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SUMMARY As tertiary gene pools of wheat, Aegilops comosa and Ae. caudata contain many excellent genes/traits and gradually become important and noteworthy wild resources for wheat improvement worldwide. However, the lack of molecular markers and cytological probes with good specificity and high sensitivity limits the development and utilization of Triticum aestivum–Ae. comosa (Ta. Aeco)/Ae. caudata (Ta. Aeca) introgression lines. Using specific‐locus amplified fragment sequencing, two Ae. comosa and one Ae. caudata accessions, Chinese Spring, and three Ta. Aeco and Ta. Aeca introgression lines each were sequenced to develop new molecular markers and cytological probes. After strict sequence comparison and verification in different materials, a total of 39 molecular markers specific to three chromosomes in Ae. comosa (nine, seven, and 10 markers for 1M, 2M, and 7M, respectively) and Ae. caudata (two, six, and five markers for 3C, 4C, and 5C, respectively) and 21 fluorescence in situ hybridization (FISH) probes (one centromeric probe with signals specific to the M chromosomes, two centromeric probes with signals in all the tested genomes, and six, eight, and four FISH probes specific to the M, C, and M, C, and U chromosomes, respectively) were successfully exploited. The newly developed molecular markers and cytological probes could be used in karyotype studies, centromere evolutionary analyses of Aegilops, and had the ability to detect the fusion centromeres and small‐fragment translocations in introgression lines. [ABSTRACT FROM AUTHOR]

SUMMARY: Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes‐mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red‐violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots‐to‐shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2‐ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2‐ipt combination. Then, Wus2‐ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene‐edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome‐editing genetic improvement of other vegetable species. Significance Statement: We successfully explored an economic and highly efficient genetic transformation method for radish by Agrobacterium rhizogenes‐mediated transformation with the help of the Wus2‐ipt‐RUBY system, which did not need any hormone supplement for shoot regeneration and could achieve a transformation efficiency of 29.21–40.00% in less than 4 months and be used to generate knockout mutants through CRISPR/Cas9 genome editing. These findings have the potential to be widely applied in genetic transformation and genome‐editing genetic improvement of other vegetable species. [ABSTRACT FROM AUTHOR]

SUMMARY Plants direct substantial amounts of carbon toward the biosynthesis of aromatic amino acids (AAAs), particularly phenylalanine to produce lignin and other phenylpropanoids. Yet, we have a limited understanding of how plants regulate AAA metabolism, partially because of a scarcity of robust analytical methods. Here, we established a simplified workflow for simultaneous quantification of AAAs and their pathway intermediates from plant tissues, based on extraction at two alternative pH and analysis by Zwitterionic hydrophilic interaction liquid chromatography coupled to mass spectrometry. This workflow was then used to analyze metabolic responses to elevated or reduced carbon flow through the shikimate pathway in plants. Increased flow upon expression of a feedback‐insensitive isoform of the first shikimate pathway enzyme elevated all AAAs and pathway intermediates, especially arogenate, the last common precursor within the post‐chorismate pathway of tyrosine and phenylalanine biosynthesis. Additional overexpression of an arogenate dehydrogenase enzyme increased tyrosine levels and depleted phenylalanine and arogenate pools; however, the upstream shikimate pathway intermediates remained accumulated at high levels. Glyphosate treatment, which restricts carbon flow through the shikimate pathway by inhibiting its penultimate step, led to a predictable accumulation of shikimate and other precursors upstream of its target enzyme but also caused an unexpected accumulation of downstream metabolites, including arogenate. These findings highlight that the shikimate pathway and the downstream post‐chorismate AAA pathways function as independently regulated modules in plants. The method developed here paves the way for a deeper understanding of the shikimate and AAA biosynthetic pathways in plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Genome‐wide association study (GWAS) with single nucleotide polymorphisms (SNPs) has been widely used to explore genetic controls of phenotypic traits. Alternatively, GWAS can use counts of substrings of length k from longer sequencing reads, k‐mers, as genotyping data. Using maize cob and kernel color traits, we demonstrated that k‐mer GWAS can effectively identify associated k‐mers. Co‐expression analysis of kernel color k‐mers and genes directly found k‐mers from known causal genes. Analyzing complex traits of kernel oil and leaf angle resulted in k‐mers from both known and candidate genes. A gene encoding a MADS transcription factor was functionally validated by showing that ectopic expression of the gene led to less upright leaves. Evolution analysis revealed most k‐mers positively correlated with kernel oil were strongly selected against in maize populations, while most k‐mers for upright leaf angle were positively selected. In addition, genomic prediction of kernel oil, leaf angle, and flowering time using k‐mer data resulted in a similarly high prediction accuracy to the standard SNP‐based method. Collectively, we showed k‐mer GWAS is a powerful approach for identifying trait‐associated genetic elements. Further, our results demonstrated the bridging role of k‐mers for data integration and functional gene discovery. Significance Statement: Through genetically dissecting five maize traits (cob color, kernel color, leaf angle, oil content, and flowering time) and identifying a novel gene responsible for leaf angle, our study illustrates the potent capability of pinpointing genetic elements associated with traits. Our study also demonstrates a bridging role of k‐mers in data integration for facilitating functional gene discovery, as well as the use of k‐mers in phenotypic prediction. [ABSTRACT FROM AUTHOR]

