Plant Physiology

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.

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.

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.

Expression of the chloroplast AAA+ chaperone CLPD gene increases during senescence and drought, but its functional role in chloroplast proteostasis is poorly understood. This study provides a comprehensive analysis of Arabidopsis CLPD protein accumulation across development from early seedlings to senescence, and compares results to its homologs CLPC1,2, as well as CLPB3 and cpHSP90. The developmental consequences of complete loss of CLPD expression (clpd-1), as well as overexpression of functional CLPD or CLPD impaired in ATP hydrolysis (CLPD-TRAP), were determined in Arabidopsis. clpd-1 has accelerated seedling development while functional CLPD overexpression lines, but not CLPD-TRAP, have delayed development. To determine if CLPD is a bona fide CLP chaperone associating with the CLPPRT protease and to identify in vivo candidate substrates, we employed the CLPD-TRAP line during the vegetative and flowering (senescent) growth stages. Affinity purification of CLPD-TRAP followed by mass spectrometry showed high enrichment of the CLP protease complex, thus providing direct support for the role of CLPD in substrate delivery to the CLP protease. CLPC1,2 were also highly enriched in the CLPD-TRAP interactome, suggesting hetero-oligomerization and cooperation between the three chaperones is likely. Nine chloroplast candidate substrates were identified in the CLPD-interactomes, including: FHY2 involved in riboflavin synthesis, THI1 and THIC involved in thiamin metabolism, and four proteins of unknown function. Several of these have been previously identified as potential CLPC1 substrates; however, others appear to be specific to CLPD. CLPD acts in substrate selection within a heteromeric CLPC-CLPD hexamer, likely to make unique contributions through its divergent N-terminus.

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.

Understanding plant growth dynamics requires imaging across day-and-night cycles to quantify growth, movement and development in the aerial plant body and to capture the rhythmic nature of these processes. This requires imaging in light during the day and in darkness at night without perturbing plant physiology. Nighttime imaging has typically depended on infrared (IR) illumination, producing monochrome datasets that require specialised hardware and separate analysis pipelines when combined with daytime RGB imaging. Here, we evaluated very low-intensity green (dimG) illumination from standard LEDs as a practical alternative for colour-consistent nighttime imaging and assessed its physiological impact in Arabidopsis thaliana and Lactuca sativa (lettuce). We show that high resolution colour images can be obtained under dimG using low- cost cameras, with sufficient consistency between full-spectrum and dimG images to allow direct comparison and unified image analysis. We show that very low-fluence green light (<0.5 mol m-2 s-1) does not sustain circadian oscillations of gene activity under continuous exposure and does not perturb rhythms when applied during the dark phase of diel cycles. DimG imaging enabled accurate detection of diel leaf movement profiles in Arabidopsis circadian mutants, revealing genotype-specific phase differences under varying photoperiods. In lettuce, dimG pulses and continuous dimG enabled accurate quantification of diel leaf movement without affecting growth, stomatal opening, electron transport rate or chlorophyll content. Motion profiles under continuous dimG mirrored those under darkness. Our findings establish dim green illumination as a cost-effective solution for night-time imaging, simplifying phenotyping workflows with minimal impact on physiology.

Soil salinization threatens global agriculture reducing yields, yet the metabolic signals controlling salt-sensitive root plasticity in alfalfa remain unclear. We hypothesize that salinity transiently uncouples the sucrose-trehalose-6-P (Tre6P)- Sucrose non-fermenting kinase 1 (SnRK1) nexus, aligning with a biphasic root metabolic response and altered root architecture. Alfalfa seedlings were grown in a hydroponic system and exposed to 200 mM NaCl, with root samples collected from 1 h to 7 d. While primary root growth and biomass remained unchanged, lateral root development was enhanced under salinity. Early response (1 h-1 d) was characterized by reduced carbon metabolites, low Tre6P, increased malondialdehyde, and SnRK1 activation, with a decline in glycolytic and TCA intermediates. During this phase, sucrose was negatively correlated with both Tre6P and SnRK1. Late response (3-7 d) showed a SnRK1 reactivation, Tre6P recovery, and osmoprotectant accumulation, including increased antioxidant capacity (+75% at 3dpt), proline (+178%), and sucrose (+18%) and starch depletion (-57%) at 7dpt respect to control. These metabolic changes coincided with the enhanced lateral root emergence. These findings indicate a two-phase response: early metabolic downscaling with transient Suc-Tre6P-SnRK1 disruption, followed by recovery with Tre6P restoration, SnRK1 reactivation, osmoprotection, and sustained root plasticity under salinity. HighlightSalinity triggers a temporary metabolic shift in alfalfa roots: plants first conserve energy, then adapt to stress, maintaining lateral root growth and flexible root architecture.

