species. Transcriptome analysis revealed extensive transcriptional reprogramming in aldoxime-overaccumulating tomato plants, including upregulation of stress- and defense-related genes. Despite the observed reduction in phenylpropanoid levels, transcript levels of most phenylpropanoid biosynthetic genes were not decreased, suggesting possible post-transcriptional regulation of this repression. Together, our findings demonstrate that aldoximes can serve as intermediates in auxin biosynthesis in tomato and reveal that aldoxime-mediated repression of phenylpropanoid metabolism extends beyond Brassicaceae.

Rapid Alkalinization Factor (RALF) peptides regulate plant growth and cell wall signaling, but their roles in fruit development remain unclear. Here, we characterized tomato (Solanum lycopersicum) fruit-associated RALF peptides and their interactions with leucine-rich repeat extensins (LRXs). Expression analyses identified SlRALF5, SlRALF7, and SlRALF10 as the main fruit-expressed RALFs. SlRALF10 was associated with early fruit development, whereas SlRALF5 and SlRALF7 remained expressed during ripening. Sequence analyses showed that SlRALF5/7 retain conserved motifs of canonical RALFs, while SlRALF10 displays divergent structural features and altered charge distribution. Synthetic SlRALF5 and SlRALF7 inhibited root growth and induced extracellular alkalinization, whereas SlRALF10 lacked both activities. Co-immunoprecipitation assays showed that all three peptides interact with the fruit-expressed proteins SlLRX2 and SlLRX5. Structural modeling predicted distinct electrostatic properties for the SlLRX5/SlRALF10 complex compared with SlRALF5. These results reveal structural and functional specialization among tomato fruit RALF peptides and suggest that distinct SlRALFs may differentially respond to cell wall remodeling during fruit development and ripening.

Carotenoid accumulation underlies fruit color and nutritional quality in squash (Cucurbita pepo). One pair of dominant genes, B and L-2, have been long known to interact epistatically, substantially boosting carotenoid accumulation and producing intensely orange-fleshed fruit. However, their molecular identities and regulatory mechanism are unknown. Here, we show that B encodes a truncated H subunit of magnesium chelatase (CpCHLHB) and L-2 encodes a homolog of Arabidopsis Pseudo-Response Regulator 2 (CpAPRR2-A). Significantly, expression of phytoene synthase (CpPSY-A), which encodes the major rate-limiting enzyme in carotenoid biosynthesis, was dramatically upregulated in fruit of B/B L-2/L-2 plants compared with b/b L-2/L-2 or B/B l-2/l-2, showing that the B and L-2 interaction affects CpPSY-A transcription. A similar upregulation was also observed in Arabidopsis gun5 L-2 transgenic plants, where gun5 is a genetic mimic of the C. pepo B gene. The wild-type CpCHLHb physically interacted with CpAPRR2-A, attenuating the CpAPRR2-A-mediated activation of CpPSY-A. In contrast, the truncated CpCHLHB lost its ability to interact with CpAPRR2-A, enabling CpAPRR2-A to activate CpPSY-A and produce intensely orange fruit. These findings uncover the mechanism underlying the epistatic interaction through which B and L-2 act synergistically to boost carotenoid production, offering novel mechanistic insights and key targets for improving crop quality. One-sentence summarySynergistic epistasis between B and L-2 arises from loss of interaction between their encoded proteins, resulting in dramatically upregulating the key rate-limiting enzyme in carotenoid biosynthesis pathway to produce intensely orange-fleshed fruit in squash.