SUMMARY: Plant hormones are chemical signals governing almost every aspect of a plant's life cycle and responses to environmental cues. They are enmeshed within complex signaling networks that can only be deciphered by using broad‐scale analytical methods to capture information about several plant hormone classes simultaneously. Methods used for this purpose are all based on reversed‐phase (RP) liquid chromatography and mass spectrometric detection. Hydrophilic interaction chromatography (HILIC) is an alternative chromatographic method that performs well in analyses of biological samples. We therefore developed and validated a HILIC method for broad‐scale plant hormone analysis including a rapid sample preparation procedure; moreover, derivatization or fractionation is not required. The method enables plant hormone screening focused on polar and moderately polar analytes including cytokinins, auxins, jasmonates, abscisic acid and its metabolites, salicylates, indoleamines (melatonin), and 1‐aminocyclopropane‐1‐carboxylic acid (ACC), for a total of 45 analytes. Importantly, the major pitfalls of ACC analysis have been addressed. Furthermore, HILIC provides orthogonal selectivity to conventional RP methods and displays greater sensitivity, resulting in lower limits of quantification. However, it is less robust, so procedures to increase its reproducibility were established. The method's potential is demonstrated in a case study by employing an approach combining hormonal analysis with phenomics to examine responses of three Arabidopsis ecotypes toward three abiotic stress treatments: salinity, low nutrient availability, and their combination. The case study showcases the value of the simultaneous determination of several plant hormone classes coupled with phenomics data when unraveling processes involving complex cross‐talk under diverse plant‐environment interactions. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants produce a staggering array of chemicals that are the basis for organismal function and important human nutrients and medicines. However, it is poorly defined how these compounds evolved and are distributed across the plant kingdom, hindering a systematic view and understanding of plant chemical diversity. Recent advances in plant genome/transcriptome sequencing have provided a well‐defined molecular phylogeny of plants, on which the presence of diverse natural products can be mapped to systematically determine their phylogenetic distribution. Here, we built a proof‐of‐concept workflow where previously reported diverse tyrosine‐derived plant natural products were mapped onto the plant tree of life. Plant chemical‐species associations were mined from literature, filtered, evaluated through manual inspection of over 2500 scientific articles, and mapped onto the plant phylogeny. The resulting "phylochemical" map confirmed several highly lineage‐specific compound class distributions, such as betalain pigments and Amaryllidaceae alkaloids. The map also highlighted several lineages enriched in dopamine‐derived compounds, including the orders Caryophyllales, Liliales, and Fabales. Additionally, the application of large language models, using our manually curated data as a ground truth set, showed that post‐mining processing can largely be automated with a low false‐positive rate, critical for generating a reliable phylochemical map. Although a high false‐negative rate remains a challenge, our study demonstrates that combining text mining with language model‐based processing can generate broader phylochemical maps, which will serve as a valuable community resource to uncover key evolutionary events that underlie plant chemical diversity and enable system‐level views of nature's millions of years of chemical experimentation. Significance Statement: This study introduces a novel workflow to map the distribution of natural products onto a phylogeny. It also demonstrates the effectiveness of combining text mining of the literature with large language model evaluation for constructing a "phylochemical" database that can organize vast portions of the chemical diversity present across the plant tree of life. [ABSTRACT FROM AUTHOR]

SUMMARY: RNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4–HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms. Significance Statement: Cas endonuclease systems have revolutionized genome editing but rapid quantitative assays for the optimization of this process that circumvent the need for DNA extraction from individual plants are rare or lacking. Here, we describe two quantitative reporter switch‐on assays for the direct side‐by‐side comparison of the editing efficiency of different Cas systems, like Cas9 and Cas12a, the principles of which can be applied for optimizing gene editing in any plant species. [ABSTRACT FROM AUTHOR]

SUMMARY: Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium‐mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene‐editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1‐VIR2 virulence helper plasmid to improve T‐DNA delivery, and/or expressing a fusion protein between a GROWTH‐REGULATING FACTOR (GRF) and GRF‐INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1‐VIR2 helper vector with no effect on event quality regarding T‐DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5‐ to 6.5‐fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR‐/Cas9‐mediated gene editing. Our results demonstrate how a GRF‐GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene‐editing applications and molecular biology studies in maize. [ABSTRACT FROM AUTHOR]

SUMMARY: Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real‐time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real‐time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies. Significant Statement: Monitoring dynamic changes in pyridine nucleotides (e.g., NADPH and NADH/NAD+ ratio) in plants at the subcellular level is a major obstacle in plant bioenergetics research. Genetically encoded fluorescent biosensors are considered to be the best option so far because they provide fast, accurate, and real‐time readouts. Here, we present a detailed study on the application of pyridine nucleotide biosensors, and we anticipate that these transgenic lines will be useful in plant redox biology studies. [ABSTRACT FROM AUTHOR]

SUMMARY: CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4‐ to 7‐plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher‐order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well. [ABSTRACT FROM AUTHOR]

SUMMARY: Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant–pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)‐GFP vector and Agrobacterium‐mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV‐GFP system, we initially tested it with well‐described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV‐GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV‐GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV‐GFP system in identifying apoplastic effectors. The easy operation, time‐saving nature, broad effectiveness, and low technical requirements of the TMV‐GFP system make it a promising approach for high‐throughput screening of effectors with immune interference activity from various pathogens. Significance Statement: The development of a robust high‐throughput functional screening assay for pathogen effectors will facilitate the study in plant‐pathogen interactions. We established a novel screening system using the TMV‐GFP vector, enabling the rapid identification of effectors that interfere with plant immunity by observing GFP fluorescence under a UV lamp within a few days. The TMV‐GFP system is a promising approach for high‐throughput screening of effectors from diverse pathogens. [ABSTRACT FROM AUTHOR]

SUMMARY: Next‐generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR‐based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG‐seq primer set (MIG‐seq being a PCR method enabling library construction with low‐quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG‐seq (dpMIG‐seq), enabled a streamlined protocol for constructing dpMIG‐seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG‐seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG‐seq protocol for advancing genetic analyses across diverse plant species. Significance Statement: Given that plants contain impurities in their leaves, DNA purification is required to construct NGS libraries with reduced genomic complexity using restriction enzymes, which poses a hinderance to rapid genotyping. We developed degenerate oligonucleotide primer MIG‐seq and its accompanying protocol, a method for constructing NGS libraries with varying degrees of complexity reduction from unpurified plant DNA using PCR, and demonstrated the feasibility of expeditious genetic analysis across various crop species. [ABSTRACT FROM AUTHOR]

SUMMARY: Confocal microscopy has greatly aided our understanding of the major cellular processes and trafficking pathways responsible for plant growth and development. However, a drawback of these studies is that they often rely on the manual analysis of a vast number of images, which is time‐consuming, error‐prone, and subject to bias. To overcome these limitations, we developed Dot Scanner, a Python program for analyzing the densities, lifetimes, and displacements of fluorescently tagged particles in an unbiased, automated, and efficient manner. Dot Scanner was validated by performing side‐by‐side analysis in Fiji‐ImageJ of particles involved in cellulose biosynthesis. We found that the particle densities and lifetimes were comparable in both Dot Scanner and Fiji‐ImageJ, verifying the accuracy of Dot Scanner. Dot Scanner largely outperforms Fiji‐ImageJ, since it suffers far less selection bias when calculating particle lifetimes and is much more efficient at distinguishing between weak signals and background signal caused by bleaching. Not only does Dot Scanner obtain much more robust results, but it is a highly efficient program, since it automates much of the analyses, shortening workflow durations from weeks to minutes. This free and accessible program will be a highly advantageous tool for analyzing live‐cell imaging in plants. Significance Statement: Live‐cell imaging analyses of fluorescently tagged particles in biological systems often require significant manual input that is time‐consuming and subject to bias. We have developed a free Python program, Dot Scanner, to allow users without any programming experience to analyze large datasets rapidly, efficiently, and with reduced bias. [ABSTRACT FROM AUTHOR]

