Cold Spring Harbor protocols最新文献_第6页

Flavonoids and Derived Anthocyanin Pigments in Plants-Structure, Distribution, Function, and Methods for Quantification and Characterization. 植物中的类黄酮和衍生花青素色素--结构、分布、功能以及定量和表征方法。

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.top108516

Nan Jiang, Erich Grotewold

引用次数: 0

Eliciting Presynaptic Homeostatic Potentiation at the Drosophila Larval Neuromuscular Junction. 在果蝇幼虫神经肌肉接头处诱发突触前稳态电位。

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.prot108424

Tingting Wang, C Andrew Frank

{"title":"Eliciting Presynaptic Homeostatic Potentiation at the Drosophila Larval Neuromuscular Junction.","authors":"Tingting Wang, C Andrew Frank","doi":"10.1101/pdb.prot108424","DOIUrl":"10.1101/pdb.prot108424","url":null,"abstract":"The Drosophila melanogaster neuromuscular junction (NMJ) is an easily accessible synapse and an excellent model for understanding synapse development, function, and plasticity. A form of plasticity called presynaptic homeostatic potentiation (PHP) operates at the NMJ and keeps synapse excitation levels stable. PHP can be induced rapidly in 10 min by application of a pharmacological antagonist of glutamate receptors (philanthotoxin-433) or chronically by deletion of the gene encoding the postsynaptic glutamate receptor subunit GluRIIA. To assess PHP, electrophysiological recordings of spontaneous miniature excitatory postsynaptic potentials and evoked excitatory postsynaptic potentials are usually performed at the NMJ of muscle 6 at abdominal segments A2 and A3. This protocol describes steps for larval dissection to access the NMJ, use of mutant lines to assess PHP, application of philanthotoxin-433 to the NMJ, and electrophysiological recordings following drug application. Collectively, these steps allow for analysis of the acute induction and expression of PHP. Recording chamber preparation, electrophysiology rig setup, larval dissection, and current clamp recording steps have been described elsewhere.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108424"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522017/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140852892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Using Electrophysiology to Study Homeostatic Plasticity at the Drosophila Neuromuscular Junction. 利用电生理学研究果蝇神经肌肉接头处的同态可塑性

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.top108393

Tingting Wang, C Andrew Frank

{"title":"Using Electrophysiology to Study Homeostatic Plasticity at the Drosophila Neuromuscular Junction.","authors":"Tingting Wang, C Andrew Frank","doi":"10.1101/pdb.top108393","DOIUrl":"10.1101/pdb.top108393","url":null,"abstract":"The Drosophila melanogaster neuromuscular junction (NMJ) is a superb system for studying synapse function. Beyond that, the NMJ is also great for studying forms of synaptic plasticity. Over the last 25 years, Drosophila NMJ neuroscientists have pioneered understanding of a form of plasticity called homeostatic synaptic plasticity, which imparts functional stability on synaptic connections. The reason is straightforward: The NMJ has a robust capacity for stability. Moreover, many strategies that the NMJ uses to maintain appropriate levels of function are mirrored at other metazoan synapses. Here, we introduce core approaches that neurophysiologists use to study homeostatic synaptic plasticity at the peripheral Drosophila NMJ. We focus on methods to study a specific form of homeostatic plasticity termed presynaptic homeostatic potentiation (PHP), which is the most well-characterized one. Other forms such as presynaptic homeostatic depression and developmental forms of homeostasis are briefly discussed. Finally, we share lists of several dozen factors and conditions known to influence the execution of PHP.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108393"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522024/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Extraction and Quantification of Phenolic Compounds in Maize. 玉米中酚类化合物的提取与定量

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.prot108576

Lina Gomez, Nan Jiang, Erich Grotewold

{"title":"Extraction and Quantification of Phenolic Compounds in Maize.","authors":"Lina Gomez, Nan Jiang, Erich Grotewold","doi":"10.1101/pdb.prot108576","DOIUrl":"10.1101/pdb.prot108576","url":null,"abstract":"Plants accumulate hundreds of thousands of specialized metabolites that participate in their interactions with the environment. Among these compounds, phenolics represent a large class, and they play important physiological roles, such as providing a first barrier against pathogens, cues to pollinators, and radiation protection. Maize is one of the most important crops worldwide for food, animal feed, and biofuels, and it has the potential to accumulate different phenolics in vegetative tissues as well as in seeds. Recent studies have identified a large number of phenolic compounds-with a diversity of chemical decorations-in different maize tissues, but these likely represent just a fraction of the metabolic diversity of maize. In this protocol, we describe a specific method for the extraction and quantification of maize phenolic compounds by ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM-MS/MS) analysis. We provide detailed instructions for the extraction of phenolics using acidic methanol, and for the quantification of 33 different compounds in maize stems, including flavonoids, phenolic acids, and lignin precursors.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108576"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142072223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Single-Cell and Spatial Transcriptomic Analysis of Maize Embryo Development. 玉米胚发育的单细胞和空间转录组学分析。

