Plant Physiology

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

O_LIResearch and rationale: This study investigates whether tissue-specific ethylene biosynthesis regulates stomatal conductance (gs) responses to changing [CO2] in Arabidopsis thaliana. While guard cells sense [CO2], mesophyll-derived signals are also implicated in stomatal control. We aimed to determine if ethylene production in guardcells or mesophyll is the primary driver of CO2-induced gs regulation. C_LIO_LIMethods: An acs octuple mutant with severely reduced ethylene production was complemented with tissue-specific ACS8/ACS11 transgenes driven by guard-cell, spongy-mesophyll, dual palisade/spongy-mesophyll, or whole-leaf promoters. Tissue-specific complementation in the different transgenic lines was confirmed and evaluated by qPCR, tissue-specific NEON expression, microscopic imaging, and ethylene production measurements. Gas-exchange measurements on intact plants recorded gs kinetics, CO2 assimilation, and water-use efficiency, across CO2 shifts. C_LIO_LIKey results: Guard-cell complementation nearly fully restored wild-type gs responses and reversed the mutants aberrant leaf phenotype. Spongy-mesophyll complementation failed to rescue either trait, while dual palisade- and spongy-mesophyll complementation yielded only partial recovery. C_LIO_LIConclusion: Ethylene produced in guard cells is the dominant regulator of CO2-induced stomatal conductance regulation, with mesophyll-derived ethylene contributing secondarily via long-distance signaling or by augmenting the overall ethylene pool. These findings underscore the importance of spatially regulated ethylene biosynthesis in balancing carbon assimilation and transpiration. C_LI

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.

Cyclic electron transport (CET) around photosystem I (PSI) is essential for maintaining photosynthetic efficiency by balancing ATP/NADPH production and protecting PSI from photoinhibition. Although the PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET pathway is known to be critical under high or fluctuating light conditions, its role under fluctuating low light remains poorly understood. In natural environments, plants frequently experience prolonged low irradiance interspersed with brief sunflecks, making fluctuating low light a physiologically relevant condition. Here, we investigated Arabidopsis thaliana lines with graded PGR5 expression levels to evaluate the dose-dependent contribution of PGR5 to CET activity, photosynthetic regulation, and growth performance under both low light and fluctuating low light conditions. Moderate increase in the PGR5 protein level enhanced CET activity, accelerated photosynthetic induction, improved PSI protection and increased biomass accumulation under fluctuating low light. In contrast, excessive PGR5 accumulation impaired photosynthetic performance and reduced plant growth, indicating that optimal CET capacity requires precise tuning of PGR5 abundance. These results reveal a non-linear relationship between PGR5 protein levels and photosynthetic performance and demonstrate that moderate enhancement of CET improves plant productivity under fluctuating low light. Our findings highlight the importance of optimizing CET capacity to match dynamic light environments and suggest that fine-tuning PGR5 expression could be a promising strategy for improving crop performance under natural canopy conditions. Significance statementModerate increase in the PGR5 improves plant productivity, whereas excessive PGR5 accumulation impaired photosynthetic performance and reduced plant growth. Therefore, optimizing CET capacity by the fine-tuning PGR5 expression is important for improving crop productivity.

ATP-binding cassette (ABC) transporters mediate substrate translocation across membranes by using energy from ATP hydrolysis. While ABC peptide exporters have been characterized in the mitochondria of metazoans and yeast, corresponding chloroplast peptide transport systems in plants remain uncharacterized. Using in silico and experimental approaches, we identify three previously uncharacterized Arabidopsis thaliana ABCB half-transporters - TAP1, NAP8, and ATH12 - that localize to the chloroplast inner envelope and form homodimers. These proteins are phylogenetically related to known peptide exporters, and functional complementation in Saccharomyces cerevisiae demonstrates that each plant transporter can rescue the heat sensitivity of a{Delta} mdl1 mutant, indicating conserved peptide export activity. In chloroplast peptide efflux assays, peptide export upon heat stress was strongly reduced only in the tap1 nap8 ath12 triple mutant, but not in single or double mutants, indicating functional redundancy. Furthermore, mass spectrometry of chloroplast supernatants revealed an abundance of thylakoid-derived hydrophilic peptides in the wild type, predicted to have antioxidant activity. Under heat stress, the triple mutant displayed increased sensitivity, characterized by reduced biomass, chlorophyll and carotenoid content, and compromised photosynthetic efficiency. Comprehensive analyses revealed altered redox homeostasis in the triple mutant, including modified antioxidant dynamics, differential antioxidant enzyme activities, and distinct gene expression profiles compared with the wild type. Our findings demonstrate that TAP1, NAP8, and ATH12 constitute a chloroplast peptide export system required for efficient peptide release under heat stress, with a role in Arabidopsis thermotolerance. These results provide new insights into organellar peptide transport and its integration with stress mitigation mechanisms in plants.