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.

O_LIIn fluctuating environments, the kinetics of stomatal opening and closing influence the balance between carbon gain and water loss. Smaller guard cells may respond faster to fluctuating environmental conditions because of their greater surface area for osmolyte flux relative to cell volume. A related hypothesis is that operational stomatal conductance (gop) is often well below its theoretical maximum (gmax) because at this stomatal aperture, guard cell volume is poised to change rapidly with small changes in turgor pressure. C_LIO_LIWe analyzed 2,124 estimates of stomatal closure kinetics in response to an abrupt increase in vapor pressure deficit (VPD) among 29 diverse wild tomato populations in the genus Solanum. C_LIO_LILeaves with small guard cells and a lower gop to gmax ratio (fgmax) closed faster, but explained variation in kinetic parameters at different levels of biological organization. Guard cell size had high phylogenetic heritability and varied relatively little within populations, whereas fgmax varied mostly among individuals and between light intensity treatments. C_LIO_LISmaller stomata can be speedier, but only if stomata are held at an aperture where they are responsive to changing turgor pressure. Selection on stomatal speed may influence not only anatomical traits like guard cell size, but also physiological controls on gop. C_LI

Plant receptor-like kinases (RLKs) are involved in diverse processes, ranging from growth and reproduction to interactions with microbes. Variation in the extracellular domains delineates several RLKs subfamilies, including the malectin-like domain leucine-rich repeat receptor-like kinases (MLD-LRR-RLKs). Symbiosis Receptor-like Kinase (SymRK) is the prototypical member of MLD-LRR-RLKs and is required for microbial accommodation in host roots during root endosymbiosis. Yet, comparative phylogenetic analysis of SymRK orthologs in the broader context of MLD-LRR-RLK subfamily evolution remains limited. In this study, we examined the inventory, phylogeny and clade-specific evolutionary and transcriptional characteristics of this receptor group. SymRK and its closest homologs are present in most land plant lineages and group into four major clades and six additional species-specific clades. These clades can be distinguished by their evolutionary characteristics as either conserved with reduced gene copy number changes (including SymRK) or expanded and diversified, as observed in clade IV. Clade IV dynamics are largely driven by tandem gene duplications, which often arise within gene clusters. We further analysed the evolutionary characteristics of MLD-LRR-RLKs at the population level in Arabidopsis thaliana accessions. We found that some genes are conserved across accessions and are therefore likely to be functionally important, whereas a subset of genes, often located within tandem clusters, are highly diverse and likely contribute to accession-specific adaptations. Finally, most MLD-LRR-RLKs in the A. thaliana Col-0 accession are expressed in roots and respond broadly to biotic stimuli at the transcriptional level. Notably, clustered genes frequently exhibited divergent expression profiles, suggesting transcriptional diversification. Together, we revealed two contrasting evolutionary characteristics among members of the MLD-LRR-RLK subfamily, potentially associated with their functions in plants.

A global nuclear war could inject soot into the stratosphere, blocking sunlight and causing rapid cooling. Assessments of the resulting agricultural collapse rely on crop models never validated under such conditions. We grew wheat, canola, and potato in growth chambers simulating the light and temperature of an extreme nuclear winter at tropical and temperate sites. In the tropical chamber (18-20 {degrees}C, 200 mol m-2 s-1 PAR), all three crops produced viable yields. Wheat yielded 2.1-2.3 t/ha (n=3 well-watered, n=3 water-stressed pots), 60% of the global average, and single-pot observations of canola and potato suggested biological yields comparable to global averages. In the temperate chamber simulating nuclear winter irradiance (60-360 mol m-2 s-1), wheat stems collapsed under their own weight. Although hand-harvesting recovered 0.6-2.8 t/ha of grain, mechanical field harvest of a flat canopy would recover substantially less. This failure mode was not observed in a higher-light control chamber and is not captured by existing crop models, which may therefore overestimate temperate cereal production under nuclear winter. Canola produced comparable yields under both temperate light regimes without lodging. Empirical screening of additional staples is needed to identify which remain viable under nuclear winter.

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.

