Plant Direct

Recent studies have demonstrated the presence of kynurenine (KYN) and kynurenic acid (KYNA) in several plant species, but the metabolic function of these metabolites remains undefined. We hypothesized that KYN and KYNA are metabolites of auxin and play a role in plant morphogenesis. To test our hypothesis, we developed a plant tissue-culture-based bioassay using Hypericum perforatum (St. Johns wort; SJW), a model system for auxin and indoleamine metabolism and pharmacological inhibitors (PF-04859989, RO-61-8048, and KMO inhibitor II, JM6) of human kynurenine pathways enzymes. SJW is an interesting model system because explants root in the absence of plant growth regulators but supplementation of the culture media with 10 M IAA induces a callus response without de novo root organogenesis. Supplementation of the culture media with 10 M KYN increased root number and internodal length relative to basal media. We used a previously validated high-resolution mass spectrometry analytical method to quantify KYN, KYNA, and 3-hydroxyanthranilic acid (3-HAA). KYN, KYNA and 3-HAA were quantified in roots and shoots of SJW grown on basal media. Supplementation of the culture media with 10 M KYN increased the concentration of KYN, KYNA and 3-HAA in roots and shoots. Treatment with 10 M IAA increased KYN and 3-HAA concentration in shoots. Three pharmaceutical candidates that are kynurenine pathway inhibitors in humans were taken up into the tissues from the culture media and increased KYN content as compared to basal control. Together, these data propose a role for KYN in IAA metabolism, shoot and root organogenesis. HighlightsO_LIKynurenine metabolites are detected and accumulate in H. perforatum tissue culture C_LIO_LIIAA redirects metabolism towards accumulation of KYN and 3-HAA in shoots C_LIO_LIExogenous KYN promotes KYNA accumulation C_LIO_LIPharmacological inhibition alters kynurenine pathway metabolite profiles in a tissue-specific manner C_LIO_LIKynurenine and IAA differentially regulate root development C_LI

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

Drought and heat are key abiotic stresses that severely affect crop production. Sorghum (Sorghum bicolor L. Moench), a drought-resilient cereal, serves as a model for studying abiotic stress responses in cereals. To elucidate the molecular responses to individual drought and heat stress, we conducted a transcriptome analysis of sorghum seedlings subjected to individual drought and heat treatment for 1h and 6h. Our analysis revealed distinct as well as overlapping patterns of gene expression between the two stresses at two time points, with a larger transcriptional shift observed at 6h of drought and heat treatment. We identified 410 and 4,136 differentially expressed genes (DEGs) in response to 1 and 6 h of drought treatments, respectively, whereas 1,807 and 2,776 DEGs were identified under 1 and 6 h of heat treatments. Among all four stress conditions, 32 common DEGs were identified. Genes encoding ion transporters were enriched among DEGs common in both 1h stress treatments. Genes involved in ribosome biogenesis were enriched among the common DEGs in both 6 h stress treatments. Specifically, drought-regulated genes were involved in ribosome biogenesis, whereas heat-induced genes were involved in protein folding and histone modifications. Enrichment of ribosome biogenesis in different sets of DEGs suggests that maintaining a balance between growth and survival by regulating protein synthesis may play a role in defining early stress response in sorghum. 6h of drought resulted in strong upregulation of abscisic acid and jasmonate-associated genes. Genes encoding bZIP, MYB, and HSF transcription factors displayed both stress-specific and common temporal regulation, suggesting vital regulatory roles of these transcription factors in mediating responses to drought and heat treatments. Extensive downregulation of genes encoding core histone proteins, in response to both 6h of drought and heat stress, was detected, indicating possible roles of chromatin structure and accessibility in mediating early responses to drought and heat treatments in sorghum.