Abscisic acid (ABA) plays a critical role in regulating plant responses to abiotic stress by modulating various physiological processes through complex molecular networks. TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) transcription factors are known developmental regulators, but their roles in ABA signaling and abiotic stress responses remain poorly understood. We addressed this question by analyzing the closely related Arabidopsis thaliana class I TCPs TCP14 and TCP15 using a variety of genetic and molecular approaches. We found that TCP14 and TCP15 negatively influence ABA and salt stress responses. They function by directly activating genes encoding negative regulators of ABA signaling, such as ABA-INSENSITIVE FIVE-BINDING PROTEIN 2 (AFP2), thereby suppressing the expression of ABA-INSENSITIVE 5 (ABI5) and downstream ABA-responsive genes to inhibit ABA responses under non-stressful conditions. Notably, the TCPs are targets of ABA-mediated regulation, as ABA negatively affects TCP14 and TCP15 protein abundance, providing a mechanism to limit TCP-dependent transcriptional growth responses and de-repress ABA-signaling pathways during times of stress. We propose that the antagonistic interplay between class I TCPs and ABA may serve to fine-tune plant growth and stress responses according to environmental conditions, positioning TCP14 and TCP15 as crucial players in balancing plant developmental progression and stress adaptation.

Electrons energised by light energy at photosystem I (PSI) primarily enter the linear photosynthetic electron transfer (LET) or cyclic electron transfer (CET) pathway. The balance between LET and CET activity is a crucial factor in the regulation of photosynthesis, since CET increases the stoichiometry of proton to electron transfer. The additional transmembrane proton gradient ({Delta}pH) triggers feedback control of light harvesting and electron transfer photoprotection via non-photochemical quenching (NPQ) and photosynthetic control (PCON), maintaining the balance between the output of the light reactions and the downstream metabolism. Previously, it was found that Arabidopsis mutants lacking the stromal-facing membrane-extrinsic PSAE subunit of PSI (psae1-3) show enhanced CET activity, decreased LET and lower PSI oxidation in excess light. Here we show that high CET activity in psae1-3 primarily depends on the Proton Gradient Regulation 5 (PGR5)-dependent CET pathway rather than the NDH-dependent pathway. High CET is abolished in the psae1-3 pgr5CAS double mutant. In the psae1-3 ndho double mutant the elevated proton flux and CET are largely maintained; however, CO2 fixation and growth are significantly worsened, indicating that NDH still makes a physiologically meaningful contribution in the psae1-3 background. Biochemical analysis revealed that PGRL1 is redistributed from its normal mixed membrane distribution to become predominantly PSI-associated in psae1-3, providing a physical basis for the enhanced PGR5-dependent CET. These results underscore the primary importance of the PGR5-dependent CET pathway for optimal photosynthesis and CO2 fixation in Arabidopsis and establish the organisation of the PSI acceptor side as a key regulatory determinant of the CET/LET balance. HighlightLoss of the PSI acceptor-side subunit PSAE enhances predominantly PGR5-dependent cyclic electron transfer and redirects PGRL1 to PSI in Arabidopsis, while NDH contributes to maintaining CO2 fixation when the PSI stromal side is disrupted.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative pentose phosphate pathway, generating NADPH to sustain redox metabolism and signaling. However, whether individual G6PDH isoforms directly regulate oxidative stress signaling remains unclear. To determine the contribution of the different Arabidopsis G6PDH isoforms to oxidative stress signaling, we introduced single T-DNA mutants into the catalase-deficient cat2 background, a genetic system in which intracellular H2O2 production activates salicylic acid (SA)-dependent cell death and defense pathways. Interestingly, impairment of cytosolic, but not chloroplastic G6PDH activity suppressed cat2-triggered phenotypes, with loss of G6PD5 function fully abolishing lesion formation. The cat2 g6pd5 double mutant phenocopied the SA biosynthesis-deficient mutant cat2 sid2 and showed reversion of defense responses as well as metabolomic and transcriptomic profiles to the wild-type state. Strikingly, despite the suppression of SA-dependent lesions, loss of G6PD5 activity does not appear to reduce stress intensity. On the contrary, cat2 g6pd5 plants exhibit increased glutathione synthesis and oxidation, elevated expression of oxidative stress marker genes, and enhanced accumulation of reactive nitrogen species relative to cat2. Protein-protein interaction analyses revealed that G6PD5 associates with several redox and defense-related proteins. In particular, we confirmed a physical interaction between G6PD5 and thioredoxin h5, a key component of redox-dependent SA signaling. However, analysis of cat2 trxh5 and cat2 npr1 lines indicated that this interaction alone cannot explain the G6PD5-dependent control of SA responses. Our work reveals that cytosolic G6PD5 integrates redox metabolism with immune signaling to control plant responses to oxidative stress.