Plants acclimate to mechanical stimuli such as touch and wind via thigmomorphogenesis, a suite of developmental responses that alter their growth and architecture. However, the early signaling mechanisms translating mechanoperception into long-term morphological changes remain incompletely understood. We investigated the role of the rapidly touch-induced transcription factor RRTF1 (REDOX RESPONSIVE TRANSCRIPTION FACTOR 1) in these processes. Phenotypically, under aggressive mechanical stimulation, rrtf1 mutant exhibited attenuated stunting (less height reduction). This suggests a key role for RRTF1 in promoting thigmomorphogenic responses under severe mechanical stimuli, though the rrtf1 mutant responded similarly to wild-type under gentle, repeated brushing. The alleviation of growth stunting in rrtf1 was largely jasmonic acid (JA)-independent. Transcriptome analysis at 10 minutes post-touch revealed that rrtf1 mutant maintained approximately 86% of wild-type touch-responsive gene expression. Nevertheless, RRTF1 modulated specific regulons, partly through an interplay with WRKY transcription factors, as evidenced by altered TF binding motif enrichment in RRTF1-specific differentially expressed genes. We conclude that RRTF1 acts as a modulator of early touch signaling in Arabidopsis shoots. It is not essential for the bulk of the initial transcriptional response but fine-tunes specific gene sets and plays a crucial role in calibrating long-term thigmomorphogenic development, particularly by promoting growth inhibition under severe mechanical stimulation. This study provides insights into the alleviation of touch-induced growth inhibition in rrft1 mutant, which might be relevant to breeding for crops that are planted in high density and experience constant physical contact with neighboring plants.

Under natural growth conditions, plants are not usually exposed to the high-energy ultraviolet C range (UV-C, 100-280 nm) of the solar spectrum, as this is absorbed by the ozone layer. However, low doses of UV-C radiation can trigger stress responses in plants. Nevertheless, it is not yet fully understood how UV-C light affects plant development at the hormonal level. Here we show that a single one-min UV-C light pulse (20 W/m2) alters gibberellin (GA) homeostasis in Arabidopsis in two phases: initially, the level of GA12 - a key precursor of the final part of gibberellin biosynthesis - is reduced. Consistent with this, the transcript levels of the CPS, KS and KAO2 genes, which encode enzymes involved in the initial parts of gibberellin biosynthesis, decrease. The level of the plant hormone GA4 also decreases initially, probably due to the reduced GA12 precursor levels. However, in a second phase, the endogenous GA4 levels rise in UV-C treated plants relative to control plants. This increase leads to an early onset of flowering, as well as increased growth and fertility, in UV-C-treated Arabidopsis plants. The GA signalling mutant gdella does not exibit wild-type phenotypic responses to UV-C treatment, indicating that GA signalling is essential for the UV-C response. To further narrow down the responsible steps in the GA-signalling pathway, we tested the kao1 and kao2 mutants, which are both impaired in early gibberellin biosynthesis. Neither mutant displays phenotypic responses to the UV-C treatment, indicating that both genes are required for mediating the UV-C response. In contrast, the quintuple 2-oxidase mutant C19--2oxqM exhibits responses to UV-C treatment similar to the wild-type, suggesting that the five catabolic 2-oxidases that act on C19-GAs play a negligible role in regulation GA-hormone levels for growth and development in this case. HighlightUV-C pulse triggers biphasic gibberellin dynamics, delaying early development but ultimately enhancing growth and fertility in Arabidopsis thaliana.