Lipids are the predominant building blocks of plant membranes and are essential for plant growth and development. They are crucial for survival during times of stress as lipids are involved in multiple signaling pathways, and their relative abundances can change in response to environmental factors. To better characterize the lipid composition of the vital food crop maize, we generated a comprehensive glycerolipid atlas using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. We surveyed the lipid profiles of three different maize genotypes: B73, a temperate inbred; CML312, a subtropical inbred; and Palomero Toluqueno, an open-pollinated variety from the Mexican highlands. We collected leaf samples from 4 developmental stages and 6 leaves. From one growth stage, we also sampled along with three leaf zones: base, center, and tip. The genotype and leaf number were the major drivers of lipid differences. Phosphatidylcholine, lysophosphatidylcholine, and triacylglycerol genotypic differences were particularly high. We generated an eFP browser to be integrated into the maize genome browser, as well as a separate web interface to easily browse and compare lipid levels across tissues and genotypes, available at https://rrellan.shinyapps.io/Zea-Lip/. SIGNIFICANCE STATEMENTThis work creates a spatial map of lipids in maize leaves across four growth stages for three genotypes: a lowland, a sub-tropical, and a highland. The resources generated here will directly benefit both the maize and lipid communities by creating a large dataset that can be used to generate new hypotheses in understanding lipid metabolism and environmental responses in maize.

Creeping bentgrass (Agrostis stolonifera) is a turfgrass species established on golf course surfaces but is criticized for high irrigation requirements. While genetic variation for water deficit stress tolerance exists between cultivars, the lack of defined critical soil water potential thresholds (Soil {Psi}crit) for this species complicates precise irrigation strategies and benchmarks for plant breeding. This study utilized a polyethylene glycol (PEG) infused agar-based system to simulate water potential reductions and determine the water potential threshold ({Psi}crit) for seedling root elongation. Creeping bentgrass cv Pure distinction seedlings were subjected to six water potentials ({Psi}) ranging from -0.36 MPa (no PEG applied) to -1.72 MPa. Daily digital imaging was used to measure root elongation over 5 days. Results across two experiments demonstrated that creeping bentgrass seedlings are highly sensitive to mild reductions in {Psi}. A reduction to -0.61 MPa significantly decreased root length and growth rates by over 50% compared to the control. Regression models predicted that a {Psi}crit of approximately -0.45 MPa reduced daily root growth by 25%, while upwards {Psi} of -1.0 MPa resulted in a 75% reduction of seedlings root growth. Furthermore, seedlings exposed to the lowest water potentials were predicted to require an additional 30 to 46 days to achieve the same root length as control plants. These findings establish specific {Psi}crit benchmarks for water deficit stress tolerance using a PEG-based system to induce dehydration. These methods can be used in breeding programs, and will help develop more accurate experiments examining the mechanisms of water deficit stress tolerance.

Cotton (Gossypium hirsutum) is an economically important crop, but it faces increasing pest outbreaks, especially in non-irrigated areas. In this study, 20 cotton farms using center-pivot irrigation were sampled in southern Georgia to assess chemical differences between non-irrigated and irrigated areas. Proton (1H) nuclear magnetic resonance (NMR) data were obtained from cotton leaves, and Principal Component Analysis (PCA) was performed to assess differences in chemical composition. Across all samples, farm site accounted for most of the variability, but within each farm site, the PCA scores plots showed clear separation between non-irrigated and irrigated conditions in 10 sites. Inspecting the PCA loadings revealed significant resonances resembling a lipid-like signal. After reverse-phase fractionation, we observed that many of these resonances appeared together in later fractions, suggesting a lipid, specifically a fatty acid such as linoleic acid. We hypothesized that differences in net lipid saturation level may drive separation between non-irrigated and irrigated samples. Six farm sites had a significantly or marginally significantly higher degree of unsaturation in irrigated samples, while one farm site had significantly higher unsaturation in non-irrigated samples. Our results indicate that drought stress likely affects lipid profile composition, which could be driving higher herbivorous pest densities in drought-stressed crops.

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii (C. richardii). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii (CrKAI2), as well as the signaling components CrMAX2 and CrSMXL. We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii. Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.