SUMMARY: Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation‐based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)‐based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co‐expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA‐INSENSITIVE, GAI; REPRESSOR‐of‐ga1‐3, RGA; RGA‐like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA‐oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta. Significance Statement: Using the degradation‐based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)‐based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. [ABSTRACT FROM AUTHOR]

Inventive step is the sine qua non for the grant of a patent. Arguably, this subjective evaluation of the claimed invention ensures a sufficient leap forward to the existing technology. The footnote appended to Article 27 of the TRIPS Agreement contemplates that inventive step may be deemed tobe synonymous with "non-obvious". Thus, the footnote has left it open-ended for the WTO members to speculate on may or may not situations or entirely rule out this apparent inventive step/non-obviousness equivalence suggestion. TRIPS Agreement does not define inventive step or lay down the standards to be followed in construing the person from whose perspective the claimed invention has to be looked into for inventive step/non-obviousness determination. In the U.S., Congress and the Courts have consistently interpreted "non-obviousness" as technical advancement to the concerned discipline. When it comes to Indian law, there is an apparentclarity, but when delved deep, the outcome lacks crucial elements of predictability. Section 2(1)(ja) of the Indian Patents Act, 1970 is framed in such a way so that it gives an impression to the reader that technical advancement and being non-obvious to a person skilled in the art are two distinct concepts. The US Courts always gave economic considerations a secondary status in the determination of the non-obviousness of a given invention. Indian patent statute specifically mentions economic significanceand places it as an alternative to technical advancementwithoutclarifying the interconnection of the two connotations. The authors intend to understand, in what way technical advance can be considered as a completely separate standalone criterion for patentability in conjunction with non-obviousness. Furthermore, as a step forward, the authors want to expound on whether economic consideration alone, without even requiring non-obviousness, can fulfil the inventive step criterion in India. [ABSTRACT FROM AUTHOR]

SUMMARY: Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time‐consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time‐lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open‐source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof‐of‐concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response. Significance Statement: PIRO addresses the main bottleneck preventing widespread use of many fantastic custom‐made phenotyping platforms: feasibility to replicate the system without access to engineering skills. SPIRO is designed to be built and operated with no training in engineering or programming, it enables continuous imaging within standard plant growth cabinets under both day and night conditions, seamlessly integrating into existing laboratory workflows; provides exceptional data quality, thereby facilitating a new research standard for all laboratories. [ABSTRACT FROM AUTHOR]

SUMMARY: The cytosol‐facing outer membrane (OM) of organelles communicates with other cellular compartments to exchange proteins, metabolites, and signaling molecules. Cellular surveillance systems also target OM‐resident proteins to control organellar homeostasis and ensure cell survival under stress. However, the OM proximity proteomes have never been mapped in plant cells since using traditional approaches to discover OM proteins and identify their dynamically interacting partners remains challenging. In this study, we developed an OM proximity labeling (OMPL) system using biotin ligase‐mediated proximity biotinylation to identify the proximity proteins of the OMs of mitochondria, chloroplasts, and peroxisomes in living Arabidopsis (Arabidopsis thaliana) cells. Using this approach, we mapped the OM proximity proteome of these three organelles under normal conditions and examined the effects of the ultraviolet‐B (UV‐B) or high light (HL) stress on the abundances of OM proximity proteins. We demonstrate the power of this system with the discovery of cytosolic factors and OM receptor candidates potentially involved in local protein translation and translocation. The candidate proteins that are involved in mitochondrion–peroxisome, mitochondrion–chloroplast, or peroxisome–chloroplast contacts, and in the organellar quality control system are also proposed based on OMPL analysis. OMPL‐generated OM proximity proteomes are valuable sources of candidates for functional validation and suggest directions for further investigation of important questions in cell biology. [ABSTRACT FROM AUTHOR]

SUMMARY: Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by‐effects, we investigated the impact of GRF4‐GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4‐GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM‐WUS. As agrobacterium effectors increase the ability of T‐DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4‐GIF1 with helper plasmid pVS1‐VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71‐fold of the original method. Compared with BBM‐WUS, overexpressing GRF4‐GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene‐editing tool based on this GRF4‐GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation. Significance Statement: An accelerated and efficient transformation strategy of sorghum was established which has 38.28% efficiency in less than 2 months. The regenerated plants showed no growth defect. [ABSTRACT FROM AUTHOR]

SUMMARY: Anionic phospholipids (PS, PA, PI, PIPs) are low‐abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at defined spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin‐layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive, and accurate quantification of all anionic phospholipid species. Here we developed a method based on high‐performance liquid chromatography (HPLC) combined with two‐dimensional mass spectrometry (MS2) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peak, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way for new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses. Significance Statement: While anionic phospholipids have key functions in plant cellular processes, their low concentration in biological samples and their low stability during the analysis complicate their quantification. Here, we present the first one‐shot analytical method for the profiling and quantification of all anionic phospholipid classes and species from plant tissues with unprecedented sensitivity. This method opens the way to future studies requiring a fine quantification of anionic phospholipids to understand their role in plant cell processes. [ABSTRACT FROM AUTHOR]

SUMMARY: Generation of mutant populations with high genetic diversity is key for mutant screening and crop breeding. For this purpose, the single‐seed descent method, in which one mutant line is established from a single mutagenized seed, is commonly used. This method ensures the independence of the mutant lines, but the size of the mutant population is limited because it is no greater than the number of fertile M1 plants. The rice mutant population size can be increased if a single mutagenized plant produces genetically independent siblings. Here, we used whole‐genome resequencing to examine the inheritance of mutations from a single ethyl methanesulfonate (EMS)‐mutagenized seed (M1) of Oryza sativa in its progeny (M2). We selected five tillers from each of three M1 plants. A single M2 seed was selected from each tiller, and the distributions of mutations induced by EMS were compared. Surprisingly, in most pairwise combinations of M2 siblings from the same parent, ≥85.2–97.9% of all mutations detected were not shared between the siblings. This high percentage suggests that the M2 siblings were derived from different cells of the M1 embryo and indicates that several genetically independent lines can be obtained from a single M1 plant. This approach should allow a large reduction in the number of M0 seeds needed to obtain a mutant population of a certain size in rice. Our study also suggests that multiple tillers of a rice plant originate from different cells of the embryo. [ABSTRACT FROM AUTHOR]