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

Symbiotic nitrogen fixation (SNF) relies on aerobic respiration, yet the key enzyme, nitrogenase, is extremely oxygen labile. Leghemoglobin (Lb) resolves this "oxygen paradox" by buffering and facilitating O2 transport. However, the dynamic regulation of Lb during nodule development remains poorly understood. Earlier results from our laboratory demonstrated that site-specific serine phosphorylation of Lb reduces its oxygen sequestration capacity. Here, we investigated the spatio-temporal regulation of Lb with the progress of rhizobial load during SNF. Fluorescence immunohistochemistry (FIHC) using anti-Lb antibody revealed that its localization gradually shifted from the plasma membrane to the cytoplasm of infected cells as nodules mature. Using phospho-peptide (Lb) specific antibodies, we found that serine phosphorylation triggers this translocation. Furthermore, FIHC in conjunction with immunoprecipitation followed by immunoblotting with phospho- and non-phospho-peptide specific antibodies demonstrated that the non-phosphorylated form is detectable as early as 9 dpi, whereas the phosphorylated forms were first detected at 11 dpi and progressively accumulated during nodule maturation. This spatio-temporal transition coincides with increasing rhizobial colonization and is accompanied by a decline in the non-phosphorylated pool. Therefore, the increased cytoplasmic pool of phosphorylated Lb, which exhibits reduced oxygen sequestration capacity, likely functions in promoting oxygen transport to sustain elevated rhizobial respiration. Together, these findings demonstrate that site-specific serine phosphorylation represents one of the key regulatory mechanisms linking Lb localization dynamics with progression of rhizobial infection, thereby contributing to the maintenance of oxygen homeostasis during SNF.

In nature, plants are subjected to multiple environmental stress factors simultaneously or sequentially. Recent studies revealed that when three or more stress factors impact a plant simultaneously (termed multifactorial stress combination; MFSC), plant survival declines, even if the intensity of each individual stress involved in the MFSC is low. We previously identified RAP2.3 as a key transcription factor (TF) required for Arabidopsis thaliana survival, specifically under a MFSC of salt+excess light+heat stress (i.e., S+EL+HS). Here we report that RAP2.3 is required for the expression of SIGMA3, a nuclear-encoded factor that directs plastid RNA polymerase to specific plastid promoters, and MYB51, a key stress response TF involved in glucosinolate metabolism and oxidative stress responses, specifically during a MFSC of S+EL+HS. Like rap2.3 mutants, myb51 and sig3 mutants display significantly low survival rate specifically under the MFSC of S+EL+HS. Based on MYB51 gene regulatory network analysis and characterization of jasmonic acid (JA) mutants, we further reveal that suppression of JA signaling could play an important role in promoting plant survival under conditions of S+EL+HS. Our findings uncover an additional layer of the response of plants to MFSC, as well as identify potential targets for breeding crops with enhanced tolerance to climate change.

Adaptation of plant photosynthesis to dynamic light conditions experienced in natural environments is achieved through specific protective mechanisms. Energy-dependent non-photochemical quenching (qE), regulated by Photosystem II Subunit S (PsbS), is a key process facilitating acclimation to fluctuating light in C3 plants, which operate conventional photosynthesis. C4 plants, which include some of the world's most productive and agriculturally important crops, have evolved a distinct high-efficiency photosynthetic pathway. Little is known about the role of specific processes, like qE, in acclimation of C4 plants to dynamic light environments. We generated gene-edited lines of the model C4 grass Setaria viridis lacking PsbS, which were found to be deficient in qE. This deficiency resulted in a modest increase in PSII photoinhibition and a CO2 assimilation penalty under light stress in short-term experiments, but photosynthesis and growth under fluctuating light were unaffected. Instead, keeping Photosystem I oxidised through photosynthetic control, negative feedback regulation of the Cytochrome b6f complex, was critical. Therefore, unlike in C3 plants, qE does not provide a significant adaptive advantage to C4 plants under dynamic light conditions. These findings provide important insights into the biology of C4 plants and help prioritise future strategies for improving the productivity and resilience of C4 crops.