Strigolactones (SLs) are known to regulate shoot architecture and to be involved in plant responses to environmental stress, whereas their specific contributions to drought adaptation in barley remain incompletely defined. In this study, we analysed transcriptional, hormonal, and physiological responses to water deficit in barley SL mutants affected in early biosynthesis (Hvd10 and Hvd17), late biosynthesis (Hvmax1a), or signalling (Hvd14). The Hvd10, Hvd17, and Hvd14 mutants exhibited the typical high-tillering phenotype of SL deficiency, whereas Hvmax1a displayed characteristics similar to the wild type (WT), indicating functional differences within the SL biosynthetic pathway. Transcriptome analysis showed a clear overlap in gene expression among the high-tillering SL mutants under both control and drought conditions. We also used computational methods to identify potential transcription factors that might regulate SL-dependent gene expression. A drought experiment showed that SL mutants exhibited reduced biomass, relative water content, and photosynthetic efficiency, with the most pronounced effects observed in the high-tillering lines. Drought also activated the abscisic acid (ABA) pathway in all genotypes, with particularly high accumulation of ABA metabolites in the high-tillering SL mutants. Notably, Hvmax1a resembled the mutant-like metabolic profile, despite maintaining a wild-type-like architecture. Taken together, these results provide new insights into the roles of SL pathway components in drought responses and highlight functional differences among individual genes influencing both plant architecture and stress-related transcriptional programmes. Furthermore, the mutants generated in this study using Cas9-mediated genome editing represent a valuable genetic collection for future research into SL-mediated development and stress responses in barley.

Accurate verification of transgenic plant materials is essential for maintaining scientific integrity and ensuring experimental reproducibility. As the number and diversity of transgenic constructs continue to expand, there is a growing need for practical and scalable methods that enable routine confirmation of transgene presence and identity. Reliable detection systems are particularly important for laboratories handling large numbers of genetically modified lines or distributing materials across research groups. To address this need, we developed two complementary methods for efficient detection of commonly used transgenes. The first method, fDET, is a higher-throughput system capable of simultaneously detecting 15 transgenes and three endogenous genes in a single multiplex PCR reaction followed by capillary electrophoresis. This approach provides rapid, high-resolution detection suitable for high-volume or time-sensitive applications. The second method, DET, offers a more accessible workflow that detects 10 transgenes and one endogenous gene using four multiplex PCR reactions followed by agarose gel electrophoresis. Because DET requires only standard molecular biology equipment, it can be readily implemented in a wide range of laboratory environments without specialized instrumentation. Together, these methods provide flexible and practical solutions for verifying the genetic status of both transgenic and non-transgenic plant materials. By enabling efficient and comprehensive transgene detection, they support reproducible experimentation, facilitate quality control in plant research, and streamline the management and exchange of genetically modified lines. These approaches contribute to more reliable and transparent use of transgenic resources across the plant science community.

Heavy metals act as essential metalloprotein cofactors in numerous physiological processes but can become toxic when non-essential metals accumulate or when essential metals are in excess. As plants continuously encounter heavy metals through their roots, they have evolved complex homeostatic mechanisms to regulate metal uptake and distribution. The Heavy Metal ATPase (HMA) gene family encodes a group of heavy metal transporting P-type ATPases that have been linked to stress resistance and nutrient supply. Here, we used a bioinformatics approach to identify and characterise 13 HMA genes containing characteristic P1B-type ATPase domains and motifs in the barley Morex V3 reference genome. The genes are located on five of the seven barley chromosomes. Phylogenetic analysis revealed that they cluster into five sub-clades, including one clade unique to barley. Expression profiling across multiple datasets showed distinct temporal and tissue-specific expression patterns among HvHMAs, with several members exhibiting significant transcriptional responses to specific biotic and abiotic stresses. By utilising recently available pan-transcriptomic and pan-genomic resources, we have identified substantial allelic diversity and inter-accession variation in HvHMAs. Our findings suggest that HvHMAs have functions extending beyond canonical heavy metal homeostasis and warrant further investigation for their potential roles in broader physiological and stress-related processes.