Accurate quantification of leaf lesion severity is essential for plant disease research and phenotyping but is often limited by subjective visual scoring and time-intensive manual image analysis. We present LIME, a fully automated, open-source image analysis pipeline for high-throughput quantification of leaf lesions from disease assay images. LIME integrates zero-shot leaf segmentation using the Segment Anything Model with a convolutional neural network for lesion area estimation. Applied to Arabidopsis thaliana leaves infected with Sclerotinia sclerotiorum, the proposed approach achieved a mean absolute percentage error of 12.9%, comparable to observed intrarater variability in manual scoring. Stratified evaluation across lesion-size groups demonstrated consistent prediction accuracy for small, intermediate, and large lesions, and comparative analysis showed that the deep learning-based model substantially outperformed color-based baseline methods. Under GPU-accelerated execution, LIME processed complete assays containing approximately 200 leaves in 15 minutes, representing an approximate 13-fold reduction in processing time relative to manual annotation. Together, these results indicate that LIME enables objective, reproducible, and scalable quantification of leaf lesion severity in standardized plant pathology assays. The pipeline is released as an open-source tool to support quantitative phenotyping studies.

3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) is an essential component of rhamnogalacturonan II (RG-II), a complex pectic polysaccharide required for plant growth and development. While most steps of the KDO biosynthetic pathway have been characterized in plants, KDO-8-phosphatase (KDO8Pase), the phosphatase responsible for converting KDO 8-phosphate (KDO8P) to KDO, remained unidentified. To identify this missing component, we performed gene co-expression analysis and identified At5g57440 (GPP2) as the primary candidate in Arabidopsis (Arabidopsis thaliana L.). Recombinant GPP2 protein exhibited KDO8P-specific phosphohydrolase activity in vitro. A GFP-tagged GPP2 protein was predominantly localized to mitochondria, consistent with the compartmentation of the subsequent step in KDO biosynthesis. Null mutants of GPP2 exhibited significant growth retardation under boron-limited conditions, in which expression of GPP2 and other KDO biosynthetic genes was up-regulated. The growth retardation was also observed in liquid culture in normal media, a condition that induces rapid growth and thus likely increases metabolic demand for KDO. Despite this growth defect, the KDO content per unit cell wall in gpp2 remained equivalent to that in wild-type plants. These results are consistent with the identification of GPP2 as the elusive plant KDO8Pase and suggest a model where KDO availability becomes the rate-limiting factor for cell wall production. Our findings complete the plant KDO biosynthetic pathway and provide new insights into the physiological significance of RG-II in cell wall biosynthesis. Significance statementThis study identifies the previously unknown plant KDO-8-phosphatase, thereby completing the biosynthetic pathway for KDO in rhamnogalacturonan II. Our findings demonstrate that KDO synthesis is up-regulated under boron deficiency, and its supply becomes a rate-limiting factor for cell wall formation.

Micro-dwarf tomato cultivars are increasingly considered for urban and controlled-environment agriculture due to their compact architecture and suitability for high-density planting. However, optimal canopy management strategies for these cultivars remain poorly defined. In this study, we evaluated the effects of different leaf removal intensities on leaf-level physiological performance, fruit yield, and fruit quality in three micro-dwarf tomato cultivars (Mohammed, Hahms Gelbe Topftomate, and Red Robin) grown under contrasting seasonal light conditions. Plants were subjected to low (15%), moderate (30%), or severe (90%) leaf removal, and leaf-level gas exchange was measured across canopy layers, along with yield and fruit quality assessments. Severe leaf removal (90%) increased carbon assimilation, transpiration, and stomatal conductance in middle and lower canopy leaves by up to approximately twofold compared with control plants, indicating improved light availability at the leaf level. However, these physiological enhancements did not consistently translate into higher yield, reflecting reduced whole-plant source capacity under excessive leaf removal. Low to moderate leaf removal (15-30%) generally increased or maintained yield and fruit number, whereas severe leaf removal reduced yield in Hahms Gelbe and Red Robin, particularly under low seasonal radiation. In contrast, Mohammed exhibited yield increases of up to 220% under low leaf removal and maintained increased yield even under severe leaf removal under high-light conditions. Fruit quality was largely unaffected by leaf removal, except for total soluble solids, which declined by approximately 12% under severe leaf removal across cultivars, consistent with sugar dilution under source limitation. Overall, these results demonstrate that optimal leaf removal in micro-dwarf tomatoes requires balancing improved canopy light distribution with maintenance of sufficient leaf area for carbon assimilation. Leaf removal thresholds are strongly cultivar- and light-dependent, emphasizing the need for cultivar-specific canopy management strategies in compact tomato systems and controlled-environment agriculture.

