Environmental and Experimental Botany

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.

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.

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.

Climate change is increasing the frequency and intensity of heatwaves and droughts, threatening medicinal and aromatic plants (MAP) production. Temporary shading during the hottest part of the day may reduce heat and radiative stress while limiting the reduction in light availability. We evaluated this strategy in a field experiment using three MAP species with contrasting ecological requirements and harvested organs: lavandin (Lavandula x intermedia), curly parsley (Petroselinum crispum) and valerian (Valeriana officinalis). Vertical shade nets provided midday shading and effects on crop microclimate, leaf temperature, water status, PSII photochemical efficiency, plant growth and biomass and essential oil production were assessed over two growing seasons. Midday shading reduced daily photosynthetically active radiation by 19.9-55.9 %, with weak effects on air temperature and vapor pressure deficit. In contrast, leaf temperature at solar noon decreased by 6.9 degrees C, 5.6 degrees C and 3.0 degrees C in lavandin, valerian and parsley, respectively. Midday shading improved light-adapted and maximum PSII photochemical efficiencies in all three species and reduced branch dehydration in lavandin during heatwave periods. However, agronomic responses were species-dependent. Floral biomass decreased in lavandin, whereas parsley leaf biomass and valerian root biomass increased. Essential oil concentration was unaffected in lavandin and valerian. Overall, our results show that midday shading primarily reduced leaf-level thermal and radiative stress rather than modifying the surrounding air microclimate, and suggest that its agronomic effects may reflect a balance between stress alleviation and reduced carbon acquisition, that varies with species ecology and production objectives.

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.

High temperatures during grain filling stage are becoming increasingly frequent, compromising both grain and eating quality and thereby driving demand for heat-resilient cultivars. Such conditions are known to reduce the expression of granule-bound starch synthase I (GBSSI) and starch branching enzyme IIb (BEIIb), which are involved in starch biosynthesis, resulting in a decrease in amylose content and an increase in long-chain amylopectin. Thus, the present study introduced functional mutation in starch synthase IIa (SSIIa) that increases the proportion of short amylopectin chains to genetically compensate for the high-temperature-induced increase in amylopectin long chain. Rice lines carrying the ss2a mutation were grown at two locations with cooler (Akita) and warmer (Okayama) temperatures. Their grain traits, starch structure, and eating quality were compared. The ss2a mutant lines showed an increased proportion of short amylopectin chains as well as an increased apparent amylose content. Furthermore, these alterations in starch structure varied with the grain-filling temperature of the cultivation sites, ultimately affected eating quality. These results suggest that enriching short amylopectin chain via the ss2a mutation can counteract the increase in long amylopectin chain caused by high temperatures during grain filling, thereby maintaining a desirable starch structure and eating quality.

Although iron (Fe) is abundant in soils, its low solubility, particularly under alkaline conditions, often limits plant Fe acquisition. Under these conditions, coumarin-mediated Fe mobilization becomes increasingly important. Arabidopsis thaliana FE DEFICIENCY-INDUCED KELCH REPEAT PROTEIN 1 (FDK1, AT3G07720) is strongly induced by Fe deficiency and regulated by the central transcription factor FIT, but its function is unknown. We investigated FDK1 using genetic, physiological, metabolite, and transcriptomic analyses. Loss of FDK1 altered root growth, whereas this phenotype was restored in the FDK1-Ox overexpression line. Under alkaline Fe deficiency, growth medium from FDK1-Ox contained strongly increased levels of the Fe-mobilizing coumarins fraxetin and esculetin compared with wild type, whereas their induction was attenuated in fdk1-1. RNA-seq analysis linked FDK1 abundance to coumarin biosynthesis and iron-homeostasis gene expression. FIT and many FIT-associated genes were highly expressed in FDK1-Ox, whereas several upstream iron-homeostasis regulators, including bHLH Ib factors, were highest in fdk1-1. These findings identify FDK1 as a component of the alkaline Fe-deficiency response that promotes iron-mobilizing coumarin accumulation and is associated with coordinated regulation of iron-homeostasis genes.