The composition and quantity of root exudates are strongly influenced by physiological and environmental conditions, reflecting dynamic changes in plant metabolism. Although many studies report that root exudate metabolite profiles vary with plant phenology, few have disentangled the effects of phenological stage from those of nutrient availability. We collected root exudates from white lupin (Lupinus albus) grown in a fine phosphorus (P) gradient (5, 10, 20, 30 and 50 {micro}M P) in hydroponics at three developmental stages, performed untargeted metabolomics using GC-MS, and measured 10 above and belowground traits. Our results show that plant phenological stage exerts a stronger influence on exudate metabolomic profiles than variation in P supply. During leaf development, exudates were dominated by metabolites associated with carbon metabolism, whereas flowering was characterized by compounds related to secondary metabolism and cell wall turnover. Phosphorus influenced exudate profiles only at the flowering stage, with distinct profiles observed at 5 and 10 {micro}M P compared with 20-50 {micro}M P. These findings provide new insights into the temporal regulation of root exudation and demonstrate that plant developmental stage is a primary determinant of metabolic responses to phosphorus availability and, potentially, rhizosphere functioning under nutrient limitation. HighlightRoot exudate quantity and composition in white lupin is shaped more by plant phenological stage than phosphorus supply, with phosphorus effects emerging only during flowering.

Auxin is a key phytohormone that regulates all aspects of plant growth, development, and environmental responses, making the precise analysis of its distribution and signaling essential for understanding plant adaptation and physiological processes. However, despite the agricultural importance of oilseed rape (Brassica napus), the lack of robust, species-specific molecular tools limits detailed studies of hormone signaling in this crop. Here, we developed and characterized reporter systems for the sensitive visualization and quantification of auxin distribution and signaling in B. napus. The DR5cc auxin signaling reporter and a novel synthetic auxin-responsive reporter, BIP3, assembled from promoter fragments of three oilseed rape IAA genes, were generated to drive GUS expression. In hairy roots, both reporters showed auxin-responsive expression in the root apical meristem that became broader after auxin treatment. In transgenic seedlings, flowers at anthesis, and 12-day-old embryos, DR5cc exhibited a more defined expression pattern than BIP3. To monitor real-time auxin dynamics under abiotic stress, DR5cc fluorescent reporters were employed in hairy roots. Mannitol and NaCl treatments induced a time-dependent increase in fluorescence, peaking at 6-12 h before returning to basal levels after 24 h. Furthermore, dual-reporter assays enabled simultaneous monitoring of auxin and cytokinin signaling, revealing distinct hormone-specific spatial responses in hairy roots. Finally, we established a quantitative DII (qDII) reporter system using degron domains from B. napus Aux/IAA proteins, providing a high-resolution quantitative readout of auxin depletion. Together, these reporter systems enable spatial, temporal, and quantitative analyses of auxin dynamics during development and stress adaptation in oilseed rape.

Leaf shape varies widely across plant taxa and has repeatedly been shown to affect ecophysiology, interspecific interactions, and fitness. We used population genomics, genome wide association studies (GWAS), and comparative genomics to determine the genetic basis and evolutionary history of an uncharacterized Mendelian leaf shape polymorphism in Ipomoea hederacea. To do so, we assembled a reference genome and generated whole genome sequencing for 123 individuals from 55 populations. We identified a 117 kb indel that perfectly co-segregates with leaf shape by conducting a GWAS and assessing differences in coverage. Syntenic orthologs for genes on the indel were present in five other Ipomoea species with various leaf shapes, indicating the indel is newly arisen deletion in I. hederacea despite similar leaf shape phenotypes in the other species. More broadly, these results illustrate how a range of leaf shape phenotypes can be produced by distinct genetic mechanisms even in closely related species. Although none of the genes on the indel itself are known leaf shape candidates, there are multiple leaf shape candidate genes in close proximity that are involved in the auxin biosynthesis pathway. Additionally, the genes within the indel have gene functions that could affect other potentially ecologically relevant traits that have previously been shown to be associated with leaf shape in I. hederacea. Therefore, the pleiotropic effect of the indel polymorphism can have important implications for understanding the ecological mechanisms influencing a well-documented leaf shape latitudinal cline in I. hederacea. Significance StatementIpomoea hederacea has been used to investigate the ecological and evolutionary effects of leaf shape because of a well-documented latitudinal leaf shape cline governed by an uncharacterized Mendelian polymorphism in this species. We identified a 117 kb indel that perfectly co-segregates with leaf shape in I. hederacea. The indel appears to be a distinct genetic mechanism than those governing similar leaf shapes in other closely related Ipomoea species. Additionally, possible pleiotropic effects of the indel on other traits has important implications for understanding the ecology and evolution of leaf shape variation in I. hederacea. Therefore, both the results and the new genomic resources that we developed will help facilitate future work understanding the genetic, developmental, and ecological mechanisms governing leaf shape variation.

