Physiologia Plantarum

Plants frequently experience recurrent drought events separated by periods of rehydration. Although drought imposes strong constraints on plant physiology, prior exposure may alter subsequent stress responsiveness through memory-based mechanisms. Here, we investigated whether recurrent drought at distinct developmental stages establishes stress memory in sugarcane and whether this response persists across vegetative propagation. Two genotypes contrasting in drought tolerance and productivity (IACCTC07-8008 and IACSP95-5000, respectively) were grown under greenhouse conditions and subjected to three drought cycles imposed either at tillering or maturation stage. Gas exchange, photochemical performance, leaf water status, primary metabolite profile, and growth traits were assessed across cycles, and vegetative propagules were subsequently evaluated under renewed drought. The first drought cycle imposed strong limitations on carbon assimilation and photochemistry in both genotypes and developmental stages. However, subsequent cycles resulted in attenuated reductions in A and g, improved intrinsic water use efficiency, and partial stabilization of PSII performance, indicating a modified stress-response trajectory. Young plants displayed earlier improvements (from the second cycle), whereas in mature plants this shift was evident mainly during the third cycle. Recurrent drought promoted sustained reorganization of amino acid, carbohydrate, organic acid and polyol metabolism alongside increased root biomass and higher relative water content during later cycles. Importantly, propagules derived from drought-conditioned plants exhibited faster recovery of photosynthetic performance and reduced cumulative physiological impairment under renewed drought, despite showing similar stress sensitivity at maximum water deficit. This persistence of enhanced recovery capacity across vegetative propagation indicates that drought-induced memory was maintained beyond the initially stressed plants. Together, our findings demonstrate that recurrent drought establishes a metabolically imprinted state in clonal sugarcane, integrating physiological adjustment, metabolic reprogramming, and whole-plant acclimation. These results highlight the potential of stress memory as a mechanism supporting resilience in perennial crops exposed to increasingly recurrent drought events.

Understanding the genetic architecture of ecophysiological traits related to carbon gain and water loss is crucial to develop cultivars tolerant to abiotic stresses and that maintain productivity in the context of climate change. This can be achieved both through experiments in the greenhouse on potted plants under controlled conditions and in the field. However, the stability of genetic determinism of ecophysiological traits across environmental conditions has rarely been evaluated. We studied a grapevine diversity panel grown both in a greenhouse and in the field, to investigate the impact of growing conditions on leaf ecophysiological traits related to leaf morphology, fluorescence, gas exchange and water use efficiency. The correlation structure between traits was conserved within each environment even though, for most traits, cultivars ranking was not conserved between both environments. However, a set of cultivars, sharing the same functioning pattern in terms of gas exchange and fluorescence in both environments, was identified. Single- and multi-environment Genome-Wide Association Studies (GWAS), based on a dense genotyping matrix, were used to dissect the genetic architecture of leaf traits. The Quantitative Trait Loci (QTLs) identified through single-environment analyses differed substantially from those detected using multi-environment ones. Multi-environment approaches highlighted shared QTLs across environments for gas exchange and fluorescence traits. Candidate genes associated with carbon- and water-related functions were further identified in these genomic regions. Despite the weak correlation between greenhouse and field trait values, combining both environments enabled the identification of robust genetic regions associated with ecophysiological traits of interest.

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.

Cannabis sativa L. cultivation in tropical regions or under indoor conditions requires supplemental lighting to maintain plant yields. Night-break (NB) photoperiods offer a cost-effective alternative to conventional day extension, although genotypic responses have not been clearly described. This study evaluated the effects of continuous and pulsed NB treatments on the growth, phenology, physiology, productivity, and cannabinoid accumulation of three Cannabis sativa genotypes representing two chemotypes. Plants were exposed to a commercial 18-h photoperiod (T1), a continuous 1-h NB (T2), or four 15-min NB pulses (T3) with equal total duration. Continuous NB produced responses comparable to the commercial photoperiod. In contrast, pulsed NB accelerated phenological progression by two to three weeks and reduced final plant height by 15-28%, depending on genotype, without impairing photosynthetic performance or decreasing floral dry biomass. Harvest index increased under both NB treatments, indicating enhanced biomass allocation to reproductive structures, while cannabinoid accumulation remained primarily genotype-dependent with only minor treatment-specific modulation. These findings demonstrate that NB efficacy depends not only on the duration of nocturnal light but also on its temporal distribution, highlighting temporal organization of nocturnal light as a key regulator of photoperiodic responses and a promising strategy for optimizing cannabis cultivation.