The unicellular green alga Chlamydomonas reinhardtii provides a tractable model for investigating how carbon availability influences metabolic organization and cell-cycle control in photosynthetic eukaryotes. Its capacity for autotrophic (light, CO2), mixotrophic (light, CO2, acetate), and heterotrophic (acetate, dark) growth enables systematic analysis of trophic-state-dependent regulation. We performed comparative transcriptomic analyses of strain 21gr grown under these three regimes at 30 {degrees}C. Mixotrophy resulted in the highest biomass accumulation and was associated with earlier cell-cycle commitment compared with autotrophy, whereas heterotrophy displayed delayed commitment and reduced growth. Transcriptomic profiling revealed coordinated upregulation of central carbon metabolic pathways under mixotrophy, including photorespiration, glycolysis, the oxidative pentose phosphate pathway, and tricarboxylic acid cycle functions, consistent with enhanced carbon flux and biosynthetic capacity. In contrast, heterotrophy preferentially induced acetate assimilation and glyoxylate cycle genes and was accompanied by elevated expression of cell-cycle regulators, including the CDK-inhibitory kinase WEE1. Together, these findings indicate that trophic mode modulates the coupling between carbon metabolism and cell-cycle progression, with mixotrophy supporting integrated metabolic and proliferative activity, whereas heterotrophy is associated with delayed cell-cycle timing and transcriptional signatures of metabolic adjustment.

The chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport (CET) around photosystem I (PSI) and contributes to photosynthetic regulation and photoprotection under various environmental stresses. Although NDH function has been extensively characterized under controlled conditions, NDH-deficient mutants often show only subtle phenotypes in such environments, leaving its physiological importance under naturally fluctuating field conditions poorly understood. Here, we evaluated growth, yield, and photosynthetic performance of NDH-deficient rice cultivated under field conditions. Mutant plants exhibited reduced biomass accumulation and grain yield compared with wild type. Detailed physiological analyses revealed that NDH deficiency markedly decreased PSI electron transport and CO2 assimilation, particularly under low temperature and sub-saturating irradiance. At moderate and high temperatures, reductions in carbon fixation were largely confined to low-light conditions, whereas at low temperatures, impairment extended across nearly the entire light response range. Under repetitive fluctuating light regimes, NDH-deficient plants showed progressive declines in photosynthesis accompanied by a selective decrease in PSI photochemical capacity without changes in PSII maximum efficiency, indicating PSI-specific photoinhibition. These findings demonstrate that NDH-dependent CET plays a crucial role in sustaining photosynthetic efficiency and crop productivity in dynamic field environments by stabilizing PSI redox balance and maintaining long-term carbon gain. Summary StatementNDH-dependent cyclic electron transport supports photosynthesis and yield in field-grown rice by maintaining PSI function under fluctuating light, low temperature, and sub-saturating irradiance.

Translation initiation factors of the eIF4E family play a crucial role in regulating translation and the cellular metabolism of mRNAs. Research has addressed the role of canonical 4E family isoforms in development, stress response, and during viral infection. Nevertheless, the class-II eIF4E family member cap-binding protein 4EHP (nCBP), has remained poorly characterized in plant stress responses. In this study, we show that loss of 4EHP confers enhanced basal and acquired thermotolerance and causes a mild flowering delay without major root defects. Under heat stress, 4EHP-GFP re-localizes from a diffuse cytosolic pattern to cytoplasmatic foci, co-localizing with canonical stress granule (SG) markers. Transcriptomic analysis under control, acclimation and heat stress conditions reveals that 4EHP limits the accumulation of heat-responsive mRNAs, especially those encoding heat shock proteins (HSPs), which remain constitutively expressed in 4ehp-1 mutant under control and heat stress conditions. Proteomic analysis also indicates that the absence of 4EHP alters the repertoire of HSPs compared with wild type (Col-0), especially upon heat stress, without significantly impairing the recruitment of corresponding mRNAs to translationally active polysomes. Together, our results indicate that 4EHP negatively modulates the accumulation of a specific subset of heat-responsive mRNAs fine-tuning chaperone production, via heat-responsive SG regulatory pathways.

Understanding the regulation of enzyme activity involved in photosynthesis is essential for engineering enhanced carbon fixation in crops. In C4 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is one of the most abundant leaf enzymes and plays an essential role in photosynthetic carbon dioxide (CO2) fixation. The enzyme also plays a key role in central metabolism (e.g., providing intermediates to the citric acid cycle) and therefore must be highly regulated to coordinate its activity. The regulation of PEPC activity can occur allosterically by glucose 6-phosphate activation and malate inhibition, which is in part influenced by reversible phosphorylation. A specific light-dependent phosphorylation of PEPC at an N-terminal serine residue by the PEPC-protein kinase (PEPC-PK) can regulate its sensitivity to this allosteric regulation. However, the impact of this PEPC phosphorylation has not been tested in a C4 crop. Therefore, we created PEPC-PK mutant lines in Zea mays to assess the impact of PEPC phosphorylation on its allosteric regulation, photosynthesis, and growth. While the maximum PEPC activity was unchanged, PEPC in the PEPC-PK mutant plants was not phosphorylated under light and was more sensitive to malate inhibition. However, gas exchange, electron transport, and field biomass analyses showed no differences in the PEPC-PK mutant plants. These results demonstrate that in Z. mays PEPC phosphorylation affects enzyme sensitivity to malate in vitro but does not substantially alert photosynthetic performance or growth under field conditions suggesting additional regulation of PEPC activity in planta.