The selection of drought-resistant cotton genotypes with high productivity and improved water-use efficiency is an increasingly pressing challenge in arid and semi-arid cotton-growing regions, where climate variability is intensifying water scarcity. This study evaluated two widely cultivated cotton varieties in Texas, NG 4190 B3XF and ST 4990 B3XF, under full and moderate deficit irrigation to identify physiological markers associated with contrasting yield responses under water limitation. A comprehensive set of physiological traits was assessed, including plant water status, leaf gas exchange, carbon isotope composition ({delta}13C), total nitrogen and C/N ratio, chlorophyll fluorescence, light and CO{square} response curves, and canopy temperature. While no yield differences were observed under full irrigation, moderate water deficit resulted in stable seed and fiber yield in NG 4190, but significant yield reduction in ST 4990. Yield differences were not explained by instantaneous gas exchange or direct biochemical limitations of photosynthesis, but rather by integrated physiological behavior over time. Key discriminating traits included midday relative water content (RWC), photosynthetic light-response parameters ( and Pm), stomatal optimization parameter (g{square}), and {delta}13C. NG 4190 exhibited higher RWC, more negative {delta}13C, and higher g{square} values, indicating a less conservative stomatal regulation strategy that supported sustained carbon assimilation under water stress. These findings provide insight into the physiological mechanisms underlying cotton performance under drought and support the use of integrated physiological markers for the selection of resilient genotypes in water-limited environments.

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

Sorghum (Sorghum bicolor) is a climate-resilient crop whose acclimation to nutrient limitation and low temperature likely involves extensive lipidome reconfiguration. Lipids are key membrane components, carbon and energy stores, and mediators of stress signaling, yet population-scale lipidomics data for sorghum are limited. We present the Sorghum Lipid Database (SoLD), a curated lipidomics resource from the Sorghum Association Panel grown under two field regimes: (i) a nutrient-sufficient with usual planting date environment (control) and (ii) a low-input treatment with reduced nitrogen and phosphorus, earlier planting, and no application of insecticides, herbicides, or pesticides (low-input). Using high-resolution LC-MS, we quantified 244 lipid species and detected broad, largely conserved compositional shifts across field trials. However, there were four major low-input-associated lipid signatures relative to control: (i) depletion of sulfoquinovosyldiacylglycerol, (ii) triacylglycerol enrichment, (iii) phospholipid redistribution centered on phosphatidylserine, and (iv) coordinated lysophospholipid remodeling, reflected in altered lysophosphatidylcholine-to-lysophosphatidylethanolamine ratios. Analyses of lipid chemical space and lipid ontology enrichment supported these compositional changes. GWAS of lipid species, class sums, and class ratios revealed recurrent, environment-specific loci. Control-associated loci were enriched for genes involved in lipid and isoprenoid metabolism, developmental regulation, and cell-wall biosynthesis and modification. Low-input-associated loci were enriched for genes involved in nutrient-stress signaling, cell-wall remodeling, defense, developmental control, and cold-related barrier formation and proteostasis. Thus, SoLD provides a framework connecting sorghum lipid diversity with environmental and genetic variation. All information regarding the database and the experiment is freely accessible through a Shiny application: https://nirwan.shinyapps.io/SAP-Lipidomics-Database/. The database enables users to move from lipid-class to individual molecular species and associated candidate loci, for hypothesis generation, comparative analyses, and prioritization of targets for functional validation.