The COP1/SPA ubiquitin ligase is a key repressor of photomorphogenesis that is inactivated by photoreceptors to initiate light signalling. The four SPA proteins (SPA1-SPA4) confer functional specificity to COP1 during plant growth, yet the underlying molecular mechanisms remain unclear. Here, we used a domain-swap approach in transgenic seedlings to address the functional divergence of SPA2 and SPA3. We show that the respective N-terminal kinase domain determines the contrasting protein stabilities of SPA2 and SPA3 in light-grown seedlings. The instability of SPA2 correlates with a specific ability of the SPA2 N-terminal domain to bind phytochrome A in the light, suggesting that phytochrome A promotes the CUL4DET1/COP1-dependent degradation of SPA2 but not of SPA3. We uncover that the coiled-coiled and WD-repeat domains of SPA2 and SPA3 substantially differ in their activity in repression of photomorphogenesis, with those of SPA2 being more active repressors than those of SPA3. Thus, SPA2 combines a potent repressor activity with light-induced instability. We conclude that the evolution of SPA2 instability in response to light counterbalances its inherent strong repressor activity, thereby allowing seedling etiolation in darkness followed by rapid reduction in COP1 activity through SPA2 degradation upon light-exposure as seedlings emerge from soil to initiate photosynthetic growth. HighlightThe repressor of light signaling SPA2 combines a phytochrome A-interacting instability domain with a potent repressor domain to allow greatly contrasting activities of COP1 in skoto- and photomorphogenesis.

The shikimate pathway provides precursors for phenolic metabolites in plant primary and secondary metabolism. Carbon flux measurements in intact Arabidopsis leaves using a novel isotopologue MS/MS methodology revealed unexpected dynamics between chorismate and isochorismate pools. 13CO2 labeling kinetics point to chloroplast-derived shikimate as a major bifurcation point, with approximately one third diverted away from the chloroplast. In contrast, the small pool of chorismate was almost exclusively chloroplast-localized and turned over rapidly, reaching more than 70% labeling within minutes. Isochorismate, a precursor to salicylic acid (SA) in the Brassicales, was present at 50-fold molar excess over chorismate but labeled much more slowly and appeared primarily extra-chloroplastic. Total isochorismate declined by 90% in the Arabidopsis enhanced disease susceptibility mutant, which lacks the isochorismate exporter. Non-aqueous fractionation further supported a primarily chloroplast-localized chorismate pool but extra-plastidic isochorismate. Populus trichocarpa and Nicotiana benthamiana leaves contained chorismate but only trace isochorismate, consistent with their use of the benzoyl-CoA route to SA. Carbon commitment calculations indicated that one third of the total chorismate pool in Arabidopsis leaves is diverted to isochorismate. Global leaf calculations based on elemental analysis-isotope ratio mass spectrometry and targeted isotope recovery indicate that only 0.03% of total assimilated carbon (5.27 pmol{middle dot}mg-1 D.W.{middle dot}min-1) enters the shikimate pathway in photosynthetically active mesophyll. For comparison [~]0.05% (7.75 pmol{middle dot}mg-1 D.W.{middle dot}min-1) enters the 2-C-methyl-D-erythritol 4-phosphate pathway, which provides precursors for photosynthetic pigments and electron carriers. The compartmentalization and turnover dynamics of shikimate, chorismate, and isochorismate suggest continuous demand for aromatic precursors in mesophyll tissue is comparable to demand for MEP-pathway derived pigments.

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

In plant cytokinesis, the partitioning membrane is made by homotypic fusion of secretory vesicles, progressing in a centre-to-periphery direction. In Arabidopsis, this process is mediated by a cytokinesis-specific fusion machinery involving Qa-SNARE KNOLLE which is made during G2/M phase and degraded at the end of cytokinesis. Here we analyse how the turnover of KNOLLE protein is regulated. KNOLLE is ubiquitinated, which is best detected after combined treatment with inhibitors of endocytosis and de-ubiquitination. Site-directed mutagenesis of three clustered lysine residues prevented ubiquitination and internalisation, resulting in stable accumulation of KNOLLE at the plasma membrane in all cells of the seedling root. This is in stark contrast to the transient accumulation of wild-type KNOLLE in dividing cells only. Partial-substitution mutant lines revealed redundancy of lysine residues in both KNOLLE ubiquitination and turnover. KNOLLE ubiquitination resulted in K63-linked ubiquitin chains known to be involved in endocytosis whereas K48-linked chains were not detected. To explore the spatio-temporal conditions, we analysed KNOLLE ubiquitination in cis-SNARE and trans-SNARE complexes during membrane traffic and cell-plate formation. Our findings suggest that KNOLLE protein turnover is caused by a ubiquitination process that depends on successful membrane fusion generating the cell plate.