Microalgae are photosynthetic organisms capable of autotrophic growth. Their applicability in multiple industrial fields has been largely studied, thanks to their ability to fixate CO2 into high added value organic products like fatty acids and carotenoids. However, our understanding of the cellular signaling networks that control carbon flux and acclimation to environmental stress remains incomplete. In this study, we used the Chlamydomonas reinhardtii mutant strain vip1-1, which carries a loss-of-function mutation in the hexakisphosphate kinase re-sponsible for the synthesis of inositol pyrophosphates InsP7 and InsP8 (PP-InsPs), to investi-gate the role of these molecules during high-light acclimation. Our results indicate that PP-InsPs participate in the regulation of carbon storage in the form of starch and their deficiency increases TAGs levels in the algal cells. They also impact chloroplast-specific lipid remodeling by modifying membrane composition and fluidity through fatty acid desaturations and glycer-olipid composition. In addition, our findings suggest that PP-InsPs are involved in chloroplast-nucleus communication, where they coordinate transcriptional repression of photosynthesis associated nuclear genes (PhANGs), fatty acid desaturases and lipid synthases, contributing to cellular acclimation to high light. We also found that PP-InsPs modulating effect extended to protein synthesis and accumulation of Calvin-Benson-Bassham cycle intermediates. Therefore, we propose that PP-InsPs function as integratory molecules that balance carbon allocation between storage and structural pools, in response to environmental cues such as high light. These data uncover a novel function of PP-InsPs in high light acclimation and po-tentially in chloroplast-nucleus communication, providing new insights that may help engineering more resilient and efficient strains.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) of Gram-negative pathogenic bacteria recognized by plants, triggering typical pattern-triggered immunity (PTI) responses. However, a LPS sensing receptor for the recognition of plants remains largely undefined. A plant receptor for lipopolysaccharide (LPS) has not yet been identified. Here, we identify a plant protein, OsML1, with homologies to animal MD-2, which is capable of binding LPS. Furthermore, it may act as a molecular chaperone to assist CK1 in perceiving LPS signals. Our results show that OsML1 functions as an LPS-binding protein recognizing LPS and participates in downstream rice immune response activation. Structural modeling and sequence analysis revealed that OsML1 contains both a typical ML domain and a conserved three-dimensional {beta}-barrel structure as mammalian MD-2 proteins. Microscale thermophoresis assays confirmed that OsML1 binds LPS with high affinity. Functional analyses further demonstrated that OsML1 knockout plants show reduced resistance to the rice bacterial blight pathogen, as well as attenuated ROS bursts upon LPS treatments, whereas overexpression plants show enhanced immune responses. Metabolomic profiling indicated significant metabolic changes in OsML1 knockout plants, particularly in immune-related pathways involving lipids, amino acids, and antimicrobial compounds. OsML1 is consequently a structurally conserved and functional LPS-binding protein linking lipid metabolism, LPS perception, immune activation, and metabolic regulation. Phylogenetic and structural analyses revealed that OsML1 likely arose from a duplication of OsML2, forming an independently functional subgroup within the PITP family. Our study identifies OsML1 as a LPS recognition factor involved in LPS sensing and downstream ROS bursts activation, callose deposition, and broad-spectrum gene expression of resistance. These findings expand our knowledge of bacterial LPS perception and immune regulation in plants, offering novel targets and strategies for disease-resistant breeding.

