Plant Direct

Sterol biosynthesis underlies significant physiological functions in plants, including the production of membrane structural sterols and hormones such as brassinosteroids and cytokinins. Inhibition of sterol biosynthesis has been shown to disrupt multiple aspects of Arabidopsis thaliana development. Here, the effects of lovastatin, an inhibitor of HMG-CoA reductase, on root development were investigated, focusing on auxin-cytokinin distribution and transport. Lovastatin inhibited primary root growth, especially cell elongation, in a dose-dependent manner. Additionally, lateral root density was considerably increased and lateral root primordia (LRP) emerged ectopically. In accordance to the above defects, auxin/cytokinin imbalance was recorded by the ectopic presence of the synthetic auxin marker DR5 and a significant decrease of cytokinins, as revealed by depletion of the TCS (two-component signaling) marker. Because auxin distribution appeared disturbed, auxin transport impairment was further examined. Plasma membrane localization of PIN auxin efflux carriers declined significantly, showing additional diffuse cytoplasmic localization in LRP cells. However, the cell-specific localization patterns of several PINs and their abundance at the transcript and protein level appeared unaffected or slightly increased. Fluorescence recovery after photobleaching (FRAP) analysis regarding membrane kinetics of PIN2 revealed altered PIN2 membrane dynamics and transmission electron microscopy (TEM) observations showed structural defects at the plasma membrane-cell wall interface. Together, these results support that sterol biosynthesis is essential for maintaining plasma membrane organization, which, in turn, is key factor for the distribution of hormones that control root development. HighlightsLovastatin treatment inhibits root growth and causes deregulated formation of lateral roots. Consistently, lovastatin causes altered patterns of auxin distribution relevant to PIN protein mis-localization and decreases cytokinin levels. These changes could be attributed to reduced structural sterols as exemplified from alteration in PIN2 membrane dynamics.

Small signaling peptides (SSPs) are critical regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 S. bicolor genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, and Brachypodium distachyon. The 219 sorghum genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Expression of the 219 SSP encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stems and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for [~]80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP encoding sorghum genes in several families (CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate cambial activity and vascular bundle cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF, and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of 5 SbRALFs in the stem nodal plexus. Cell type specific expression of many SSP encoding sorghum genes was also observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP functions in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.

The nucleus is the characteristic organelle for eukaryotic organisms. Unlike the classic textbook view of static two-dimensional nuclei, nuclear shape is dynamic inside the live cell. The alteration or deformed nuclear shape is the hallmark of cancer in animal cells and environmental stress in plants. The nuclear envelope proteins interact with chromatin to regulate gene expression. Unfortunately, we have limited knowledge about the impact of abiotic stress on nuclear shape, movement, and chromatin dynamics. To circumvent this issue, we are utilizing a dual fluorescently tagged marker lines - nuclear envelope protein and chromatin - to perform live cell imaging in the model plant Arabidopsis thaliana root. The live cell imaging was performed in control and salt-stressed conditions. We utilized these captured movies to analyze through open-source image processing software Fiji/ImageJ with the help of the TrackMate plugin. Using this method, we have demonstrated that chromatin velocity is decreased in salt-treated conditions. This method will be widely applied to quantitative live cell imaging of nuclear shape and chromatin dynamics during plant development and environmental stress. SummaryThis process aims to simultaneously record nucleus and chromatin dynamics in Arabidopsis thaliana roots and investigate changes in these dynamics in response to developmental and environmental cues.

Plant-specific membrane receptor kinases with structurally diverse extracellular domains regulate key processes in plant growth, development, immunity and symbiosis. Structural studies of these glycoproteins are often hampered by the limited quantities in which they can be obtained. Here, we describe the LRR crystallization screen, which has enabled the successful crystallization and structure determination of multiple receptor kinase ectodomains, including ligand-and co-receptor-bound complexes. As an example, we report the 1.5 [A] resolution crystal structure of the leucine-rich repeat (LRR) domain of STRUBBELIG-RECEPTOR FAMILY 6 (SRF6) from Arabidopsis thaliana. The SRF6 ectodomain contains seven LRRs and a disulfide-bond-stabilised N-terminal capping domain but lacks the canonical C-terminal cap and the N-glycosylation pattern typically observed in other family members. Previously reported protein-protein interactions between the SRF6 and SRF7 ectodomains and the receptor kinases BRI1, BRL1, BRL3, SERK3 and BIR1-3 could not be confirmed by quantitative isothermal titration calorimetry and grating-coupled interferometry assays, suggesting that these structurally conserved LRR receptor kinases may have signalling functions outside the brassinosteroid pathway. SynopsisA crystallisation screen that has enabled the structural analysis of various extracellular domains of plant membrane receptor kinases is described together.