Photoperiod, the daily duration of light, is a key environmental cue that varies with season and latitude. Photoperiod signals profoundly influence plant growth and development, and play a central role in crop adaptation across latitudes. Deciphering how plants perceive these seasonal light cues at the molecular level is essential for understanding crop evolution and shaping their geographical distribution. In this study, we investigated how photoperiod influences gene expression dynamics in tomato (Solanum lycopersicum) by performing an RNA-seq time-course across three photoperiod regimes, sampling at two-hour intervals. We use isogenic lines segregating for wild alleles of genes involved in circadian rhythm domestication in tomato to describe their contributions to the circadian clock and flowering time pathways. The high temporal resolution of our experiment allowed precise characterization of transcriptional dynamics and their responses to photoperiod, including shifts in phase, amplitude, and waveform. We found that most transcripts time their expression to dawn or approximately 12 hours later, resulting in a systematic misalignment between evening transcripts and the actual timing of dusk. Morning- and evening-phased transcripts differ markedly in expression levels and waveform responses to changes in photoperiod. Together, these patterns suggest that morning transcripts sense photoperiod transcriptionally, and serve as zeitgeber references for evening transcripts, that could measure photoperiod length by coincidence with external cues. Together, our results provide new insight into the molecular basis of photoperiodic adaptation in plants.

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.

Soil salinization is a growing global threat that limits crop productivity. To cope with sodium (Na) stress, plants have evolved tolerance mechanisms, including excluding Na from shoot tissues and tolerating elevated Na within shoots through tissue- and cellular-level mechanisms. Most current knowledge of Na accumulation comes from organ- or whole-plant measurements that lack the spatial resolution needed to resolve cellular tolerance mechanisms. Here, we used histological approaches to map leaf Na distribution in tomato (Solanum) species with contrasting salt-tolerance strategies. In the Na-excluding domesticated tomato (cv. M82), Na was largely confined to the bundle sheath, whereas Na-including wild relatives accumulated Na throughout the blade mesophyll. Consistent with these cell population-specific Na patterns, M82, but not S. pennellii, exhibited reduced symplastic transport and plasmodesmal permeability under salt stress. A genetic screen combined with transcriptome profiling implicated Plasmodesmata-Located Protein 1 (PDLP1), a regulator of callose-mediated plasmodesmal closure, in establishing symplastic domains in M82 that restrict Na movement into the mesophyll. Moreover, PDLP1 expression negatively correlated with mesophyll Na+ levels across wild and domesticated tomatoes. Collectively, these results link cellular Na enrichment patterns to symplastic connectivity and suggest that PDLP1-mediated regulation of plasmodesmata contributes to leaf-level salt-tolerance strategies. HighlightsO_LICell type-specific Na accumulation differs between domesticated tomato (Solanum lycopersicum cv. M82) and its wild relative S. pennellii. C_LIO_LIAdditional salt-tolerant wild tomato relatives exhibit leaf Na enrichment patterns similar to S. pennellii. C_LIO_LISalt stress reduces symplastic transport and plasmodesmal permeability in M82 leaves but not in S. pennellii. C_LIO_LIAn introgression line (IL6-4) between the two tomato species, which carries S. pennellii Plasmodesmata-Located Protein 1 (SpPDLP1), shows S. pennellii-like Na enrichment patterns. C_LIO_LIPDLP1 expression shows a negative correlation with mesophyll Na+ levels across tomato species. C_LI