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 and waterlogging are prominent climate change challenges that, alone or in sequence, pronouncedly constrain agricultural crop production. A better understanding of relationships between water availability and nutrition regarding plant functioning is needed to optimise fertilisation under these challenges. We investigated impacts of drought, waterlogging and drought followed by waterlogging under two fertilisation regimes (low, normal) on various traits of wheat (Triticum aestivum). Next to biomass and photosynthesis measurements, cross-platform metabolomics was applied for in-depth metabolic analyses of flag leaves. We found detrimental effects of drought and drought-waterlogging on shoot and root biomass, especially at normal fertilisation, while photosynthetic traits were not or only slightly affected. Foliar nitrogen contents were higher in plants exposed to drought or to drought-waterlogging. Numerous primary and specialised metabolites were modulated by fertilisation and/ or watering. In particular, amino acids, benzoxazinoids and flavonoids were affected by fertilisation. Due to watering especially several amino acids changed in concentrations, with the induction pattern partly depending on fertilisation. Our study deepens the understanding of the interplay between water and nutrient availabilities as well as nutritional demands, growth and metabolism of wheat. This is highly relevant to balance sustainable agricultural nutrient management under the challenges of ongoing climate change.

Plant encounters with saline conditions trigger a phase of osmotic stress, followed by ionic stress as salt accumulates in plant tissue. Jasmonate (JA) signalling plays a vital role in both phases, but its role for adaptation depends on timing. Using wild-type rice (Oryza sativa L. cv. Kitaake) and a JA-deficient allene oxide cyclase (aoc) mutant, we followed whole-plant physiology (growth, biomass, photosynthesis, ion distribution) with expression analysis for genes of jasmonate signalling, ion transporters and redox balance to separate adaptive from maladaptive JA effects. The aoc mutant sustained shoot and root growth more effectively than the wild type, maintaining cell expansion in the shoot and cell proliferation in the root under saline conditions. Likewise, stomatal conductance in the mutant was more persistent under salinity, accompanied by improved shoot K/Na homeostasis and reduced sodium in young leaves. In the wild-type, prolonged saline conditions were linked with induction of OsJAZ13 and OsNHX1, stomatal closure and chlorophyll degradation. We discuss these findings in the context of a model, where the function of JA signalling does not merely depend on its accumulation, but on its temporal dynamics. Here, OsHAK16 emerges as a marker of early adaptive responses, whereas OsNHX1 and OsJAZ13 characterize later maladaptive stress states, emphasising the potential of modulating JA dynamics to improve salt resilience in rice breeding.

Groundnut (Arachis hypogaea) is an important oilseed crop, and its production is severely affected by drought worldwide. Improving the drought-tolerance of groundnut without compromising its agronomic traits is a major challenge for researchers. Here, a meta-transcriptome analysis identified tandemly duplicated G{beta} subunits (AhG{beta}1 and AhG{beta}3) as early signaling genes under water deficit conditions. Heterotrimeric G-protein {beta}-subunit is a major signaling element that can regulate diverse responses in plants. Heterologous complementation studies in Arabidopsis G{beta} mutant (agb1-2) demonstrated evolutionary conservation of AhG{beta} function, while the transgenic overexpression and RNAi knockdown in groundnut established AhG{beta}1 as a positive regulator of plant growth, yield, and drought tolerance. Integrated transcriptomic, transcription factor regulatory network, and metabolomic analyses demonstrated that AhG{beta}1 coordinates drought-responsive transcriptional and metabolic reprogramming characterized by suppression of cell wall-associated processes and activation of phenylpropanoid, flavonoid, and isoflavonoid biosynthesis. This reprogramming resulted in enhanced accumulation of antioxidant flavonoids, including quercetin and rutin, reduced ROS accumulation, and improved drought tolerance. Furthermore, exogenous application of these flavonoids enhanced drought recovery, highlighting their role as protective downstream metabolites. Collectively, our findings establish AhG{beta}1 as a conserved regulator linking early G-protein signaling with transcriptional and metabolic reprogramming to maintain redox homeostasis during drought stress and identify promising molecular and metabolic targets for improving drought resilience in groundnut.

Palladium (Pd), a technology critical platinum group metal, is increasingly lost to the environment through anthropogenic activity, yet the molecular basis of Pd uptake and tolerance in plants remains poorly understood. Here, we examine the role of the Arabidopsis thaliana (Arabidopsis) COPT2 copper transporter in Pd transport and plant stress responses. Expression of COPT2 in Saccharomyces cerevisiae conferred Pd sensitivity, indicating a capability for Pd transport. Arabidopsis copt2 loss-of-function mutants accumulated less Pd in shoots and exhibited reduced reactive oxygen species (ROS) production, supporting a direct role for COPT2 in Pd distribution and associated oxidative stress. Our subsequent transcriptome profiling revealed extensive changes under Pd exposure, including downregulation of HMA2 and aquaporins, and upregulation of HMA7, glutathione transferases, and glutamine synthetase GLN1;1, consistent with the activation of metal detoxification and redox homeostasis pathways. Together, these findings identify COPT2 as a key transport protein mediating the uptake of Pd in Arabidopsis and suggest that Pd induces similar detoxification mechanisms as zinc (Zn), copper (Cu) and cadmium (Cd), providing a foundation for engineering plants for precious metal recovery.