Course-based Undergraduate Research Experiences (CUREs) have emerged as a transformative approach to science education, expanding access to authentic research opportunities beyond the traditional undergraduate research assistant (URA) training. By embedding research into a curriculum, CUREs engage a broad and diverse population of students in a classroom environment that emphasizes experimental design, data analysis, and scientific communication. However, this has been difficult to develop for fields such as plant synthetic biology due to the long timescales of plant transformation. One avenue around this problem is to utilize a recent innovation that enables high throughput and rapid screening of gRNA efficacy by leveraging viral-based delivery of guide RNAs (gRNAs). In this work, we develop and validate a CURE with undergraduate students at Colorado State University (CSU). Students worked in teams to design and test efficacy of gRNAs targeting a Cas9-based transcriptional repressor to different regions of the promoters of the three GIBBERELLIN INSENSITIVE 1 genes (GID1a, GID1b, and GID1c) in Arabidopsis thaliana. Over the semester, students generated and analyzed gene expression data to understand the efficiency of twelve new gRNAs. We further validated CURE student-identified gRNAs with an undergraduate research assistant (URA) that assessed target gene expression and phenotypic outcomes in stable transgenic lines expressing SynTF constructs with the strongest gRNAs from the class. We further describe the curriculum structure to facilitate adoption at other institutions and present student-generated datasets demonstrating the utility of ViN-based screening for identifying effective SynTF gRNAs for plant functional genomics and engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/715601v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@13869f5org.highwire.dtl.DTLVardef@b469feorg.highwire.dtl.DTLVardef@9aa51borg.highwire.dtl.DTLVardef@cdc129_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Glufosinate-ammonium (GA) has been widely used in Midwestern fields, and in recent years a growing number of failures to control waterhemp [Amaranthus tuberculatus (Moq.) Sauer] have raised concerns about the potential evolution of resistance. The goal of this study was to investigate four independent cases of suspected resistance to GA in A. tuberculatus from Illinois using greenhouse, field, and transcriptomics studies. Greenhouse dose-response experiments revealed resistance ratios ranging from 2.2- to 3.4-fold based on survival and from 1.3- to 2.8-fold based on dry biomass relative to a susceptible population. A subsequent field study where one of the populations originated confirmed that twenty percent of treated plants survived the labeled GA field-recommended rate. Screening for other herbicide sites of action revealed that most populations showed reduced sensitivity to atrazine, glyphosate, and imazethapyr, surviving up to three times the field-recommended rates, and to a lesser extent, lactofen and fomesafen. Transcriptomic analysis of plants surviving GA revealed no resistance-associated mutations or differential transcript abundance in the plastidic and cytosolic isoforms of glutamine synthetase. Among the four suspected resistant populations, there were 182 genes differentially expressed relative to two susceptible populations. Different sets of genes were differentially expressed among the populations studied, with only one gene (upregulated relative to two susceptible populations) shared among all four. Many of the differentially expressed genes, including cytochrome P450s, glutathione S-transferases, glycosyltransferases, transporters, and transcriptional regulators, are commonly associated with metabolic resistance. Gene ontology enrichment analyses indicated significant overrepresentation of stress response, defense regulation, and secondary metabolism categories across the populations. Together, these findings provide evidence for the evolution of GA resistance in populations of A. tuberculatus in Illinois. While more in-depth studies are needed to fully characterize the underlying mechanisms, the consistent differential expression of metabolism-related genes and no indication of target-site mechanisms points to a potential metabolic basis for resistance.

Under climate change, understanding how plants and crops respond to drought is essential for basic research in ecology and evolution, and improving agricultural resilience. One common method of simulating drought in experimental conditions is by applying polyethylene glycol (PEG) to plants. We investigated drought growth responses in Medicago lupulina (black medic) using PEG to simulate drought stress. We grew Medicago lupulina plants inoculated with Sinorhizobium meliloti in Magenta boxes under controlled conditions and randomly assigned them to one of three treatments: a control, PEG applied to the bottom (PEG added to the bottom-watering container of a magenta box), or PEG applied from the top (PEG poured over the growth media). After 60 days, we measured true leaf number, nodule count, and below- and above-ground dry biomass. PEG treatments significantly reduced above-ground growth, including total biomass and leaf number, but unexpectedly increased nodulation. Our results suggest that while PEG effectively simulates drought stress on above-ground growth parameters, it may not accurately simulate drought effects on rhizobial symbiosis. PEG treatments had no effect on below-ground biomass, suggesting that increased nodulation is not a result of increased plant investment in below-ground growth under simulated drought. We hypothesize that PEG, as a persistent liquid that plants do not absorb, created conditions favorable for nodulation. Overall, these results highlight the importance of interpreting PEG-simulated drought experiments with caution when assessing mutualistic interactions.