The JAGGED transcription factor family regulates lateral organ development across angiosperms. In soybean (Glycine max Merr.), a D9H mutation in the EAR repression motif of GmJAG1 causes a narrow leaflet phenotype and explains over 70% of phenotypic variance in leaf shape. Because this mutation does not affect the zinc finger DNA-binding domain, both alleles bind identical targets but differ in repressor recruitment. Previous studies mapped GmJAG1 binding sites, but the functional targets controlling leaf morphology are uncharacterized. Here, we used comparative transcriptomics across four soybean genotypes with contrasting leaf shape, spanning a developmental time series from shoot apex to mature leaf, and identified 1,567 candidate target genes. GmJAG1 expression was confined to the shoot apex, yet 99.1% of candidate targets maintained differential expression throughout development. We found that neither Kip-Related Protein (KRP) cell cycle inhibitors nor Cyclin-Dependent Kinases (CDKs) showed differential expression despite binding evidence in Arabidopsis. However, D-type cyclins were upregulated in narrow-leaf genotypes suggesting cyclin-mediated rather than KRP-mediated cell cycle regulation in soybean. Pathway analysis revealed enrichment of auxin (1.8-fold, P = 0.02) and salicylic acid (4-fold, P = 0.016) genes among JAG1D9H targets. Filtering by differential expression, binding data, phenotype correlation, and co-expression network membership identified 79 high-confidence targets, including orthologs of NPH3 (phototropin-mediated leaf flattening), MIK2 (cell wall integrity sensing), RD22 (ABA-responsive stress signaling), and SCL23 (GRAS transcription factor in bundle sheath development). These candidates provide targets for functional validation and breeding in legumes.

Starch synthesis in wheat endosperm involves the initiation of large A-type starch granules during early grain development, followed by small B-type granules in later grain development. It is established that MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1) plays an important role in granule initiation in Arabidopsis chloroplasts, but how it influences A- and B-type initiations in wheat amyloplasts is not known. We discovered that due to a gene duplication in cereals, wheat contains two MFP1 paralogs, MFP1.1 and MFP1.2, which are both expressed in the developing endosperm. We generated a series of durum wheat mutants defective in all homoeologs of either MFP1.1 or MFP1.2, or both. While starch granule size distributions and granule morphology of mfp1.1 and mfp1.2 mutants were identical to those of the wild-type, the mfp1.1 mfp1.2 mutants had fewer, but larger B-type granules - suggesting that the two paralogs play redundant roles in B-type granule initiation. Consistent with this, both paralogs interacted with B-GRANULE CONTENT 1 (BGC1), a key protein required for proper B-type granule initiation in wheat, and both paralogs could partially complement defects in starch initiation in the Arabidopsis mfp1 mutant. Our work demonstrates that MFP1 is required for establishing correct starch granule number in non-photosynthetic amyloplasts, but its role in wheat is limited to B-type granule initiation. One-sentence summaryWheat has two MFP1 paralogs that interact with the granule initiation protein, BGC1 and influence B-type granule initiation in non-photosynthetic amyloplasts of endosperm.

Rapid shoot growth (flushing) phenology is a fundamental developmental process in perennial woody plants such as citrus. In a separate study, we identified physiological shifts from photosynthesis to mobilization of nitrogen and carbohydrate to support new shoot growth. However, the underlying molecular and biochemical signals remain largely unknown. Here, we integrated proteomic and metabolomic analyses to investigate carbohydrate and hormone dynamics across three flush stages in Citrus sinensis: quiescent period (stage 1), new shoot initiation (stage 2), and full expansion (stage 3). Sucrose, maltose, and trehalose accumulated in apical leaves during early shoot initiation and declined during subsequent shoot expansion, indicating depletion of carbohydrate reserves and enhanced resource remobilization. These changes were accompanied by coordinated regulation of starch-metabolizing enzymes, including ADP-glucose pyrophosphorylase, -amylase, and isoamylase, supporting a transition from carbon storage to carbon export during active shoot growth. Indole-3-acetic acid increased continuously across stages, while trans-zeatin and gibberellin A{square} showed opposite trends in apical versus basal leaves before jointly increasing at stage 3. Hormone analysis revealed dynamic and coordinated signaling changes during flush development. Abscisic acid declined from stage 1 to 2, whereas jasmonoyl-isoleucine and salicylic acid increased from stage 2 to 3. Some hormone-responsive proteins, including Gretchen Hagen 3 and Gibberellin-insensitive dwarfing 1, exhibited expression patterns consistent with hormonal fluctuations. Together, these results support a stage-specific regulatory framework in which carbohydrate metabolism and hormone signaling are tightly coordinated to regulate rapid source-sink transitions during citrus flush development. HighlightWe reveal how carbohydrate metabolism and hormone signaling are spatiotemporally coordinated during citrus shoot growth phenology, and we develop an integrated metabolic-hormonal model that connects carbon allocation to developmental transitions.