Cassava produces starch-filled tubers which are a crucial food source for many people, especially in Africa. This plant species is famous for drought tolerance and resilience, but changing conditions due to climate change require genetic improvements to ensure sustainable cassava cultivation. In this context, a comprehensive understanding of the genetic basis responsible for a variety of traits is beneficial. Here, we present insights into the anthocyanin metabolism, responsible for coloration and protection of cassava structures during stress. Intraspecific variation in the transcriptional control of the anthocyanin biosynthesis was characterized through comparative genomics and phylogenetic analyses. A member of the R2R3-MYB transcription factor family, orthologous to crucial activators of anthocyanin biosynthesis in other plant species, was identified as a genetic hotspot for pigment accumulation capacity between cultivars. Orthologs of this gene appeared absent from three cassava cultivars without pronounced anthocyanin pigmentation, while being present in cultivars known for anthocyanin formation. This discovery corroborates previous reports about the importance of transcriptional regulators in the intraspecific diversity of anthocyanin pigmentation.

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.

Background and aims Alpine ecosystems are highly sensitive to climate change, with rising temperatures threatening ecosystem structure and function. This study examined the short-term responses of alpine grassland ecosystems (Mont Blanc, Italy) to projected climate scenarios using an Ecotron mesocosm experiment. Methods Ecosystem monoliths were exposed to current (~420 ppm CO2) and future CO2; concentrations (~550 and ~800 ppm) under RCP 4.5 and RCP 8.5 scenarios, incorporating changes in temperature, precipitation, humidity, and radiation. CO2; fluxes (NEP, GPP, Reco), soil chemistry, vegetation coverage, above- and belowground biomass, and plant functional traits were assessed throughout the experiment. Results Alpine grassland ecosystems withstood moderate warming (RCP 4.5), while under severe warming (RCP 8.5), GPP, vegetation cover and biomass increased with a shift toward aboveground allocation, although daytime NEP remained stable as gains were offset by higher Reco, with no detectable water and nutrients limitations. On a diel basis, higher GPP was offset by enhanced Reco, indicating a potential towards net C loss. Salix herbacea exhibited functional trait adjustments-higher LMA and Na, and elevated delta15N-reflecting greater leaf longevity, water-use efficiency, and altered nitrogen metabolism, suggesting potential community reorganization. Conclusion Over a single growing season, alpine ecosystems appeared relatively stable under moderate warming. However, under the more severe RCP 8.5 scenario, biomass allocation shifted toward greater aboveground and reduced belowground investment, leading to altered C fluxes that, if sustained over the long-term, could drive the ecosystem C balance from a sink to a source. Responses were plant species-specific, indicating uneven acclimation within the community.

DNA methylation dynamics regulate diverse plant stress responses, but the mechanisms governing arsenic (As) tolerance through epigenetic pathways remain largely unknown. Here we report that Arabidopsis mutants lacking the DNA demethylase, REPRESSOR OF SILENCING (ROS1), show substantially enhanced As resistance. Integrating transcriptome and methylome data, we found that PIN-FORMED 2 (PIN2), encoding an auxin efflux carrier that also transports As out of cells, represents a key ROS1 target. In ros1, we observed increased methylation in the last exon of PIN2, a region functioning as the promoter for an antisense long non-coding RNA (lncRNA). Hypermethylation of the promoter suppresses the antisense lncRNA, which follows reduction of sense PIN2 transcription. As a result, PIN2 expression increases in ros1 mutants during As exposure, enhancing As tolerance. Analysis of ros1 pin2 double mutants confirmed that PIN2 acts downstream of ROS1 in As tolerance. Our study demonstrates an epigenetic pathway in which ROS1 regulates As tolerance by controlling PIN2 expression through antisense lncRNA-mediated transcriptional interference, offering new epigenetic strategies using plants in heavy metal contaminated environments.

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.

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.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.