Cold Spring Harbor protocols Pub Date : 2025-04-23 DOI: 10.1101/pdb.top108468

Hao Wu, Michael J Scanlon

{"title":"Single-Cell and Spatial Transcriptomic Analysis of Maize Embryo Development.","authors":"Hao Wu, Michael J Scanlon","doi":"10.1101/pdb.top108468","DOIUrl":"https://doi.org/10.1101/pdb.top108468","url":null,"abstract":"Plant embryogenesis encompasses the biological processes wherein the zygote (fertilized egg) undergoes cell division, cell expansion, and cell differentiation to develop histological tissue layers, meristems, and various organs comprising the primordial body plan of the organism. Studies of embryogenesis in the agronomically important maize crop advance our understanding of the fundamental mechanism of plant development, which, upon translation, may advance agronomic improvement, optimization of conditions for somatic embryogenesis, and plant synthetic biology. Maize embryo development is coordinated temporally and spatially and is regulated by interactive genetic networks. Single-cell RNA sequencing (RNA-seq) and spatial transcriptomics are powerful tools to examine gene expression patterns and regulatory networks at single-cell resolution and in a spatial context, respectively. Single-cell technology enables profiling of three-dimensional samples with high cellular resolution, but it can be difficult to identify specific cell clusters due to a lack of known markers in most plant species. In contrast, spatial transcriptomics provide transcriptomic profiling of discrete regions within a sectioned, two-dimensional sample, although single-cell resolution is typically not obtained and fewer transcripts per cell are detected than in single-cell RNA-seq. In this review, we describe the combined use of these two transcriptomic strategies to study maize embryogenesis with synergistic results.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955290","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Single-Cell and Spatial Transcriptomic Analysis of Maize Embryo Development: a Sample Preparation Protocol. 玉米胚发育的单细胞和空间转录组学分析：样品制备方案。

Cold Spring Harbor protocols Pub Date : 2025-04-23 DOI: 10.1101/pdb.prot108645

Hao Wu, Michael J Scanlon

{"title":"Single-Cell and Spatial Transcriptomic Analysis of Maize Embryo Development: a Sample Preparation Protocol.","authors":"Hao Wu, Michael J Scanlon","doi":"10.1101/pdb.prot108645","DOIUrl":"https://doi.org/10.1101/pdb.prot108645","url":null,"abstract":"Maize is an important crop that contributes to the modern economy in various ways, including use for human consumption, as animal feed, and in industrial products. Research on maize is crucial for understanding plant development, which in turn provides valuable insight into improvement of maize crops to meet the food demands of a growing population. Maize embryogenesis, which is the primordial stage of the corn life cycle, determines the fundamental body plan and developmental programs that organize the tissue patterning and subsequent growth and reproduction of the corn plant. Investigating maize embryogenesis at high cellular resolution can enhance our understanding of the homology, ontogeny, and developmental genetic mechanisms of embryonic organ morphogenesis. However, until recently, no published studies have used methods for analyzing maize embryo development at single-cell resolution. This protocol describes single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic analyses, which are powerful, combinatorial tools that can be used to study maize embryogenesis at the single-cell level within a spatial context. These tools have the power to reveal transcriptomic relationships between tissues/organs, and to provide insight into the gene regulatory networks operating during embryogenesis. In this protocol, we describe a detailed procedure to prepare maize embryo samples for construction of scRNA-seq and Visium spatial transcriptomic libraries that are suitable for massively parallel sequencing. Our protocol borrows from prior published studies and manufacturer's instructions and is optimized for studies of the maize embryo.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143986133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sterol and Brassinosteroid Hormone Quantification by LC/MS of Picolinyl Ester Derivatives. Picolinyl酯衍生物的LC/MS定量分析甾醇和油菜素类固醇激素。

Cold Spring Harbor protocols Pub Date : 2025-04-17 DOI: 10.1101/pdb.prot108646

Brian P Dilkes, Norman B Best

引用次数: 0

Analysis of Polar and Nonpolar Small Plant Growth Hormones and Quantification by LC/MS. 极性和非极性小型植物生长激素的分析及LC/MS定量。

