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

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.

Pennycress (Thlaspi arvense L.) is an intermediate winter oilseed crop that has only recently been domesticated for agronomic use. Improving agronomic traits requires sources of genetic variation, and mutagenesis is frequently used to help overcome the limitations of natural populations. We investigate the impact of Ethyl methanesulfonate (EMS) on genetically effective cells (GECs) to characterize the intra-individual genetic variation of EMS mutagenesis in pennycress. We identified that pennycress contains at least 4 GECs which, when treated with EMS, create unique mutations across different branches within the same individual plant. We then propagated the M2 plants for whole genome sequencing, providing extensive characterization of the EMS mutation profile and developing a gene index as a resource for future reverse genetic screenings. Article SummaryPennycress is an emerging winter oil seed crop in the American Midwest. Domestication efforts have advanced rapidly through a combination of genetic techniques. One of the most successful methods has been the use of a mutant gene index, a large collection of pennycress seed where new genetic variation has been created through Ethyl methanesulfonate (EMS). EMS mutations are not uniform however, and a single treated seed can have wide genetic variation within the resulting plant. We investigate the role of genetically effective cells on EMS variation, and present the full EMS population as a resource for further pennycress domestication efforts.

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.

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.

Spinach (Spinacia oleraceae) is a principal vegetable crop commercially grown in Controlled Environment Agriculture (CEA). Recent research suggests that root morphological and architectural differences among crop species influence yield, resource use efficiency, and environmental stress tolerance. These root traits may be exploited to increase yield, promote efficient nutrient use, and mitigate environmental stressors. This study measured differences between various spinach cultivars in CEA systems to reveal morphological and anatomical variation. We grew three spinach cultivars with different reported growing rates ( Income, Darkside, and El-Majestic) under NFT hydroponic and substrate-based systems in a controlled greenhouse environment over 45 days with destructive harvests at days 15, 30, and 45. Supplemental light (250 {micro}mol/m2/s) with 12-hour photoperiod and periodic fertigation was used. Harvests included the collection of leaf and root biomass, and scanning of root systems in WinRhizo software, measuring ten variables. On day 45, root cross-sections from orders 1-5 were embedded in JB-4 resin, sectioned, stained, and analyzed for diameter, vasculature, and rhizodermis characteristics. Results indicate that in spinach, differences in root system morphology are linked to cultivation systems over cultivar identity. Vascular and root anatomical alterations are minor compared to morphological differences in response to the cultivation system. Hydroponic-style growth systems are associated with the proliferation of fine-root ideotypes compared with substrate-based conditions. Such findings affirm previous studies, which suggest plastic root morphology in response to growth systems, and may be used to help create more resilient, resource-efficient cultivars. HighlightsO_LIIn spinach, root system morphology differences are linked to cultivation systems. C_LIO_LIRoot vascular and anatomical alterations are minor in response to cultivation system. C_LIO_LIHydroponic growth systems are linked to fine-root ideotype proliferation in spinach. C_LIO_LIFine-root ideotype proliferation may be a breeding target for CEA spinach. C_LI

Members of the HD-ZIP transcription factor family play an important role in plant processes, including growth, development, metabolism, and stress response regulation. Among these, the sub-family IV members regulate epidermal cell differentiation, trichome development, and secondary metabolism. Monarda citriodora, an aromatic plant, produces economically important essential oils enriched in thymol. Thymol and other related monoterpenes are primarily biosynthesized and stored in glandular trichomes. Despite its significant economic value, the comprehensive identification of the transcription factor families has not been studied in this plant species. Given the importance of HD-ZIP-IV members in regulating trichome development and secondary metabolism, we identified these members in M. citriodora in the present study. To this end, firstly, we carried out transcriptome sequencing of M. citriodora flowers, and the resulting reads, along with previously sequenced reads, were used to reconstruct a transcriptome assembly. The assembled transcripts represented all major plant parts. Using this improved assembly, HD-ZIP-IV members were identified. Their expression profiles and phylogenetic positions, in conjunction with those of known regulators, identified candidate genes involved in the secondary metabolism and/or trichome development in M. citriodora. Furthermore, through gene co-expression analysis, several McHD-ZIP-IV members were found to be co-expressed with McDXS and McTPS genes. These McHD-ZIP-IV members may serve as key candidate genes for functional analysis to determine the regulation of trichome development in M. citriodora. Taken together, the present study provides a resource for improving M. citriodora using molecular tools.