SUMMARY: The modification of photosynthesis‐related genes in plastid genomes may improve crop yields. Recently, we reported that a plastid‐targeting base editor named ptpTALECD, in which a cytidine deaminase DddA functions as the catalytic domain, can homoplasmically substitute a targeted C to T in plastid genomes of Arabidopsis thaliana. However, some target Cs were not substituted. In addition, although ptpTALECD could substitute Cs on the 3′ side of T and A, it was unclear whether it could also substitute Cs on the 3′ side of G and C. In this study, we identified the preferential positions of the substituted Cs in ptpTALECD‐targeting sequences in the Arabidopsis plastid genome. We also found that ptpTALECD could substitute Cs on the 3′ side of all four bases in plastid genomes of Arabidopsis. More recently, a base editor containing an improved version of DddA (DddA11) was reported to substitute Cs more efficiently, and to substitute Cs on the 3′ side of more varieties of bases in human mitochondrial genomes than a base editor containing DddA. Here, we also show that ptpTALECD_v2, in which a modified version of DddA11 functions as the catalytic domain, more frequently substituted Cs than ptpTALECD in the Arabidopsis plastid genome. We also found that ptpTALECD_v2 tended to substitute Cs at more positions than ptpTALECD. Our results reveal that ptpTALECD can cause a greater variety of codon changes and amino acid substitutions than previously thought, and that ptpTALECD and ptpTALECD_v2 are useful tools for the targeted base editing of plastid genomes. Significance Statement: The preferred positions of cytosines that are substituted by a plastid‐targeted base editor, named ptpTALECD, were determined for more precise base editing in the Arabidopsis plastid genome. We also compared the substitution frequencies between ptpTALECD and the developed version of ptpTALECD (ptpTALECD_v2) and found that both ptpTALECD and ptpTALECD_v2 can be used to edit the plastid genome of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

SUMMARY: Both stable and transient protein interactions play an important role in the complex assemblies required for the proper functioning of living cells. Several methods have been developed to monitor protein–protein interactions in plants. However, the detection of dynamic protein complexes is very challenging, with few technologies available for this purpose. Here, we developed a new platform using the plant UBIQUITIN promoter to drive transgene expression and thereby to detect protein interactions in planta. Typically, to decide which side of the protein to link the tags, the subcellular localization of the protein fused either N‐terminal or C‐terminal mCitrine was firstly confirmed by using eight different specific mCherry markers. Following stable or transient protein expression in plants, the protein interaction network was detected by affinity purification mass spectrometry. These interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer and co‐immunoprecipitation assays. The dynamics of these interactions were monitored by Förster resonance energy transfer (FRET) and split‐nano luciferase, whilst the ternary protein complex association was monitored by BiFC‐FRET. Using the canonical glycolytic metabolon as an example, the interaction between these enzymes was characterized under conditions that mimic physiologically relevant energy statuses. Significance Statement: We developed a new platform using the plant UBIQUITIN promoter to drive transgene expression and thereby to detect protein interactions. Protein interaction network was detected by affinity purification mass spectrometry. Protein interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer and co‐immunoprecipitation assays. The dynamics of interactions were monitored by Förster resonance energy transfer (FRET) and split‐nano luciferase, whilst the ternary protein complex association was monitored by BiFC‐FRET. [ABSTRACT FROM AUTHOR]

SUMMARY: The application of DNA–protein interaction reporter assays for relational or ratiometric measurements within an experimental system is popular in biological research. However, the existing reporter‐based interaction assays always require special equipment, expensive chemicals, and a complicated operation. Here, we developed a DNA–protein interaction technology integrating two visible reporters, RUBY and UV‐visible GFP (eYGFPuv), which allows the expression of the cassette reporter contained cis‐acting DNA element (DE) fused upstream of TATA box and RUBY, and a constitutive promoter regulating eYGFPuv in the same construct. The interaction of transcription factor (TF) and the DE can be detected by co‐expressed the cassette reporter and TF in tobacco leaves where the cassette reporter alone serves as a control. We also revealed that eight function‐unknown bamboo AP2/ERFs interacted with the DE of ANT‐AP2R1R2 (ABE), DRE (DBE), GCC‐box (EBE), and RAV1 binding element (RBE), respectively, which are consistent with the results by dual‐luciferase reporter assays. Thus, the dual‐visible reporters offer a convenient, visible, and cost‐saving alternative to other existing techniques for DNA–protein interaction in plants. [ABSTRACT FROM AUTHOR]

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

SUMMARY: Late blight caused by the oomycete Phytophthora infestans is a most devastating disease of potatoes (Solanum tuberosum). Its early detection is crucial for suppressing disease spread. Necrotic lesions are normally seen in leaves at 4 days post‐inoculation (dpi) when colonized cells are dead, but early detection of the initial biotrophic growth stage, when the pathogen feeds on living cells, is challenging. Here, the biotrophic growth phase of P. infestans was detected by whole‐plant redox imaging of potato plants expressing chloroplast‐targeted reduction–oxidation sensitive green fluorescent protein (chl‐roGFP2). Clear spots on potato leaves with a lower chl‐roGFP2 oxidation state were detected as early as 2 dpi, before any visual symptoms were recorded. These spots were particularly evident during light‐to‐dark transitions, and reflected the mislocalization of chl‐roGFP2 outside the chloroplasts. Image analysis based on machine learning enabled systematic identification and quantification of spots, and unbiased classification of infected and uninfected leaves in inoculated plants. Comparing redox with chlorophyll fluorescence imaging showed that infected leaf areas that exhibit mislocalized chl‐roGFP2 also showed reduced non‐photochemical quenching and enhanced quantum PSII yield (ΦPSII) compared with the surrounding leaf areas. The data suggest that mislocalization of chloroplast‐targeted proteins is an efficient marker of late blight infection, and demonstrate how it can be utilized for non‐destructive monitoring of the disease biotrophic stage using whole‐plant redox imaging. [ABSTRACT FROM AUTHOR]

SUMMARY: Hemp (Cannabis sativa) is a highly versatile crop with a multitude of applications, from textiles, biofuel and building material to high‐value food products for consumer markets. Furthermore, non‐hallucinogenic cannabinoids like cannabidiol (CBD), which can be extracted from female hemp flowers, are potentially valuable pharmacological compounds. In addition, hemp has high carbon sequestration potential associated with its rapid growth rate. Therefore, the hemp industry is gaining more traction and breeding hemp cultivars adapted to local climate conditions or bred for specific applications is becoming increasingly important. Here, we present a method for the rapid generation cycling (speed breeding) of hemp. The speed breeding protocol makes use of the photoperiod sensitivity of Cannabis. It encompasses vegetative growth of the plants for 2 weeks under continuous light, followed by 4 weeks under short‐day conditions, during which flower induction, pollination and seed development proceed, and finally a seed ripening phase under continuous light and water stress. With the protocol described here, a generation time of under 9 weeks (61 days) from seed to seed can be achieved. Furthermore, our method synchronises the flowering time of different hemp cultivars, thus facilitating crosses between cultivars. The extremely short generation time will enable hemp researchers and breeders to perform crosses in a time‐efficient way and generate new hemp cultivars with defined genetic characteristics over a short period of time. [ABSTRACT FROM AUTHOR]