Advances in automation, imaging, and artificial intelligence have enabled researchers to capture large volumes of high-quality plant data for understanding crop growth, stress, and genotype-by-environment interactions. While genomics has achieved remarkable throughput, phenotypic data acquisition remains a critical bottleneck for accelerating crop improvement and biological discovery. To address this challenge, an integrated multispectral phenotyping framework was developed using imagery from the Texas A&M AgriLife Precision Automated Phenotyping Greenhouse, a fully controlled facility designed for reproducible plant monitoring throughout the entire growth cycle of most crops. The framework expands the Plant Growth and Phenotyping (PGP v2) dataset and establishes a standardized system for continuous image acquisition, segmentation, deep feature extraction, and temporal analysis across multiple crop species. The project was organized around five coordinated areas: Administration and Coordination, Imaging and Sensor Operations, Data Processing and Management, Artificial Intelligence and Analytics, and Plant Science and Discovery. This structure ensured consistent data quality, version-controlled workflows, and communication across disciplines. The analytical pipeline integrates pseudo-RGB generation, deep learning-based detection and segmentation, image stitching, and temporal (longitudinal) tracking to isolate individual plants and analyze changes in morphology, spectral reflectance, and texture over time. Beyond technical innovation, the framework provides a replicable model for interdisciplinary collaboration and administrative integration in plant phenomics. The combined dataset, workflow, and management framework enable scalable, reproducible, and data-driven plant science research that bridges engineering and biological discovery. Plain Language SummaryTemporal imaging of plants in controlled environments helps scientists better understand growth and biological processes. However, analyzing large volumes of images has been limited by a lack of automated tools. Multispectral imagery captures additional information about plant pigments, structure, and stress beyond standard color images. We developed an automated analysis pipeline that identifies individual plants, tracks their growth over time, and measures traits such as height, area, shape, texture, and vegetation indices. Using artificial intelligence, the system efficiently processes thousands of images to provide consistent and repeatable measurements. By integrating engineering and plant biology, this work supports data-driven decisions for crop improvement and agricultural research.

Virus induced gene silencing (VIGS) is a method that exploits plant antiviral defence mechanisms to downregulate endogenous genes. The technique is versatile, rapid, and widely used for functional genomics studies. Here we report a method for VIGS in the medicinal plant, Calendula officinalis (pot marigold). This species produces anti-inflammatory triterpenoids and has also been bred and cultivated as an ornamental plant. We describe a method for the injection of Agrobacterium tumefaciens cultures into leaf midribs and compare visual marker genes for tracking VIGS utilising constructs that simultaneously target visual marker and target genes. We use these tools to demonstrate that silencing a gene encoding cycloartenol synthase results in changes to leaf phytosterols. This method could be used to further investigate the genetic basis of specialised metabolism in this species and could be adapted to other members of the Asteraceae family, many of which are of economical and chemical value.

Phytohormones are key players in the regulation of plant development and metabolism. The different phytohormone classes comprise numerous chemically very diverse compounds, which are often present at very low concentrations. The chemical properties of phytohormones range from acidic to basic and from polar to non-polar. Furthermore, concentration varies strongly among different phytohormones, between plant species, tissues and developmental stages. Challenges often arise when only small amounts of plant material are available and when plant species are investigated in which the phytohormone profile has not yet been characterized. To establish a method for comprehensive phytohormone analysis we addressed these challenges by choosing and optimizing a suitable extraction method followed by optimized HPLC separation. We compared the most widely-used mass spectrometric detection methods, multiple reaction monitoring (MRM) on a triple quad instrument with high-resolution mass spectrometry (HRMS) on a Q-TOF instrument, and discuss the advantages of both methods and their limitations. O_LIWe compared various methods described in literature for the extraction of six phytohormone classes by liquid-liquid extraction and solid phase extraction purification and describe our optimizations to the selected method. C_LIO_LIWe optimized HPLC separation for 50 different phytohormones. C_LIO_LIWe evaluated the application of MRM and HRMS detection strategies. C_LI