Amaranthus tricolor is a climate-resilient orphan crop whose betalain pigments contribute to nutritional quality and stress tolerance. However, breeding efforts have traditionally relied on field yield trials due to the limited availability of genomic information and genetic resources. To address this gap, we first used the Illumina and PacBio sequencing platforms to assemble a novel reference genome of A. tricolor, using the Japanese cultivar Bayam. By combining this reference genome with a core collection developed in our previous study, we aimed to identify genomic regions associated with pigmentation-related traits across winter and summer seasonal environments. Betalains are antioxidant phytonutrients that contribute to abiotic stress tolerance, making the regulation of their biosynthesis under heat stress particularly relevant for developing climate-resilient crops such as A. tricolor. As a result, ATR1.0CH03_5131103, which is associated with ATR1.0ch03g000565, was identified under the winter seasonal environment. Meanwhile, transcriptomic profiles in this study revealed that ATR1.0ch03g000565 was differentially expressed under both control and heat stress conditions, suggesting that it may be associated with the regulation of CYP76AD1 expression under optimal conditions. Under the elevated-temperature treatment, transcript levels of CYP76AD1 and 5GT decreased in Am087, coinciding with lower betacyanin-dominated absorbance indices. cDOPA-like homologs were upregulated; however, pigmentation-related absorbance remained reduced. Further molecular experiments, such as CRISPR-Cas9 knockouts or overexpression studies, are necessary to validate the regulatory mechanisms of CYP76AD1 and 5GT under heat stress.

Bulk phloem flow is driven by pressure gradients generated by water drawn from the xylem, coupling source-sink transport with water status. Huanglongbing (HLB), a phloem-affecting bacterial disease of citrus, disrupts phloem transport, and causes canopy water deficit. Although improved water management can enhance HLB-affected tree health, the effects of HLB on water-driven source-sink relations are unknown. Fruits function as both carbon sink and water reservoir, but the role of fruit in regulating whole-tree water status remain poorly understood. We examined leaf-fruit water relations in HLB-affected citrus and evaluated how HLB exposure duration, fruit developmental stage, and source-sink manipulation shape these dynamics. Source-sink relations were manipulated in branches of symptomatic trees with long-term (four years) and recent (six months) HLB exposure. We girdled branches with a single fruit as the sink and defoliated at (0%, 50%, or 75%), and an ungirdled, non-defoliated branch served as control. We measured stomatal conductance, leaf hydraulic traits, leaf and fruit soluble sugars, diurnal fruit shrinkage (to estimate xylem backflow) and fruit water loss. Source-sink manipulation did not affect shoot total soluble sugar. HLB-exposure resulted in less negative osmotic potential, more negative turgor potential and reduced modulus of elasticity and relative water content at turgor loss point. HLB exposure also increased diurnal fruit shrinkage, with shrinkage responses to source-sink manipulation varying across fruit development stages. Collectively, these findings suggest that long-term HLB exposure promotes leaf acclimation to water deficits, however, high sink demand may diminish the leaf water status regulation at Stage II, when fruit sink carbon demand is highest.

Chlorophyll fluorescence imaging is a powerful tool used to characterize abiotic and biotic stress response of photosynthesis in plants. However, extracting metrics such as Fv/Fm (maximum quantum yield of photosystem II) from chlorophyll fluorescence images remains a substantial bottleneck in large measurement campaigns, often requiring significant manual effort. To resolve this issue, we developed fvfmPy, a user-friendly program which enables rapid analysis of chlorophyll fluorescence images of arrays of leaf discs. fvfmPy uses algorithmic image segmentation to identify regions of interest and extract fluorescence metrics from tens or hundreds of leaf samples per image with little to no manual input. fvfmPy is developed in Python and includes a graphical user interface enabling intuitive parameter adjustment and real-time quality control checking. We additionally developed fvfmR, an R wrapper package for fvfmPy. Fv/Fm values extracted with fvfmPy were indistinguishable from those obtained using manual methods (r2 > 0.99) but required 94% less time to complete (13 s vs. 200 s per 100 measurements). fvfmPy and fvfmR were built to directly integrate with existing fluorescence software packages and can greatly increase throughput of fluorescence measurements to help address key challenges in agriculture and ecology.

