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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]

• 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]

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 (MS 2 ) 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., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)