Chilling stress induces photosystem I (PSI) photoinhibition in chilling-sensitive cucumber, in which insufficient activity of the chloroplast NADH dehydrogenase-like complex (NDH) leads to PSI over-reduction and damage. However, it is not yet clear whether these findings can be generalized to other species or what the molecular mechanism underlying impaired NDH function is. In this study, we first examined whether NDH is essential for PSI protection under chilling stress using an NDH-deficient rice mutant. Compared with wild-type plants, the NDH-deficient mutant exhibited enhanced PSI over-reduction and pronounced PSI photoinhibition under chilling stress. In contrast, rice plants expressing flavodiiron protein (FLV), which functions as an alternative electron acceptor downstream of PSI, did not exhibit PSI photoinhibition under chilling stress, demonstrating that electron sink capacity of NDH is important for PSI protection under chilling stress. Furthermore, analysis of the factors responsible for NDH dysfunction under chilling stress in cucumber revealed that chilling stress destabilizes the PSI-NDH supercomplex, leading to NDH monomerization and a consequent loss of NDH activity. This NDH monomerization is likely attributable to chilling-induced damage to the light-harvesting complex Lhca, which mediates the association between PSI and NDH. Together, these results indicate that NDH is essential for protecting PSI from photoinhibition under chilling stress in both rice and cucumber, and that chilling-induced destabilization of the PSI-NDH supercomplex represents a key molecular mechanism underlying PSI over-reduction and photoinhibition.

Most proteins targeted to the organelles of endosymbiotic origin are encoded in the nuclear genome, placing them under the regulatory dominance of the nucleus. For photosynthetic eukaryotes, nuclear-encoded chloroplast proteins arise via two routes: First, genes of cyanobacterial origin were relocated to the nucleus through endosymbiotic gene transfer (EGT). Second, proteins of eukaryotic origin emerged to support chloroplast function and structure. These proteins are reimported into the chloroplast via an import machinery. Reversing the transfer of such genes from the nucleus to the chloroplast genome may offer insights into chloroplast regulation and evolution. In this study, we established a highly efficient and accessible electroporation protocol for chloroplast transformation in the green alga Chlamydomonas reinhardtii, and used it to reverse-transfer two nuclear-encoded genes encoding proteins arising via the two routes described above: the cyanobacteria-derived chloroplast division protein FtsZ1 and the Rubisco-linker EPYC1 of eukaryotic origin. Regardless of origin, both chloroplast-encoded FtsZ1 and EPYC1 showed proper localization and functionality comparable to their nuclear-encoded counterparts. Together, our study provides a robust protocol for chloroplast transformation, a platform for investigating the evolutionary drivers of EGT, and a foundation for advancing chloroplast bioengineering. SIGNIFICANCE STATEMENTO_LIEndosymbiotic gene transfer has resulted in the mass migration of genes from the chloroplast genome to the nuclear genome. Reversing the gene transfer could reveal the evolutionary significance of genome partitioning. C_LIO_LIUsing the green alga Chlamydomonas reinhardtii, this study developed an efficient, electroporation-based protocol for chloroplast transformation. Relocating the genes encoding two chloroplast-targeted proteins, FTSZ1 and EPYC1, to the chloroplast genome showed that the proteins maintained normal localization and function. C_LIO_LIThe established transformation protocol facilitates systematic testing of reverse gene transfer to elucidate the potential evolutionary advantages of genome partitioning and opens new avenues for chloroplast bioengineering. C_LI

Glucosinolates (GSLs) are major defensive compounds massively accumulated in Brassicaceae seeds, including that of the model plant Arabidopsis thaliana. While most studies have focused on the role of GSL in responses to biotic stress, the potential regulation and function of GSLs in responses to abiotic stresses have been neglected, particularly in seeds. In this study, multi-omic analyses revealed a previously uncharacterized GSL modification pathway induced by elevated temperature (ET) during A. thaliana seed development. Activation of this pathway leads to the production of several novel thioglucose-acylated GSLs, including sinapoylated and benzoylated derivatives. A reverse genetics approach demonstrated that the SERINE CARBOXYPEPTIDASE-LIKE 17 (SCPL17) and BENZOYLOXYGLUCOSINOLATE 1 (BZO1) enzymes are required for the acylation of GSL thioglucose moieties. Furthermore, the accumulation of acylated GSLs in seeds of 85 A. thaliana accessions grown under standard condition was shown to correlate with the average annual temperature of their origin site, suggesting that thioglucose-acylated GSLs production may reflect long-term thermal adaptation across natural populations. Taken together, these results demonstrate that thioglucose acylation by SCPL17 and BZO1 represents a new layer of GSL diversification in A. thaliana seeds that contributes to both ET response and long-term environmental adaptation.