BackgroundN6-Methyladenosine (m6A) regulates RNA fate in flowering plants, but the repertoire and expression patterns of m6A-related genes remain incompletely characterized in liverworts. Here, we characterized these gene families in the liverwort Marchantia polymorpha within a comparative framework spanning diverse plant lineages. ResultsA comparative survey of 119 plant genomes using sequence similarity and conserved-domain criteria identified 18 candidate m6A-related loci in M. polymorpha, comprising seven writer-related genes, five AlkB homolog genes, and six YTH- or CPSF30-related genes. Across the surveyed genomes, canonical writer components generally had low copy numbers, whereas AlkB homolog and reader-related families showed greater copy-number variation. Phylogenetic and domain analyses supported the family assignments of the M. polymorpha candidates. Eight of the 18 candidate loci showed detectable collinearity with Marchantia quadrata. Putative promoter analysis identified motifs annotated as hormone-, light-, and stress-responsive, with hormone-responsive motifs accounting for the largest share of predicted sites. Public transcriptome analysis revealed variation in candidate gene expression across developmental stages and tissues. Quantitative reverse-transcription PCR showed higher transcript abundance for all six YTH- or CPSF30-related genes in thalli than in gemmae or gemma cups. Cold, heat, osmotic, salt, and dark treatments elicited gene-specific expression responses. Darkness increased transcript abundance of several candidates, including MpMTA, MpALKBH1A, and MpECT2. A predicted protein-association network suggested links between subsets of the candidates and RNA-processing proteins. ConclusionsThese findings define a candidate m6A-related gene repertoire in M. polymorpha and identify tissue- and stress-responsive genes for targeted functional studies of RNA methylation in this liverwort.

- C2 photosynthesis reduces photorespiratory carbon loss by shuttling glycine from mesophyll (M) to bundle sheath (BS) tissues, where glycine decarboxylase (GDC) releases CO2 that accumulates around rubisco. C2 photosynthesis has been considered a constitutive character, but as a metabolic trait, it may acclimate to the rate of photorespiratory glycine production. - We grew eleven species from two eudicot and two grass lineages spanning C3 to C2 phenotypes at 32 {degrees}C days and 200 or 800 {micro}mol mol-1 CO2. Acclimation was assessed using leaf gas exchange, GDC P-protein immunolocalization, M and BS ultrastructure, photosynthetic enzyme activity, and photosynthesis modelling. - Low relative to high growth CO2 enhanced C2-related phenotypes in sub-C2 and C2 species but not C3 or proto-Kranz species. Sub-C2 species positioned chloroplasts closer to mitochondria and had lower modelled BS conductance. C2 species increased rubisco capacity and GDC allocation to the BS. Aspartate aminotransferase activity increased in both sub-C2 and C2 species. - Acclimation to greater photorespiratory conditions strengthens C2 photosynthesis, but only in species whose BS has crossed a threshold of organelle and GDC investment. We hypothesize that acclimation could expose C2-related traits to selection and facilitate the origin of C4 photosynthesis.

Efficient yield prediction under field conditions remains a key challenge for crop breeders and agronomists. YieldLearn is an interpretable machine-learning pipeline developed to estimate yield-related traits in durum wheat (Triticum durum) using physiological indicators measured by the Hansatech Instruments Pocket PEA. The workflow integrates fluorescence parameters (Area, Fo, Fm, Fv/Fm, PI Inst.) with field-measured agronomic traits (Booting, Height, Maturity, Lodging, Yield kg/plot, and Adjusted Yield kg/ha). Five supervised learning algorithms multinomial logistic regression, random forest, support vector machine (radial kernel), k-nearest neighbors, and decision tree were evaluated using five-fold cross-validation. Models achieved average classification accuracy around 0.57 with AUC values exceeding 0.58. SHAP (SHapley Additive exPlanations) visualizations revealed that high Fv/Fm and PI Inst. values were positively associated with yield potential, whereas elevated Fo indicated stress-induced yield reduction. An interactive Shiny App enables real-time prediction and feature-importance exploration. Given the small sample size (165 plots) and absence of environmental covariates such as temperature or soil moisture, YieldLearn should be regarded as a reproducible exploratory framework rather than a production-ready predictor. Nonetheless, it offers a valuable foundation for guiding experimental design, validating physiological indicators, and supporting the next generation of scalable, interpretable crop-yield models.