SUMMARY: Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T‐DNA is engineered with the gene(s) of interest. However, gene expression during 'agro‐infiltration' is intrinsically and universally impeded by the onset of post‐transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co‐expression of the tombusvirus‐encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side‐by‐side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co‐expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies – an originally unanticipated, yet increasingly popular application – and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits. Significance Statement: Since its disclosure in 2003, agro‐infiltration used in combination with the P19 RNA silencing suppressor has been a popular method to achieve high levels of transient gene expression in Nicotiana benthamiana, though the underlying molecular mechanisms have remained unclear. By shedding light on these mechanisms, the present contemporary reassessment of the method allows improvements to protein yields and further illustrates its versatility, but also exposes and rationalizes some of its limits. [ABSTRACT FROM AUTHOR]

• Technical advance has an inverted U-shape impacts on primary supply of cobalt and makes it increase by a maximum of 10 %. • Effects of technical advance in the refining, manufacturing and recycling stages are linear promotions. • By 2030, China is expected to produce 76 % of the world's refined cobalt. • The European Union will provide the largest secondary supply in the future, followed by China, the United States and Japan. • Supply-demand contradiction could be effectively alleviated with the simultaneous development of technical advance on the supply and the demand sides. By coupling dynamic material flow analysis and econometric models, the effects of technical advance on cobalt supply in the whole industrial chains are explored. Then, its supply trends are investigated at the global and national levels. The results show that: (1) the impact of technical advance on cobalt supply in the mining stage is inverted U-shape, while in the other stages, they are linear promotions. (2) Technical advance could increase global primary supply by up to 10 % from current production capacity. By 2030, secondary and total supplies will reach 79 kt and 251 kt or even more. (3) Refined cobalt supply share of China is expected to be 76 % by 2030. The European Union will produce the largest secondary supply, followed by China, the United States and Japan. (4) With the simultaneous development of technologies in each stage, supply-demand balance could be realized under some ideal scenarios. The above findings help intuitively recognize the roles of technical advance and strengthen the determination to do it. [Display omitted] [ABSTRACT FROM AUTHOR]

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

SUMMARY: The precise and accurate identification and quantification of transcriptional start sites (TSSs) is key to understanding the control of transcription. The core promoter consists of the TSS and proximal non‐coding sequences, which are critical in transcriptional regulation. Therefore, the accurate identification of TSSs is important for understanding the molecular regulation of transcription. Existing protocols for TSS identification are challenging and expensive, leaving high‐quality data available for a small subset of organisms. This sparsity of data impairs study of TSS usage across tissues or in an evolutionary context. To address these shortcomings, we developed Smart‐Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2), which identifies and quantifies TSSs and transcription termination sites. Smar2C2 incorporates unique molecular identifiers that allowed for the identification of as many as 70 million sites, with no known upper limit. We have also generated TSS data sets from as little as 40 pg of total RNA, which was the smallest input tested. In this study, we used Smar2C2 to identify TSSs in Glycine max (soybean), Oryza sativa (rice), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays (maize) across multiple tissues. This wide panel of plant TSSs facilitated the identification of evolutionarily conserved features, such as novel patterns in the dinucleotides that compose the initiator element (Inr), that correlated with promoter expression levels across all species examined. We also discovered sequence variations in known promoter motifs that are positioned reliably close to the TSS, such as differences in the TATA box and in the Inr that may prove significant to our understanding and control of transcription initiation. Smar2C2 allows for the easy study of these critical sequences, providing a tool to facilitate discovery. [ABSTRACT FROM AUTHOR]

SUMMARY: The arbuscular mycorrhizal (AM) symbiosis is characterized by the reciprocal exchange of nutrients. AM fungi are oleaginous microorganisms that obtain essential fatty acids from host plants. A lipid biosynthesis and delivery pathway has been proposed to operate in inner root cortex cells hosting arbuscules, a cell type challenging to access microscopically. Despite the central role lipids play in the association, lipid distribution patterns during arbuscule development are currently unknown. We developed a simple co‐staining method employing fluorophore‐conjugated Wheat Germ Agglutinin (WGA) and a lipophilic blue fluorochrome, Ac‐201, for the simultaneous imaging of arbuscules and lipids distributed within arbuscule‐containing cells in high resolution. We observed lipid distribution patterns in wild‐type root infection zones in a variety of plant species. In addition, we applied this methodology to mutants of the Lotus japonicus GRAS transcription factor RAM1 and the Oryza sativa half‐size ABC transporter STR1, both proposed to be impaired in the symbiotic lipid biosynthesis‐delivery pathway. We found that lipids accumulated in cortical cells hosting stunted arbuscules in Ljram1 and Osstr1, and observed lipids in the arbuscule body of Osstr1, suggesting that in the corresponding plant species, RAM1 and STR1 may not be essential for symbiotic lipid biosynthesis and transfer from arbuscule‐containing cells, respectively. The versatility of this methodology has the potential to help elucidate key questions on the complex lipid dynamics fostering AM symbioses. Significance Statement: Lipids play essential roles in the arbuscular mycorrhizal symbiosis, but the study of lipid dynamics in the association has been largely devoid of imaging techniques. A simple and versatile methodology for imaging of lipids in cells containing arbuscules using confocal microscopy was developed. Its implementation allowed unprecedented visualization of diverse patterns of lipid distribution across arbuscule development stages in different plant species and in mutants of genes described to participate in symbiotic lipid dynamics. [ABSTRACT FROM AUTHOR]

SUMMARY: Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus‐induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and can disrupt plant homeostasis by interfering with endogenous cell mechanisms. To try to circumvent this functional limitation, we have developed a silencing method based on the minimal autonomously‐infectious nucleic acids currently known: viroids, which lack proven coding capability. The genome of Eggplant latent viroid, an asymptomatic viroid, was manipulated with insertions ranging between 21 and 42 nucleotides. Our results show that, although larger insertions might be tolerated, the maintenance of the secondary structure appears to be critical for viroid genome stability. Remarkably, these modified ELVd molecules are able to induce systemic infection promoting the silencing of target genes in eggplant. Inspired by the design of artificial microRNAs, we have developed a simple and standardized procedure to generate stable insertions into the ELVd genome capable of silencing a specific target gene. Analogously to VIGS, we have termed our approach viroid‐induced gene silencing, and demonstrate that it is a promising tool for dissecting gene functions in eggplant. [ABSTRACT FROM AUTHOR]