Improving rice (Oryza sativa L.) yield requires a balanced enhancement of both sink size and source capacity. While many QTLs for sink size have been identified, only a few are known for source capacity, which is essential for achieving high yield. Here we identified qHP10 as a major QTL for increased photosynthetic rate by using chromosome segment substitution lines derived from a cross between the high-yielding indica cultivar Takanari and the average-yielding japonica cultivar Koshihikari. High-resolution mapping combined with CRISPR/Cas9-induced mutagenesis revealed that the causative gene underlying qHP10 is Mitogen-Activated Protein Kinase 4 (OsMPK4). A near-isogenic line carrying the OsMPK4Takanari allele (NIL-OsMPK4) had a 15-25% higher photosynthetic rate than Koshihikari. NIL-OsMPK4 also had higher stomatal conductance than Koshihikari but similar stomatal pore size and density, indicating that increased stomatal aperture increases photosynthetic rate. This enhancement is likely attributable to the down-regulation of OsMPK4 expression, which increases stomatal conductance and thus promotes CO2 uptake. Our findings demonstrate that OsMPK4 is a promising genetic target for increasing source capacity and, potentially, rice yield through molecular breeding. (175 words)

Background and AimsL-DOPA is an important pharmaceutical that accumulates to high levels in the legume faba bean (Vicia faba). L-DOPA is likely derived from L-tyrosine but the responsible enzyme (L-tyrosine oxidase) remains unknown. Availability of L-tyrosine may be a key factor controlling L-DOPA accumulation. In legumes, L-tyrosine is supplied via either a plastidial TyrA enzyme (ADH) or a deregulated cytosolic homolog (PDH). This study aimed at identifying L-tyrosine oxidase and TyrA genes from faba bean. MethodsWe used gene-to-metabolite correlations and homology-based searches to select fifteen L-tyrosine oxidase candidates, which were tested in yeast and in the model plant Nicotiana benthamiana. We also used isotopically labeled L-tyrosine to measure biosynthetic activity in different faba bean tissues and to test an alternative biosynthetic hypothesis. Three faba bean TyrA genes were inferred by homology and assayed in N. benthamiana by co-expression with a known L-tyrosine oxidase, CYP76AD6. Key ResultsNone of the L-tyrosine oxidase candidates produced L-DOPA upon heterologous expression. Feeding experiments showed a lack of correlation between L-DOPA accumulation and biosynthetic capacity. Feeding studies also disproved an alternative route to L-DOPA by oxidation of 4-hydroxyphenylpyruvate. Of the TyrA genes, two were able to increase L-tyrosine levels in N. benthamiana 2-3-fold (VfADH and VfPDH), and one of them was able to boost the levels of L-DOPA derivatives up to 6-fold (VfADH). ConclusionsThe faba bean L-tyrosine oxidase remains unidentified, with a possible transport of L-DOPA across tissues likely having confounded our correlation-based selection strategies. In N. benthamiana, both VfADH and VfPDH can increase the levels of L-tyrosine, while VfADH can further boost the levels of L-DOPA derivatives. Our work delivers a strategy to boost the provision of L-tyrosine in N. benthamiana and provides valuable insights in the search for the elusive L-tyrosine oxidase from faba bean.

Chloroplast genomes are invaluable resources for plant genomic research, providing insights into genome evolution and molecular adaptation. With the growing scientific and economic interest in Adansonia digitata, a comprehensive characterization of its chloroplast is timely and necessary. A complete chloroplast genome of A. digitata was assembled, annotated, and characterized. Comparative structural analysis was conducted against other Adansonia species, and the assembly was validated through phylogenetic placement within Malvaceae. The assembled genome exhibits the canonical quadripartite organization, spanning 160,061 bp with a GC content of 36.88%, 79 protein-coding genes, 32 tRNAs, and 4 rRNAs. Repeat analysis identified 100 simple sequence repeat motifs, predominantly A/T-rich mononucleotide types (76%), alongside 50 long sequence repeats dominated by forward (26) and palindromic (17) repeats. Comparative analysis with other Adansonia species revealed conserved genome structure, with minor IR boundary shifts involving the ndhF gene, and ycf1 duplication in A. gregorii and A. grandidieri. Average nucleotide identity exceeded 99% across all Adansonia species, with near-complete similarity (ANI {approx} 99.96%) observed with the putative A. kilima. All predicted RNA editing events were nonsynonymous, dominated by C[->]U conversions (55.02%). Codon usage showed non-random synonymous preferences biased toward A/U-ending codons, driven primarily by mutational pressure with detectable gene-specific translational selection. Nucleotide diversity ({pi}) was higher in intergenic spacers (0.00490 {+/-} 0.00574) than in coding regions (0.00167 {+/-} 0.00199), with the majority of genomic regions showing no sequence variation ({pi} = 0). Substitution patterns indicated pervasive purifying selection, with relatively high but insignificant signals in matK, ycf1, accD, and rpoB. Phylogenomic analyses placed the assembled A. digitata chloroplast genome within the Adansonia lineage, consistent with its established systematic position. This study provides detailed insight into the chloroplast genome of A. digitata, and the findings will contribute towards advancing its genomic research.