Pigment composition and regulation are critical for efficient photosynthesis in the ocean, where light intensity decreases and the spectrum narrows with depth. Fucoxanthin (Fx), the main carotenoid of several microalgal lineages, harvests the blue-green light prevailing in the deep euphotic zone. In pelagophytes, the acylated derivative 19'-butanoyloxyfucoxanthin (19'-BFx) is abundant, yet its function and regulation remain unclear. Here, we investigated how irradiance and spectral quality shape photoacclimation in Pelagomonas calceolata, an abundant and cosmopolitan low-light pelagophyte. Cultures were grown under blue or white light across 2-60 umol photons m^-2 s^-1. We analysed growth, PSII photophysiology, pigment composition, gene expression levels, and the impact of spectral shifts on the 19'-BFx/Fx ratio. Pelagomonas calceolata grew optimally under dim blue light and displayed low non-photochemical quenching (NPQ) under assay conditions. Transcriptomes revealed broad remodelling driven mainly by irradiance and modulated by light colour, including carotenoid-related processes. Dim blue light progressively increased the 19'-BFx/Fx ratio, whereas white light kept it low and reversed the response after a blue-to-white shift. These results identify reversible, spectrum-dependent 19'-BFx accumulation as a key component of photoacclimation in P. calceolata and separate irradiance-driven from spectrum-dependent cellular responses. This plasticity may explain the ecological success of P. calceolata in low-light oceans.

Boron toxicity constrains grapevine (Vitis) production in irrigated arid and semi-arid regions, yet most available rootstocks are sensitive to excessive boron. Although underutilized Vitis species may harbor sources of adaptation, the genetic basis of natural variation in boron exclusion remains poorly understood, limiting the development of boron-tolerant rootstocks. We screened 267 wild Vitis accessions representing 16 species for their ability to limit leaf boron accumulation under excess boron conditions. Leaf boron concentration varied 190-fold among accessions and was highly heritable (entry-mean H^2 = 0.71). Its correlation with stomatal conductance was modest (r = 0.34), indicating that transpiration rate explained only a small proportion of the variation in leaf boron accumulation. Leaf boron was correlated with leaf chloride (r = 0.36), but their largely non-overlapping association profiles supported distinct genetic control. Genome-wide association analysis identified a suggestive association on chromosome 11. Independent quantitative trait locus mapping in a full-sib F1 population (n = 158) derived from Longii-9018 (V. acerifolia) x GRN3 (commercial rootstock) identified qBEx11.1 in the same syntenic region on chromosome 11, which explained 19.5% of the phenotypic variance. Positional candidate genes near the association peak included a pectinesterase and a MATE efflux transporter, suggesting roles for cell wall modification and membrane transport. Leaf spectral reflectance was associated with leaf boron concentration in both populations, particularly at visible and red-edge wavelengths, indicating potential for high-throughput phenotyping. Together, these findings identify qBEx11.1 as a priority locus for boron exclusion and support integrated genetic and spectral approaches for breeding boron-tolerant grapevine rootstocks.

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

The Fusarium oxysporum species complex (FOSC) comprises numerous plant pathogens infecting crops such as tomato, lettuce, melon, and banana. Despite its economic and ecological importance and decades of research, the species names linked to different phylogenetic lineages within this species complex remain largely controversial. Earlier taxonomic frameworks relied on a limited number of marker genes, variable taxon sampling and inconsistent species definitions, leading to a fragmented and unstable taxonomy. To address these challenges, we here analysed nearly 2,500 conserved marker genes from 660 genomes, including genome assemblies from 22 ex-type strains that were sequenced using long-read sequencing technology, representing nearly half of the 51 currently described species within FOSC. Maximum likelihood- and coalescence-based analyses of the full phylogenomic data matrix as well as subsets yielded a robust phylogeny of FOSC, which was furthermore corroborated by whole genome comparisons. Many previously described species are redundant, while two novel lineages remain undescribed. Direct comparisons to traditional marker gene-based approaches demonstrate their inadequacy for species delimitation in FOSC. We show that our phylogenomic framework can guide the detection of novel marker genes able to distinguish the major lineages within FOSC, which will be essential for taxon delineation and classification in future studies. Thus, our phylogenomic analyses provide a stable cornerstone for revising FOSC taxonomy and offers a critical resource for the broader research community.