Coordination among cell growth, chloroplast expansion, and organelle genome dynamics is fundamental to algal physiology, yet its regulation remains unclear. We used time-resolved single-cell analyses to examine scaling relationships among cell size, chloroplast volume, nuclear dynamics, and nucleoid organization in Desmodesmus communis and Chlamydomonas reinhardtii under normal conditions and after inhibition of chloroplast DNA replication with nalidixic acid (NAL). Under control conditions, both species showed coordinated scaling among cell, chloroplast, and nuclear size, while nucleoid dynamics were driven mainly by changes in number. NAL disrupted these relationships in a species- and time-dependent manner. In C. reinhardtii, prolonged treatment uncoupled chloroplast and nuclear growth from cell expansion and led to fewer, enlarged nucleoids, consistent with impaired replication. In contrast, D. communis largely maintained coordinated scaling, with effects mainly limited to reduced nucleoid proliferation and delayed division. Temporal analyses indicated that NAL primarily affected nucleoid replication and segregation, with secondary consequences for chloroplast growth and cell-cycle progression. These findings identify chloroplast genome dynamics as a regulatory link between organelle growth and cell division.

Vacuoleless Gametophytes (VLG) is a DC1 domain-containing protein initially characterized as essential for the development of both female and male gametophytes in Arabidopsis thaliana. In addition, VLG regulates stamen development through the involvement in lignin and jasmonic acid biosynthesis pathways. In this work, we report that VLG is also involved in salt stress tolerance in A. thaliana. Under salt stress, VLG-knock-down plants exhibited reduced germination, root elongation, biomass accumulation, photosynthetic pigment content, along with diminished expression of key salt-responsive genes. Conversely, these plants accumulated higher anthocyanins, and reactive oxygen species (H2O2 and O2-) compared to wilt type, indicating impaired oxidative stress control. In contrast, VLG-overexpressing plants showed a salt stress resistant phenotype with enhanced biomass and increased expression of salt-responsive genes under saline conditions. Together, these findings uncover an unexpected role for VLG as a positive regulator of salt tolerance, expanding the functional scope of DC1 domain proteins beyond reproductive development and providing new insights into plant mechanisms of abiotic stress resilience.

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

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

Grass leaves reportedly accumulate sucrose during the day then export it to other organs at night. The control of export at night is not understood. In contrast, Arabidopsis leaves accumulate starch then convert it to sucrose for export at night. Control of starch mobilisation ensures a constant sucrose supply and exhaustion of starch around anticipated dawn. We found that that 41 species of pooid grasses differed widely in the ratio of sucrose to starch accumulation and night-time depletion (turnover) in leaf blades, regardless of growth conditions. Sucrose turnover exceeded starch turnover in most species, but in a minority of species the two were similar. In eight species spanning this diversity, the rate of sucrose depletion fell through the night. In six of the species, all with higher sucrose than starch turnover, starch depletion at night was initially slow. Later, it increased to a rate that exhausted starch around dawn. The initial lag was absent and starch depletion was linear throughout the night in the other two species, both of which had similar sucrose and starch turnover. We suggest that underlying control of starch mobilisation in grass leaf blades resembles that in Arabidopsis leaves. The initial lag in starch depletion in high-sucrose species may result from suppression of starch degradation by sucrose, acting via the sucrose signalling metabolite Tre6P. The large diel fluctuations in sucrose export from grass leaf blades may be dampened by exchange with dynamic pools of sucrose in leaf bases and sheaths prior to export to other organs.