Lignin plays a central role in the formation and function of secondary cell walls in vascular plants. However, the structural consequences of lignin modification for cell wall properties and cellular function in grasses remain poorly understood. Here, we investigated how cinnamyl alcohol dehydrogenase (CAD) deficiency alters vascular cell architecture in Sorghum bicolor, using the brown midrib-6 (bmr6) mutant as a model system. Biochemical and histochemical analyses confirmed altered lignin chemistry in bmr6, including increased incorporation of hydroxycinnamaldehyde residues and reduced tricin levels. We applied ptychographic X-ray computed tomography (PXCT) to quantify the cell wall geometry, in three dimensions, at nanometer-scale resolution. PXCT enabled measurements of wall thickness distribution and lumen shape along tracheary elements. Analyses revealed no significant differences in wall thickness between wild-type and bmr6 plants. However, three-dimensional morphometric descriptors indicated reduced lumen convexity in bmr6, suggesting localized modifications not detectable by conventional two-dimensional imaging. Water flow numerical simulations through PXCT-derived images indicated reduced vessel permeability and simulated hydraulic conductivity in bmr6, suggesting that subtle geometric changes may influence performance. These findings highlight the value of three-dimensional imaging for resolving cell wall organization and provide new insight into the architectural resilience of grass xylem in response to targeted lignin modification. HighlightThree-dimensional X-ray nano-imaging reveals alterations in the cell wall architecture that affect simulated hydraulic performance under reduced CAD activity in sorghum.

Steroidal glycoalkaloids and saponins are plant cholesterol-based steroid metabolites with antimicrobial activities and potential pharmacological value. The saponin uttroside B from black nightshade (Solanum nigrum) plays an important role in defense against herbivorous insect and exhibits anti-hepatocellular carcinoma activity. The tomato (S. lycopersicum) glycoalkaloid -tomatine has been studied because of its antinutritional effects, however, its role in protecting plants from fungal pathogens remains understudied. The biosynthetic pathway of -tomatine involves multiple clustered genes designated as glycoalkaloid metabolism (GAME) genes. In this study, we generated single knockout mutants of SlGAME4 and SlGAME2 by CRISPR/Cas9-based genome editing. The SlGAME4 mutants did not accumulate glycoalkaloids but instead redirected resources towards steroidal saponin (uttroside B) synthesis. SlGAME2 mutants contained unaltered -tomatine contents indicating that the SlGAME2 gene, previously reported to catalyze the transfer of xylose to {beta}1-tomatine, is not involved in -tomatine biosynthesis. Infection assays with four fungal tomato pathogens demonstrated that the SlGAME4 mutant plants were slightly more susceptible to Botrytis cinerea, but equally susceptible to the other three fungi. Up-regulation of -tomatine-responsive genes in B. cinerea was observed during infection on SlGAME4 mutant tomato, as well as on S. nigrum suggesting that uttroside B induces a fungal transcriptional response similar to -tomatine. Furthermore, we observed that tolerance mechanisms to plant saponins mediated by glycosyl hydrolases and a glycosyltransferase contribute to virulence of B. cinerea on SlGAME4 mutant plants and S. nigrum. This indicates that also uttroside B contributes to defense against fungal pathogens and can be detoxified by B. cinerea.

Self-incompatibility (SI), a reproductive mechanism that prevents self-pollen from fertilizing the ovule, is widespread in flowering plants, including the Brassicaceae family, where it promotes outcrossing, genetic diversity, and hybrid vigor. Although prevalent in Brassica rapa, an economically vital crop, it remains poorly characterized in widely grown varieties, such as toria and yellow sarson, with prior studies primarily focused on Brassica napus. Given its potential for hybrid breeding and crop improvement in rapeseed (B. rapa), we characterized key SI-regulatory genes, analyzing their phylogenetic relationships, structure-function dynamics, and expression patterns. Our results indicate sequence, structural, and functional homology as well as conservation with previously known candidates. This study identifies SRK, FER, and ARC1 as essential, while MLPK plays a minor role in SI for the varieties under study. Furthermore, we identified that SRK, FER, and MLPK activate ROS during the SI response, while ARC1 does not. Our findings establish a foundation for harnessing this natural system to integrate agriculturally important traits and sustain them across generations via outcrossing.