IntroductionMany plant species secrete nectar to attract beneficial animals. The chemical composition of floral nectar influences pollinator nutrition and behavior, as well as microbial growth in flowers. Yet factors that predict nectar composition across plant species, as well as chemical compounds determining microbial growth in nectar, remain poorly understood. MethodsWe used both targeted and untargeted metabolomics to compare the nectar chemical profiles across 31 phylogenetically diverse plant species that span a range of floral morphologies. We examined the common classes of compounds detected in nectar and patterns of co-occurrence among them. We combined newly collected chemical data with previously published data on microbial growth in nectar of the same plant species to examine how nectar chemistry is associated with microbial growth. ResultsPlant species and clades varied in amino acid, minor sugar, and secondary metabolite composition and concentration. Sampled rosids and lilioids generally contained higher amino acids while asterids contained greater concentrations of oligosaccharides and sugar alcohols. Across plant species, proteinogenic amino acids frequently co-occurred in nectar but many were negatively associated with sucrose concentration. Plant species with greater concentrations of amino acids and other nitrogen-containing compounds hosted greater microbial density in nectar, while some other compound groups were negatively associated with microbial diversity. ConclusionsNegative correlations between nectar amino acid and sucrose concentration across species suggest ecological tradeoffs or physiological constraints in nectar composition. Given that the growth of common nectar microbes is limited by amino acid concentration, these findings suggest an ecological cost to amino acid production in nectar. Finally, we document variation among species in nectar vitamins, non proteinogenic amino acids and secondary metabolites with hypothesized yet currently untested ecological roles.

Increasing the diversity of biofuel crops can help meet energy demands while also stabilizing the domestic biofuel market. Sorghum bicolor is a promising feedstock for bioethanol production due to its sugar accumulation and storage in the stem in addition to its cellulosic biomass. Sorghum also exhibits high tolerance to abiotic stresses like extreme temperatures and drought. However, sorghums sugar production falls short when compared to current bioethanol feedstocks like maize and sugarcane. Therefore, to improve sorghum for the bioethanol market, an autotetraploid sorghum line was induced using colchicine treatments to increase cell size for greater sugar production and storage. Induced autotetraploid sorghum lines were validated with flow cytometry and screened using stomatal prints to detect larger stomatal cells. Two separate autotetraploid sorghum lines that were derived from the same M1 plant were characterized and evaluated for sugar production in a two-year field trial. The two autotetraploid lines displayed equal or improved performance when compared to their diploid equivalents for multiple juicing traits. Altogether, the data illustrate sorghums tolerance for autopolyploidy induction in an inbred background and suggest an opportunity for further improvements through progressive heterosis. SIGNIFICANCE STATEMENTPolyploidy has played a significant role in the improvement of some crop species. The characterization of a novel autotetraploid sweet sorghum line demonstrates the potential of increased sugar production in polyploids for biofuel applications.

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

Kernel composition traits in maize, including protein accumulation, are of broad interest. The amount of the most abundant proteins in maize endosperm, the -zeins, can vary dramatically among genotypes and in response to soil nitrogen supply. Targeted reductions in -zein accumulation can improve nitrogen utilization and the nutritional quality of maize grain but have traditionally required expensive and destructive phenotyping methods. The Floury2-RFP (Fl2-RFP) reporter gene enables rapid, non-destructive visualization of -zein accumulation in individual maize kernels under white light. This feature is due to the high expression level programmed by the Fl2 promoter, the stability of zein proteins, and the use of monomeric RFP, which emits fluorescence without the need for multimerization. This study aimed to develop a method to quickly document and quantify Fl2-RFP accumulation using camera or smartphone images of either ears or shelled kernels. Results show images of shelled kernels processed with FIJI software capture the Fl2-RFP reporter phenotype better than images of ears. Fl2-RFP confirms the strong maternal control of -zein accumulation and, like grain protein concentration, responds to soil nitrogen supply. The Fl2-RFP phenotyping pipeline effectively quantified Fl2-RFP accumulation by color features from both camera and smartphone images. Smartphone imaging of Fl2-RFP in a diverse population of inbreds followed by elastic net regression of extracted image features predicted kernel protein concentration, as measured by near-infrared spectroscopy, with moderate accuracy (R2 = 0.68, MAE = 0.76, RMSE = 0.93). The spectral features that were most predictive of kernel protein concentration varied depending on whether the background endosperm color was white or yellow. The integrated analysis of Fl2-RFP intensity and grain protein concentration indicates genetic variation for kernel protein accumulation and N-responsiveness that is distinct from the well-studied -zeins. Our findings highlight the Fl2-RFP reporter gene as a valuable tool for investigating the genetic complexity of grain protein concentration and associated traits in maize.