SUMMARY: Quantification of reaction fluxes of metabolic networks can help us understand how the integration of different metabolic pathways determines cellular functions. Yet, intracellular fluxes cannot be measured directly but are estimated with metabolic flux analysis (MFA), which relies on the patterns of isotope labeling of metabolites in the network. The application of MFA also requires a stoichiometric model with atom mappings that are currently not available for the majority of large‐scale metabolic network models, particularly of plants. While automated approaches such as the Reaction Decoder Toolkit (RDT) can produce atom mappings for individual reactions, tracing the flow of individual atoms of the entire reactions across a metabolic model remains challenging. Here we establish an automated workflow to obtain reliable atom mappings for large‐scale metabolic models by refining the outcome of RDT, and apply the workflow to metabolic models of Arabidopsis thaliana. We demonstrate the accuracy of RDT through a comparative analysis with atom mappings from a large database of biochemical reactions, MetaCyc. We further show the utility of our automated workflow by simulating 15N isotope enrichment and identifying nitrogen (N)‐containing metabolites which show enrichment patterns that are informative for flux estimation in future 15N‐MFA studies of A. thaliana. The automated workflow established in this study can be readily expanded to other species for which metabolic models have been established and the resulting atom mappings will facilitate MFA and graph‐theoretic structural analyses with large‐scale metabolic networks. Significance Statement: The study provides an automated workflow for generation of atom mappings in large‐scale metabolic models. By applying the workflow to existing Arabidopsis metabolic models and employing the resulting atom maps, we simulated 15N isotope enrichment and identified metabolites that are informative in devising the nitrogen flux map in this model plant. [ABSTRACT FROM AUTHOR]

SUMMARY: High efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR‐associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR‐associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70‐nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site‐directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four‐fold higher than in untethered controls. Integrations via canonical homology‐directed repair or non‐homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN‐associated tethering to improve site‐directed DNA integration in plants. Significance Statement: An exceptionally high rate of site‐directed integration of exogenous DNA into the soybean (Glycine max) genome was achieved using a novel method to increase the local donor DNA concentration around the CRISPR target site of interest. A donor DNA was covalently tethered to a fusion protein between LbCas12a and the HUH endonuclease of Faba Bean Necrotic Yellow Virus, which was delivered into soybean protoplasts and plants as part of a ribonucleoprotein complex. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman spectroscopy (SERS) is an emerging technology that combines Raman spectroscopy and nanotechnology with great potential. This technology can accurately characterize molecular adsorption behavior and molecular structure. Moreover, it can provide rapid and sensitive detection of molecules and trace substances. In practical application, SERS has the advantages of portability, no need for sample pretreatment, rapid analysis, high sensitivity, and 'fingerprint' recognition. Thus, it has great potential in food safety detection. Alcoholic beverages have a long history of production in the world. Currently, a variety of popular products have been developed. With the continuous development of the alcoholic beverage industry, simple, on-site, and sensitive detection methods are necessary. In this paper, the basic principle, development history, and research progress of SERS are summarized. In view of the chemical composition, the beneficial and toxic components of alcoholic beverages and the practical application of SERS in alcoholic beverage analysis are reviewed. The feasibility and future development of SERS are also summarized and prospected. This review provides data and reference for the future development of SERS technology and its application in food analysis. [ABSTRACT FROM AUTHOR]

SUMMARY: Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co‐purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition‐ or cell‐specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell‐specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus‐specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi‐molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre‐existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant–pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies. Significance Statement: Translating ribosome affinity purification (TRAP) has been modified to allow with increased sensitivity the isolation of RNA from sets of cells in which the activity of condition/cellspecific promoters is weak, ribosome turnover is low, or cells whose nature is ephemeral. Based on the use of a split linker constituted of the GFP driven by a pathogen‐inducible promoter, this new TRAP system enabled efficient isolation of translated RNA from pathogen‐infected leaf cells. [ABSTRACT FROM AUTHOR]

SUMMARY: Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live‐cell labeling. It consists of the incorporation of a carbohydrate containing a bio‐orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip‐polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long‐term dynamics of labeled extracellular polysaccharides. Significance Statement: This paper reports a protocol for the metabolic‐click mediated dynamic imaging of a pollen tube, a rapidly elongating plant cell. This method results in the fluorescent labelling of cell wall polysaccharides in living cells, allowing the study of their long‐term dynamics. [ABSTRACT FROM AUTHOR]

SUMMARY: The precise determination of photosynthetic pigment content in green organisms, chlorophylls (Chls) and carotenoids (Cars), is important to investigate many photosynthetic processes such as responses to environmental fluctuations or to gene mutations, as well as to interpret biochemical and structural results obtained on purified membranes and photosynthetic complexes. The most utilized methods for determination by spectrophotometry of Chl content in solution, usually 80% acetone, are based on the use of simultaneous equations. The advantages are the easiness and speed over chromatography, which also requires less common equipment. The disadvantage is that issues in sample preparation or in the measurement are not detectable, which could lead to wrong results. Here we propose a fast, accurate and (almost) error‐proof method to measure Chl a, Chl b and also total Car content in a solution of pigments extracted from tissue, membranes or purified complexes. The method is based on the fit of the absorption spectrum of the acetone extract using the spectra of purified pigments as references. We show how this method allows a more precise and accurate estimation of pigment content as compared to classical equations, even in incorrectly prepared acetone solutions. Moreover, the method allows the discovery of artifacts in sample preparation or measurement and thus drastically reduces the risk of mistakes. Examples obtained on purified complexes are also discussed. Based on newly acquired Chl spectra, we also propose a new set of improved simultaneous equations that provide slightly different but more reliable results in comparison with the currently used equations. Significance Statement: A new method to measure Chl a, Chl b and Car content in solutions by spectrophotometry, based on the fit of the absorption spectrum with the spectra of purified pigments, is described. The method allows the discovery of artifacts, invisible using classical approaches based on simultaneous equations, drastically reducing mistakes. This method also works over a large range of incorrectly prepared solutions. We also propose new improved equations that are more reliable than the currently used equations. [ABSTRACT FROM AUTHOR]