We performed a screening to evaluate the responses to water deficit before the flowering stage in a panel of 31 genotypes composed of one commercial hybrid, one wild species, multiple ex-PVP (vintage) lines, several commercial heirlooms from the Northeast USA, Mexican landraces from hot and humid environments and genome edited lines with altered flowering and inflorescence traits. We characterized 18 different morphological, physiological and biochemical traits to assess the response to 40% water deficit. We observed diverse morphological, physiological and biochemical responses across the 31 genotypes screened, suggesting the presence of useful variation for breeding for drought tolerance in cultivated tomato. Interestingly, some heirlooms exhibited superior performance under drought, suggesting they can be used as donors to discover novel genes involved in drought responses. Unexpectedly, we found mutants in SlCLE9, a paralog of the stem cell regulator SlCLV3 known to co-regulate meristem proliferation in tomato, exhibited enhanced drought tolerance. Notably, the slcle9 mutant also conferred drought tolerance when used as rootstock with grafted scions from our reference cultivar M82, suggesting SlCLE9 influence root architecture and drought response. Consistent with this, we found SlCLE9 is transcriptionally upregulated in roots and leaves during drought stress, and exogenous application of SlCLE9 modifies root growth and response to drought. Altogether, our screening uncovered new potential trait donors for breeding against drought stress and describes a novel role for SlCLE9 in drought tolerance.

Petal coloration is a key ornamental trait in Cyclamen persicum Mill. and is closely associated with anthocyanin composition and relative accumulation, yet the major anthocyanins and candidate molecular factors associated with color variation remain unclear. In this study, anthocyanin profiling and transcriptome analysis were integrated to investigate three Cyclamen materials with distinct petal colors, BXK, FXK, and SHXK. Anthocyanin profiling showed clear separation among the three materials, indicating that petal color divergence was associated with differences in anthocyanin composition and relative abundance. Petunidin-, peonidin-, delphinidin-, malvidin-, and cyanidin-related derivatives exhibited distinct accumulation patterns. Transcriptome analysis revealed marked transcriptional divergence, with phenylpropanoid and flavonoid biosynthesis repeatedly enriched across comparisons, whereas isoflavonoid and flavone and flavonol biosynthesis were enriched in selected comparisons. Mapping differentially expressed genes onto the anthocyanin biosynthetic pathway revealed branch-specific expression patterns of structural genes, including CHS, F3H/F3'H, F3'5'H, DFR, and ANS. Notably, Cluster-26256.0, annotated as a putative anthocyanidin synthase gene, showed a progressive increase in expression from BXK to FXK and SHXK, consistent with increasing petal pigmentation. Several DFR-related transcripts also showed relatively high expression in SHXK. Co-expression analysis linked key anthocyanins, structural genes, and transcription factors, while qRT-PCR generally confirmed RNA-seq trends. These results suggest that late anthocyanin biosynthesis, particularly ANS-related transcriptional variation, may contribute to dark red petal coloration. This study provides a metabolite-transcript framework for Cyclamen petal coloration and identifies ANS-related genes as priority candidates for future functional studies and flower-color breeding.