Common bean (Phaseolus vulgaris L.) is the leading grain legume consumed directly by humans and a primary source of nutrients in many communities. This study utilized common bean genotypes with diverse seed coat phenotypes to investigate genotypic and environmental effects on pigmented seed coat area and seed macronutrient (protein, starch, fat, ash, moisture), anti-nutrient (phytate), and mineral nutrient (iron, zinc, calcium, phosphorus, magnesium, potassium, sodium) profiles. Recombinant inbred lines (RILs) that comprise six phenotypic classes for seed coat patterning and nine commercial cultivars were field-evaluated for multiple years across inland, coastal, and intermountain environments in California. A custom near-infrared spectroscopy calibration improved macronutrient prediction accuracy relative to a pre-existing calibration. Environmental effects on macronutrients were pronounced; the 2022 coastal growing environment was the most distinct, characterized by significantly higher starch and moisture content and significantly lower protein content in the RILs relative to any other environments. Across growing years in the RILs, greater consistency was observed at the inland site, where only protein was significantly different; all macronutrient traits significantly differed within the intermountain site. Certain commercial cultivars largely maintained their relative rank for protein content across environments, indicating consistency of genotypic performance, and Black Nightfall ranked among the highest for iron, zinc, phosphorus, and magnesium. Percent pigmented seed coat area was significantly negatively correlated with both calcium and magnesium concentrations. These results underscore the importance of genotype-by-environment field trials for seed coat patterning, seed nutritional composition, and their interplay, to support breeding of common bean among other grain legumes. HighlightsO_LICustom near-infrared spectroscopy (NIRS) calibration improved prediction accuracies C_LIO_LIEnvironmental effects significantly influenced common bean macronutrient composition C_LIO_LICertain cultivars ranked consistently for macronutrient traits across environments C_LIO_LISeed coat pattern was significantly associated with mineral nutrient concentrations C_LI

O_LIHerbivory is a major biotic stress for plants, triggering the induction and modulation of diverse specialized metabolites. Such induction responses are well studied for leaves and have been shown to depend on the herbivore feeding mode. Little is known about changes in flower metabolites and chemodiversity due to florivory type. Moreover, we lack an understanding of the intraspecific variation in such responses and whether these are spatially structured. C_LIO_LIThe aromatic plant Tanacetum vulgare, which shows high intraspecific chemodiversity in terpene profiles, was used to examine chemotype-specific metabolic responses of flower heads to infestation by the inflorescence-infesting aphid Macrosiphoniella tanacetaria or the flower-feeding beetle Olibrus spp. under field conditions. At peak flowering, each plant received both florivory treatments on separate stems, leaving one stem herbivore-free as a control. After four days, flower heads were harvested to analyze terpenes (GC-MS) and metabolic fingerprints (LC-MS). C_LIO_LIWe found stem-specific floral metabolic responses, with florivory altering specific chemical families and their chemodiversity. Levels of a few terpenes decreased following infestation, while none increased. Untargeted analyses revealed that aphid infestation had a lower effect on flower chemistry than beetle infestation, with aphid infestation mainly causing decreases and beetle infestation predominantly leading to increases in some metabolite intensities, but little overlap across treatments and chemotypes. C_LIO_LIOur results demonstrate that floral metabolic responses to florivory are spatially structured, florivore type-specific and shaped by plant chemotype. These findings highlight that the interplay between vascular organization, insect feeding mode, and intraspecific chemodiversity governs how flowers adjust their chemical defenses. C_LI One-sentence summaryTanacetum vulgare showed chemotype-specific responses to florivory by aphids (Macrosiphoniella tanacetaria) and beetles (Olibrus spp.), with aphids causing decreased and beetles increased levels of metabolic features within the same plant individuals, with little overlap in significant features across chemotypes.

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

BackgroundControlled-environment cultivation of medicinal cannabis (Cannabis sativa L.) typically optimizes light conditions to enhance the biosynthesis of pharmaceutically important metabolites like cannabinoids. Such experimental strategies may also influence other specialized metabolites like terpenoids, flavonoids, alkaloids, among others. Previous untargeted metabolomics studies testing short wavelength conditions like UV and blue light have shown that terpenoids and prenylated flavonoids in cannabis leaves respond differentially. However, since metabolomic studies in cannabis have so far mostly focused on floral cannabinoids, a comprehensive untargeted study into cannabis floral metabolome response to short wavelengths is currently lacking. ObjectivesOur study investigates the impact of short wavelength usage on cannabis specialized metabolism, and in particular the influence of UVB, UVA, and blue light on the cannabis floral flavonoid metabolome and associated glycosylation moieties. MethodsCannabis plants were grown under a white background light and exposed to supplemental UVB, UVA, or blue light during the generative phase of the cultivation cycle. Treatments were compared to a reference white background light without UV or blue light. Metabolites from floral tissue were extracted and analyzed via ultra-performance liquid chromatography-tandem mass spectrometry. A comparative metabolomics workflow was designed and used to characterize the floral flavonoid metabolome and associated glycosylation moieties. ResultsOur results demonstrate how short wavelengths differentially affect the metabolism of natural product compound classes including polyketides and phenylpropanoids/shikimates. Blue light induced flavonoids similarly to how UVB did, while both UVA and blue light specifically induced flavanones accumulation. UVB showed the strongest regulatory effect on flavonoids production and glycosylation patterns. ConclusionsUVB reshapes the cannabis floral flavonoid metabolome by selectively stimulating the accumulation and structural modification of flavonoids. Therefore, UVB application in cannabis cultivation represents a useful horticultural strategy to increase inflorescence medicinal quality without affecting cannabinoid levels.