Cold Spring Harbor protocols Pub Date : 2025-04-17 DOI: 10.1101/pdb.prot108647

Brian P Dilkes, Norman B Best

{"title":"Analysis of Polar and Nonpolar Small Plant Growth Hormones and Quantification by LC/MS.","authors":"Brian P Dilkes, Norman B Best","doi":"10.1101/pdb.prot108647","DOIUrl":"https://doi.org/10.1101/pdb.prot108647","url":null,"abstract":"Plant hormones are small metabolites that regulate all aspects of plant growth and development, including plant defense. The detection and quantification of these hormones are critical to understanding the mechanism of growth regulation in plants. In maize, a wealth of genetic resources has enabled progress on elucidating the genetic mechanisms underlying plant growth. Biochemical studies of growth in maize can provide insight into the physiological mechanisms of growth control by measuring endogenous levels of plant hormones, and this knowledge would be enhanced by the development of a method to analyze several hormones in a single small sample of tissue. We provide here a simple protocol to extract and accurately quantify six classes of plant hormones in a single liquid chromatography/mass spectrometry injection run using maize tissues. Those hormones include abscisic acid (ABA), 1-aminocyclopropane-1-carboxylate (ACC), gibberellic acid (GA), 3-indoleacetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA), as well as an accumulated phytoanticipin of maize, 24-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which influences the levels of IAA.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143968675","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Metabolite Profiling of Growth Regulatory Hormones from Maize Tissue. 玉米组织中生长调节激素的代谢谱分析。

Cold Spring Harbor protocols Pub Date : 2025-04-17 DOI: 10.1101/pdb.top108432

Brian P Dilkes, Norman B Best

{"title":"Metabolite Profiling of Growth Regulatory Hormones from Maize Tissue.","authors":"Brian P Dilkes, Norman B Best","doi":"10.1101/pdb.top108432","DOIUrl":"https://doi.org/10.1101/pdb.top108432","url":null,"abstract":"Plant regulatory small molecules, or phytohormones, are small regulatory metabolites in plants. Phytohormones regulate all aspects of plant growth and development. They include jasmonic acid, auxin, abscisic acid, salicylic acid, 1-aminocyclopropane-1-carboxylic acid, gibberellins, and brassinosteroids. Their activity is highly dependent on their concentration, and therefore accurate quantification is necessary to understand their biological role in regulating downstream targets. However, their low abundance results in low signal to noise ratios during detection. In addition, the chemical distinctions between the regulatory small molecule classes include a wide polarity range and differences in charge, which has previously prevented the simultaneous extraction and separation by chromatography of multiple regulatory small molecules. This review discusses the extraction of hormones from any maize tissue, followed by their purification and quantification, and the limitations of these approaches. Recent advancements in mass spectrometry and sample pretreatment have improved the sensitivity of techniques to accurately and simultaneously quantify multiple small regulatory plant hormones from maize tissue. These techniques should usher in a new era in which measurement of phytohormones will allow for more accurate evaluation of phytohormone roles in maize growth and development. We also highlight potential new plant regulatory hormones and discuss how the techniques described here may benefit future discovery of new classes of phytohormones.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143991570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Compositional Analysis of Cutin in Maize Leaves. 玉米叶中 Cutin 的成分分析

Cold Spring Harbor protocols Pub Date : 2025-04-01 DOI: 10.1101/pdb.prot108434

Richard Bourgault, Isabel Molina

{"title":"Compositional Analysis of Cutin in Maize Leaves.","authors":"Richard Bourgault, Isabel Molina","doi":"10.1101/pdb.prot108434","DOIUrl":"10.1101/pdb.prot108434","url":null,"abstract":"The cuticle is a lipid barrier that covers the air-exposed surfaces of plants. It consists of waxes and cutin, a cell wall-attached lipid polyester of oxygenated fatty acids and glycerol. Unlike waxes, cutin is insoluble in organic solvents, and its composition is typically studied by chemical depolymerization followed by monomer analysis by gas chromatography (GC). Here, we describe a method for the chemical depolymerization of cutin in maize leaves and subsequent compositional analysis of the constituent lipid monomers. The method has been adapted from protocols for cutin analysis developed for Arabidopsis, by both optimizing the amount of leaf tissue used and including a data analysis process specific to the monomers present in maize cutin. The approach uses base-catalyzed transmethylation, which produces fatty acid methyl esters, and silylation, which gives trimethylsilyl ether derivatives of hydroxyl groups for gas chromatographic analysis. For monomer identification, a few representative samples are first analyzed by GC-mass spectrometry (GC-MS). This is then followed by analysis of all replicates by gas chromatography coupled to a flame ionization detector (GC-FID) for monomer quantification, because the flame ionization detector provides a linear response over a wide mass range, is relatively simple to operate, and is more cost-effective to maintain compared to mass spectrometry detectors. Although the protocol bypasses time-consuming cuticle isolation steps by using whole-leaf samples, this means that a fraction of the compounds in the chromatographic profiles do not derive from cutin. Accordingly, we discuss some considerations for the interpretation of the resulting depolymerization products. Our protocol offers specific guidance on preparing maize leaf samples, ensuring reproducible results, and enabling the detection of subtle variations in cutin monomer composition among plant genotypes or developmental stages.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108434"},"PeriodicalIF":0.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141970789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0