In this study, the level of Rubisco protein was reduced in wheat using RNAi, to test the hypothesis that photosynthesis, growth, and grain yield could be maintained whilst improving nitrogen use efficiency. The RNAi Rubisco wheat plants, with a Rubisco activity of less than 70% of wild type (WT) plant levels, had reduced photosynthesis, reductions in leaf and stem biomass and decreased seed yield. Interestingly, in the wheat RNAi Rubisco lines that had a small (<30%) reduction in Rubisco activity, the seed number, total seed weight and harvest index were comparable to that of WT type plants. However, no improvement in photosynthetic nitrogen use efficiency (PNUE) was evident in any of the RNAi Rubisco lines. Notably, PNUE was lower than for WT wheat plants in the RNAi lines with more than a 30% reduction in Rubisco activity. This result was unexpected and caused by an accumulation of N in both the leaves and seeds. At present we do not have an explanation for this but one possible hypothesis is that it could be due to slower growth caused by a reduction in source strength in the RNAi plants, which in turn resulted in changes to carbon and nitrogen allocation. HighlightWheat RNAi plants with small reductions in the amount and activity of Rubisco had a similar biomass and total seed weight to that of untransformed controls but no improvement in nitrogen use efficiency was evident.

Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the complex system of phytohormone signaling pathways. Quantification of phytohormones with the commonly used technique liquid chromatography coupled to tandem mass spectrometry is susceptible to the influence of non-phytohormone components present in the sample, a phenomenon referred to as matrix effect. To reduce matrix effect, some phytohormone quantification methods include additional steps of cleanup of crude extracts. However, to what extent additional purification steps provide increased accuracy compared to simpler, less laborious methods is seldomly evaluated. We evaluated three previously described phytohormone extraction methods, two of which include solid-phase extraction and one that does not, in their ability to minimize matrix effect and generate accurate estimates of phytohormone species spanning six classifications, from fruit and leaf tissue of Solanum lycopersicum cv. Micro-Tom (tomato). Our results show that, while the methods that included solid phase extraction occasionally outperformed each other regarding matrix effect and/or recovery efficiency for broad range of phytohormones, they rarely outperformed the simpler single-phase extraction method. Short AbstractAccurate, simultaneous quantification of chemically diverse phytohormones by LC-MS/MS is frequently confounded by matrix effects, leading to the incorporation of additional purification steps. We systematically compared three published extraction protocols with or without solid-phase extraction in tomato tissues across six hormone classes. Solid-phase methods occasionally improved matrix suppression or recovery, but did not consistently outperform the single-phase approach, questioning the added value of extra cleanup steps, particularly when high-throughput is desired, as in the case of systems biology interrogations.

Pseudomonas putida strain KT2440 is a crucial model organism for synthetic biology and bioengineering applications, yet there currently exists no comprehensive metabolomics database comparable to those available for other model organisms. This gap hinders the use of untargeted metabolomics for exploratory analyses in this system. We developed the P. putida metabolome reference database (PPMDB v1) to address this limitation by consolidating metabolite information from multiple sources and expanding coverage through computational predictions. The database was constructed by curating metabolites from BioCyc, BiGG, and other literature sources, then computationally expanding this collection using BioTransformer environmental transformation predictions to generate additional predicted metabolites. We enhanced the databases utility for molecular annotation in metabolomics studies by incorporating analytical properties including collision cross-sections, tandem mass spectra, and gas-phase infrared spectra. These analytical properties were gathered from existing measurement data or predicted using computational tools. We further augmented the database through inclusion of reaction information and pathway annotations, facilitating biological interpretation of metabolomics data. This publicly available resource fills a critical gap in P. putida research infrastructure, supporting metabolite annotation and biological interpretation in untargeted metabolomics studies and enabling in-depth exploratory analyses of this important synthetic biology platform at the molecular level. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713193v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c8828forg.highwire.dtl.DTLVardef@1f3a5c5org.highwire.dtl.DTLVardef@1084535org.highwire.dtl.DTLVardef@1f7ca4a_HPS_FORMAT_FIGEXP M_FIG C_FIG