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)

Micro-dwarf tomato cultivars are increasingly considered for urban and controlled-environment agriculture due to their compact architecture and suitability for high-density planting. However, optimal canopy management strategies for these cultivars remain poorly defined. In this study, we evaluated the effects of different leaf removal intensities on leaf-level physiological performance, fruit yield, and fruit quality in three micro-dwarf tomato cultivars (Mohammed, Hahms Gelbe Topftomate, and Red Robin) grown under contrasting seasonal light conditions. Plants were subjected to low (15%), moderate (30%), or severe (90%) leaf removal, and leaf-level gas exchange was measured across canopy layers, along with yield and fruit quality assessments. Severe leaf removal (90%) increased carbon assimilation, transpiration, and stomatal conductance in middle and lower canopy leaves by up to approximately twofold compared with control plants, indicating improved light availability at the leaf level. However, these physiological enhancements did not consistently translate into higher yield, reflecting reduced whole-plant source capacity under excessive leaf removal. Low to moderate leaf removal (15-30%) generally increased or maintained yield and fruit number, whereas severe leaf removal reduced yield in Hahms Gelbe and Red Robin, particularly under low seasonal radiation. In contrast, Mohammed exhibited yield increases of up to 220% under low leaf removal and maintained increased yield even under severe leaf removal under high-light conditions. Fruit quality was largely unaffected by leaf removal, except for total soluble solids, which declined by approximately 12% under severe leaf removal across cultivars, consistent with sugar dilution under source limitation. Overall, these results demonstrate that optimal leaf removal in micro-dwarf tomatoes requires balancing improved canopy light distribution with maintenance of sufficient leaf area for carbon assimilation. Leaf removal thresholds are strongly cultivar- and light-dependent, emphasizing the need for cultivar-specific canopy management strategies in compact tomato systems and controlled-environment agriculture.

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.

CoverCress is a new winter annual oilseed crop developed from field pennycress within the past 20 years. Field pennycress is commonly considered to be self-pollinated but little basic research has been published and there is some misalignment of conclusions. Our experience working with pennycress plant growth in greenhouse and field conditions over the past 13 years suggests that outcrossing is uncommon. We conducted lab, greenhouse, and field experiments to strengthen the body of work. Pollen viability kinetics analysis showed that longevity of pollen viability is negatively impacted by increasing temperatures and by direct exposure to light. Samples treated at 4C declined to 50% viability in 12 hours while it took just 2.5 hrs at 37C, and 1.6 hrs in full sunlight on a cool early April day. Cross-pollination was absent among greenhouse-grown plants flowering inside an agitated plastic pollen-containment covering. Across greenhouse tests, high rates of cross-pollination occurred only in an emasculation treatment that rendered flowers male sterile and opened the pistil to cross-fertilization. Field trials designed to measure pollen flow distance using a trackable fae1 knockout reporter gene failed to show detectable movement of pollen under field conditions in two locations. This data strongly suggests that domesticated field pennycress may be considered a self-pollinated crop and managed as such.

Plant DNA metabarcoding enables the identification of plant taxa in mixed samples, with the trnL (UAA) intron and its P6 loop mini-barcode region performing as well as or better than other commonly used markers. Reliable metabarcoding requires high-quality reference databases, yet a regularly maintained trnL resource is currently lacking. Consequently, most studies use uncurated sequences downloaded directly from public repositories without essential validation. We address these gaps by providing guidance through a systematic comparison of three database curation tools - OBITools3/ecoPCR, RESCRIPt, and MetaCurator - to generate three trnL reference sequence databases and evaluate their classification performance across commonly sequenced trnL regions (CD, CH, and GH). Reference trnL sequences and taxonomy files were retrieved from public sequence repositories and curated using standardized filtering steps to reduce taxonomic errors, sequence ambiguity, and redundancy. Four simulated query datasets--two base sets and their mutated counterparts--were constructed to assess classification performance of the databases using the Naive Bayesian Classifier implemented in DADA2.- The evaluation showed that performance differed by trnL region: MetaCurator and RESCRIPt yielded higher and similar metrics for trnL CD; OBITools3/ecoPCR and RESCRIPt were comparable for trnL CH; and MetaCurator attained the highest performance for trnL GH region. All reference databases, taxonomy, and evaluation files are available at Zenodo (https://doi.org/10.5281/zenodo.17969450). The complete computational workflow and scripts are available on GitHub (https://github.com/oskuddar/trnL_DB). Although evaluation was focused on plant taxa in the United States, the resulting databases are suitable for use as global trnL reference databases.

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.

Background and AimsPlants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. MethodsLarvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC- qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key ResultsLarvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. ConclusionsInsect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Issue SectionOriginal article