Synthetic nitrogen fertilizers enhance maize (Zea mays ssp. mays) productivity but impose substantial environmental and economic costs. Some Mexican maize landraces support biological nitrogen fixation (BNF) in aerial root mucilage, raising the question of how much variation exists in this trait and how domestication, geographic spread, and improvement have shaped BNF-related traits across Zea in the Americas. We evaluated aerial root traits and BNF capacity across the maize domestication continuum using 60 Zea accessions grown in field experiments over two years under low-N conditions. Some accessions of wild Zea, landraces from Mexico and South America, and improved varieties had elevated nitrogen derived from the atmosphere (%Ndfa), ranging from 20 to 40%; some of these also had greater %Ndfa than typical improved varieties. Spread and improvement appear to have significantly reduced the number of aerial root-bearing nodes, with wild Zea and Mexican landraces consistently producing more. Nevertheless, the proportion of nitrogen derived from the atmosphere (%Ndfa) does not vary systematically across the domestication continuum. Thus, crop evolution shaped aerial root phenotypes more than associative BNF capacity. The persistence of substantial within-group variation indicates that the genetic potential for enhanced BNF remains within the Zea gene pool, providing opportunities to encourage and improve nitrogen use efficiency in maize cropping systems.

Iron (Fe) homeostasis is fundamental to plant growth and development and is centrally regulated by a well-conserved network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 regulates the expression of FER-like iron deficiency-induced transcription factor (FIT) and clade Ib bHLH38/39/100/101 genes, among others. However, its functional conservation remains unexplored, and some contradictory results have been reported regarding the effects of bHLH121 overexpression. Here, we investigated the evolutionary conservation of bHLH121 through complementary tests in the Arabidopsis bhlh121 mutant created by CRISPR/Cas9 gene editing. We also investigated the effects of ectopically expressing full-length, N-terminal, and C-terminal fragments of bHLH121 on plant responses to Fe deficiency in Arabidopsis. Transgenic plants overexpressing the full-length bHLH121 driven by the cauliflower mosaic virus 35S promoter (bHLH121 ox) resulted in shorter roots and more severe leaf chlorosis compared to wild-type plants under Fe limiting conditions. Moreover, overexpression of the N-terminal fragment (N121 ox) rendered plants highly sensitive to Fe deficiency, whereas overexpression of the C terminal fragment (C121 ox) did not. Consistent with the phenotypic observations, N121 ox plants accumulated lower Fe content, and the ferric chelate reductase activity was significantly reduced. In N121 ox plants, the induction of key Fe regulatory genes, especially FIT, was significantly inhibited under Fe deficiency. Confocal imaging showed that both N121-GFP and C121-GFP fusion proteins could localize to the nucleus. Electrophoretic mobility shift assay showed that both full-length bHLH121 and N121 could effectively bind the probes derived from the promoter of FIT. The present work reveals a conserved function of bHLH121 in Fe homeostasis across several dicot species and demonstrates that the N121 may possess a dominant nature in Fe deficiency responses.

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

Drought stress significantly reduces tomato (Solanum lycopersicum L.) productivity, and early detection is critical to minimize yield losses through timely interventions. In this study, we developed Drought-Spec-Net, a hybrid 1D convolutional neural network that integrates local and global spectral feature extraction to detect early drought stress from visible and near infrared (Vis NIR) spectra data of tomato seedlings. The model was trained on 378 samples using an 80:20 train test split, with 20% of the training set reserved for validation. DroughtSpecNet outperformed the evaluated baseline and state of the art models, achieving 97% accuracy, 95% precision, 98% recall, and an F1 score of 97%. To improve the agronomic interpretation of the model outputs, predicted drought probabilities were converted into a literature-informed potential yield impact indicator using a maximum impact level of 60%. On the test set (76 samples), mapped potential yield-impact values ranged from 0% to 60%, with an average reduction of 12.97%. We also conducted an initial experiment using our greenhouse RGB dataset, collected daily from drought treated and well-watered tomato plants at West Virginia State University (WVSU). From this dataset, 44 images were selected for ilastik-based canopy segmentation, producing plant-level drought severity indices (DSI) with a mean of 0.28, median of 0.14, and range of 0.01 to 0.91. Additionally, we trained and fine-tuned a large language model (LLM) based on PLLaMA7BInstruct, called AgriLLaMA, for automated agronomic report generation from Drought-Spec-Net outputs. The generated reports summarize predicted stress levels, mapped potential yield impacts, and preliminary management considerations. This integrated approach not only improves early drought stress detection but also delivers quantitative and interpretable estimates of potential productivity losses, providing a complete framework connecting physiological stress detection to actionable agricultural outcomes.