SUMMARY: Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. Chromosome level genome assemblies using sequence contigs generated from long read sequencing have involved the use of proximity analysis (Hi‐C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (HiFi; PacBio) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii, a genome that has been used as a model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig (six with telomere repeats at both ends) and the other six assembled from two to four main contigs. The small number of chromosome breaks appears to be the result of highly repetitive regions including ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now appears possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed. Significance Statement: De novo assembly of a near compete chromosome level plant genome is now possible using long read sequencing and assembly tools. [ABSTRACT FROM AUTHOR]

SUMMARY: While flux balance analysis (FBA) provides a framework for predicting steady‐state leaf metabolic network fluxes, it does not readily capture the response to environmental variables without being coupled to other modelling formulations. To address this, we coupled an FBA model of 903 reactions of soybean (Glycine max) leaf metabolism with e‐photosynthesis, a dynamic model that captures the kinetics of 126 reactions of photosynthesis and associated chloroplast carbon metabolism. Successful coupling was achieved in an iterative formulation in which fluxes from e‐photosynthesis were used to constrain the FBA model and then, in turn, fluxes computed from the FBA model used to update parameters in e‐photosynthesis. This process was repeated until common fluxes in the two models converged. Coupling did not hamper the ability of the kinetic module to accurately predict the carbon assimilation rate, photosystem II electron flux, and starch accumulation of field‐grown soybean at two CO2 concentrations. The coupled model also allowed accurate predictions of additional parameters such as nocturnal respiration, as well as analysis of the effect of light intensity and elevated CO2 on leaf metabolism. Predictions included an unexpected decrease in the rate of export of sucrose from the leaf at high light, due to altered starch–sucrose partitioning, and altered daytime flux modes in the tricarboxylic acid cycle at elevated CO2. Mitochondrial fluxes were notably different between growing and mature leaves, with greater anaplerotic, tricarboxylic acid cycle and mitochondrial ATP synthase fluxes predicted in the former, primarily to provide carbon skeletons and energy for protein synthesis. Significance Statement: A model of the relationship between photosynthesis and other metabolic pathways in soybean leaves was constructed by coupling a kinetic model of photosynthesis with a constraint‐based stoichiometric model of the rest of leaf metabolism. The new modelling formulation is shown, by comparison with measurements, to allow a broad prediction of leaf metabolic behaviour under different environments and will be a useful tool for guiding strategies for improving leaf carbon and energy‐use efficiency under global change. [ABSTRACT FROM AUTHOR]

SUMMARY: At the morphological and anatomical levels, the ionome, or the elemental composition of an organism, is an understudied area of plant biology. In particular, the ionomic responses of plant–pathogen interactions are scarcely described, and there are no studies on immune reactions. In this study we explored two X‐ray fluorescence (XRF)‐based ionome visualisation methods (benchtop‐ and synchrotron‐based micro‐XRF [µXRF]), as well as the quantitative inductively coupled plasma optical emission spectroscopy (ICP‐OES) method, to investigate the changes that occur in the ionome of compatible and incompatible plant–pathogen interactions. We utilised the agronomically important and comprehensively studied interaction between potato (Solanum tuberosum) and the late blight oomycete pathogen Phytophthora infestans as an example. We used one late blight‐susceptible potato cultivar and two resistant transgenic plant lines (only differing from the susceptible cultivar in one or three resistance genes) both in control and P.infestans‐inoculated conditions. In the lesions from the compatible interaction, we observed rearrangements of several elements, including a decrease of the mobile macronutrient potassium (K) and an increase in iron (Fe) and manganese (Mn), compared with the tissue outside the lesion. Interestingly, we observed distinctly different distribution patterns of accumulation at the site of inoculation in the resistant lines for calcium (Ca), magnesium (Mg), Mn and silicon (Si) compared to the susceptible cultivar. The results reveal different ionomes in diseased plants compared to resistant plants. Our results demonstrate a technical advance and pave the way for deeper studies of the plant–pathogen ionome in the future. Significance Statement: Using benchtop and synchrotron µXRF imaging and ICP‐OES quantification, we analysed the ionomic changes in potato (Solanum tuberosum) plants upon inoculation with the late blight pathogen Phytophthora infestans. Inoculation with the pathogen leads to shifts in the distribution patterns of several elements in either susceptible or resistant potato leaflets, including a particular redistribution of manganese (Mn) and silicon (Si) in R‐gene‐containing lines. These techniques open up new possibilities for plant–microbe interactions research at the ionome level. [ABSTRACT FROM AUTHOR]

SUMMARY: Single‐cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nucleus isolation combined with nanowell‐based library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as an alternative to the use of protoplast isolation, which is currently a standard methodology for plant single‐cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nucleus isolation method by using different plant materials from different species. The potential of our single‐nucleus RNA sequencing method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identified stage‐specific activities of transcription factors regulating this process, and predicted potential target genes of these transcription factors. Our nucleus isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single‐cell genomics experiments. Significance Statement: We introduce an optimized plant nucleus isolation procedure followed by single‐nucleus RNA sequencing that can be applied to different plant tissues. We exemplify the utility of this method by analyzing cellular heterogeneity in Arabidopsis seedlings and flowers. [ABSTRACT FROM AUTHOR]

SUMMARY: Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RHs show a fast apical growth, constituting a unique single cell model system for analysing cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, although high‐resolution imaging is still lacking to enable an investigation of the accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) that enables us to capture high resolution confocal imaging of Arabidopsis RH development with real‐time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, to illustrate the possibilities offered by the CMD, we have compared the real‐time variations in the circularity, area and aspect ratio of nuclei moving in growing and mature RHs. Interestingly, we observed higher aspect ratios in the nuclei of mature RHs, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development. Significance Statement: We present a robust approach using coverslip based microfluidic device that enables us to capture high resolution confocal imaging of Arabidopsis root hair (RH) growth, as well as concomitant nuclear movement and shape changes in real time. We show that the circularity, area and aspect ratio of the nuclei vary in real time. Interestingly, our results demonstrate a higher change in the aspect ratio in mature RH nuclei compared to growing RH. [ABSTRACT FROM AUTHOR]

SUMMARY: Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable 'AgroLux' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf‐induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non‐destructive, versatile and easy‐to‐use imaging tool to monitor both Agrobacterium and plant responses. Significance Statement: We generated a genome‐integrated bioluminescent Agrobacterium strain that grows normally in vitro and in planta. We illustrate its broad versatility by correlating reduced bioluminescence with protein expression upon high light stress, and by establishing the early and sensitive detection of immunity induced by the coexpression of matching Avr/Cf gene combinations. [ABSTRACT FROM AUTHOR]

Summary: Brassinosteroids (BRs) are a group of plant steroid hormones involved in regulating growth, development, and stress responses. Many components of the BR pathway have previously been identified and characterized. However, BR phenotyping experiments are typically performed in a low‐throughput manner, such as on Petri plates. Additionally, the BR pathway affects drought responses, but drought experiments are time consuming and difficult to control. To mitigate these issues and increase throughput, we developed the Robotic Assay for Drought (RoAD) system to perform BR and drought response experiments in soil‐grown Arabidopsis plants. RoAD is equipped with a robotic arm, a rover, a bench scale, a precisely controlled watering system, an RGB camera, and a laser profilometer. It performs daily weighing, watering, and imaging tasks and is capable of administering BR response assays by watering plants with Propiconazole (PCZ), a BR biosynthesis inhibitor. We developed image processing algorithms for both plant segmentation and phenotypic trait extraction to accurately measure traits including plant area, plant volume, leaf length, and leaf width. We then applied machine learning algorithms that utilize the extracted phenotypic parameters to identify image‐derived traits that can distinguish control, drought‐treated, and PCZ‐treated plants. We carried out PCZ and drought experiments on a set of BR mutants and Arabidopsis accessions with altered BR responses. Finally, we extended the RoAD assays to perform BR response assays using PCZ in Zea mays (maize) plants. This study establishes an automated and non‐invasive robotic imaging system as a tool to accurately measure morphological and growth‐related traits of Arabidopsis and maize plants in 3D, providing insights into the BR‐mediated control of plant growth and stress responses. [ABSTRACT FROM AUTHOR]

SUMMARY: A cultivation facility that can assist users in controlling the soil water condition is needed for accurately phenotyping plants under drought stress in an artificial environment. Here we report the Internet of Things‐based pot system controlling optional treatment of soil water condition (iPOTs), an automatic irrigation system that mimics the drought condition in a growth chamber. The Wi‐Fi‐enabled iPOTs system allows water supply from the bottom of the pot, based on the soil water level set by the user, and automatically controls the soil water level at a desired depth. The iPOTs also allows users to monitor environmental parameters, such as soil temperature, air temperature, humidity, and light intensity, in each pot. To verify whether the iPOTs mimics the drought condition, we conducted a drought stress test on rice (Oryza sativa L.) varieties and near‐isogenic lines, with diverse root system architecture, using the iPOTs system installed in a growth chamber. Similar to the results of a previous drought stress field trial, the growth of shallow‐rooted rice accessions was severely affected by drought stress compared with that of deep‐rooted accessions. The microclimate data obtained using the iPOTs system increased the accuracy of plant growth evaluation. Transcriptome analysis revealed that pot positions in the growth chamber had little impact on plant growth. Together, these results suggest that the iPOTs system is a reliable platform for phenotyping plants under drought stress. Significance statement: We developed the Internet of Things‐based pot system controlling optional treatment of soil water condition (iPOTs), an automatic irrigation system to mimic the drought condition, for phenotyping plants in a growth chamber. Bottom irrigation allows the user to adjust the soil water level. Aerial and underground sensors in each pot enable the assessment of the effect of microclimate on plant growth. Phenome and transcriptome analyses demonstrated that the iPOTs system is a reliable platform for plant phenotyping under artificial drought stress. [ABSTRACT FROM AUTHOR]

Summary: Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio‐Orthogonal Non‐Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click‐chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15N verified the data. We provide evidence that HPG‐based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)‐based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG‐based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post‐translationally modified residues than the same proteins from whole plant extracts, providing evidence for age‐related accumulation of post‐translational modifications in plants. Significance Statement: Non‐canonical amino acids can be incorporated as proteins are made and enable a sample of newly synthesized proteins to be identified and extracted from plant cells through click chemistry, allowing rapid plant cell responses and the ageing of proteins in vivo to be investigated. We show the non‐canonical amino acid homopropargylglycine enables these aims in Arabidopsis thaliana and its use can enrich newly synthesized proteins with few post‐translational modifications. [ABSTRACT FROM AUTHOR]

Summary: Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole‐genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high‐throughput measurements of meiotic recombination rates in barley pollen nuclei without whole‐genome amplification were performed through a Crystal Digital PCRTM‐based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high‐throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei. Significance Statement: Despite the advantages of measuring meiotic recombination events directly in gametes before fertilization, the number of analyzed samples particularly in large genome species such as barley is limited by relying on a previous template amplification (whole‐genome amplification) step. We present a reliable and rapid method to measure meiotic recombination rates in barley pollen nuclei without previous whole‐genome amplification. High‐throughput genotyping of single barley pollen nuclei is performed by Crystal Digital PCRTM. [ABSTRACT FROM AUTHOR]

Summary: Beyond facilitating transport and providing mechanical support to the leaf, veins have important roles in the performance and productivity of plants and the ecosystem. In recent decades, computational image analysis has accelerated the extraction and quantification of vein traits, benefiting fields of research from agriculture to climatology. However, most of the existing leaf vein image analysis programs have been developed for the reticulate venation found in dicots. Despite the agroeconomic importance of cereal grass crops, like Oryza sativa (rice) and Zea mays (maize), a dedicated image analysis program for the parallel venation found in monocots has yet to be developed. To address the need for an image‐based vein phenotyping tool for model and agronomic grass species, we developed the grass vein image quantification (grasviq) framework. Designed specifically for parallel venation, this framework automatically segments and quantifies vein patterns from images of cleared leaf pieces using classical computer vision techniques. Using image data sets from maize inbred lines and auxin biosynthesis and transport mutants in maize, we demonstrate the utility of grasviq for quantifying important vein traits, including vein density, vein width and interveinal distance. Furthermore, we show that the framework can resolve quantitative differences and identify vein patterning defects, which is advantageous for genetic experiments and mutant screens. We report that grasviq can perform high‐throughput vein quantification, with precision on a par with that of manual quantification. Therefore, we envision that grasviq will be adopted for vein phenomics in maize and other grass species. [ABSTRACT FROM AUTHOR]

Summary: Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole‐genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high‐throughput measurements of meiotic recombination rates in barley pollen nuclei without whole‐genome amplification were performed through a Crystal Digital PCRTM‐based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high‐throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei. Significance Statement: Despite the advantages of measuring meiotic recombination events directly in gametes before fertilization, the number of analyzed samples particularly in large genome species such as barley is limited by relying on a previous template amplification (whole‐genome amplification) step. We present a reliable and rapid method to measure meiotic recombination rates in barley pollen nuclei without previous whole‐genome amplification. High‐throughput genotyping of single barley pollen nuclei is performed by Crystal Digital PCRTM. [ABSTRACT FROM AUTHOR]