Cold Spring Harbor protocols最新文献_第4页

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

Assaying Nociception Behaviors in Drosophila Larvae During Parasitoid Wasp Attacks. 测定寄生蜂攻击果蝇幼虫时的痛觉行为

Cold Spring Harbor protocols Pub Date : 2025-04-01 DOI: 10.1101/pdb.prot108129

Stephanie E Mauthner, Lydia J Borjon, W Daniel Tracey

{"title":"Assaying Nociception Behaviors in Drosophila Larvae During Parasitoid Wasp Attacks.","authors":"Stephanie E Mauthner, Lydia J Borjon, W Daniel Tracey","doi":"10.1101/pdb.prot108129","DOIUrl":"10.1101/pdb.prot108129","url":null,"abstract":"Nociception in fruit fly (Drosophila melanogaster) larvae is characterized by a stereotyped escape behavior. When a larva encounters a noxious (potentially harmful) stimulus, it responds by curving its body into a c-shape and rolling in a corkscrew-like manner around its long-body axis. This rolling behavior may serve to quickly remove the larva from the source of the noxious stimulus, and is particularly adaptive to escape from a common natural predator of fruit fly larvae: parasitoid wasps (Leptopilina boulardi). L. boulardi completes its life cycle by using fruit fly larvae as hosts for its offspring. Female wasps sting fly larvae with an ovipositor and lay an egg within the larva. The wasp offspring hatches inside the fly larva, consumes the fly tissues during pupation, and eventually emerges from the pupal case as an adult wasp. Fruit fly larvae respond to oviposition attacks by rolling, which causes the long flexible ovipositor to be wound around the larval body like a spool. This dislodges the wasp and allows the larva to attempt to escape by crawling. Rolling behavior is triggered by the activation of sensory neurons (nociceptors) whose function can inform our understanding of the mechanisms of nociception. In this protocol, we describe a simple behavioral assay to test and measure nociceptive responses in Drosophila larvae during oviposition attacks by female parasitoid wasps. First, we discuss parasitoid wasp husbandry and culturing methods in the laboratory. We then describe how to perform the wasp nociception assay on third-instar fruit fly larvae.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108129"},"PeriodicalIF":0.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11787398/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878472","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

Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae. 光遗传刺激果蝇幼虫的痛觉逃逸行为

Cold Spring Harbor protocols Pub Date : 2025-04-01 DOI: 10.1101/pdb.prot108128

Stephanie E Mauthner, W Daniel Tracey

{"title":"Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae.","authors":"Stephanie E Mauthner, W Daniel Tracey","doi":"10.1101/pdb.prot108128","DOIUrl":"10.1101/pdb.prot108128","url":null,"abstract":"In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108128"},"PeriodicalIF":0.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11787400/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878475","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

Mechanical Nociception Assay in Drosophila Larvae. 果蝇幼虫的机械痛觉试验

Cold Spring Harbor protocols Pub Date : 2025-04-01 DOI: 10.1101/pdb.prot108125

Stephanie E Mauthner, W Daniel Tracey

{"title":"Mechanical Nociception Assay in Drosophila Larvae.","authors":"Stephanie E Mauthner, W Daniel Tracey","doi":"10.1101/pdb.prot108125","DOIUrl":"10.1101/pdb.prot108125","url":null,"abstract":"The nervous system of animals can sense and respond to noxious stimuli, which include noxious thermal, chemical, or mechanical stimuli, through a process called nociception. Here, we describe a simple behavioral assay to measure mechanically induced nociceptive responses in Drosophila larvae. This assay tests larval mechanosensitivity to noxious force with calibrated von Frey filaments. First, we explain how to construct and calibrate the customizable von Frey filaments that can be used to deliver reproducible stimuli of a defined force or pressure. Next, we describe how to perform the mechanical nociception assay on third-instar larvae. Through comparison of the responses of genotypes of interest, this assay can be useful for investigation of molecular, cellular, and circuit mechanisms of mechanical nociception. At the molecular level, prior studies have identified the importance of sensory ion channels such as Pickpocket/Balboa, Piezo, dTRPA1, and Painless. At the cellular level, the class IV multidendritic arborizing (md-da) neurons are the main mechanical nociceptor neurons of the peripheral system, but class III and class II md-da have been found to also play a role. At the circuit level, studies have shown that mechanical nociception relies on interneurons of the abdominal ganglia that integrate inputs from these various md-da neuron classes.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108125"},"PeriodicalIF":0.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11787401/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878473","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

Nociception in Drosophila Larvae. 果蝇幼虫的痛觉感应

Cold Spring Harbor protocols Pub Date : 2025-04-01 DOI: 10.1101/pdb.top108172

Lydia J Borjon, Stephanie E Mauthner, W Daniel Tracey

{"title":"Nociception in Drosophila Larvae.","authors":"Lydia J Borjon, Stephanie E Mauthner, W Daniel Tracey","doi":"10.1101/pdb.top108172","DOIUrl":"10.1101/pdb.top108172","url":null,"abstract":"Nociception is the sensory modality by which animals sense stimuli associated with injury or potential tissue damage. When Drosophila larvae encounter a noxious thermal, chemical, or mechanical stimulus, they perform a stereotyped rolling behavior. These noxious stimuli are detected by polymodal nociceptor neurons that tile the larval epidermis. Although several types of sensory neurons feed into the nociceptive behavioral output, the highly branched class IV multidendritic arborization neurons are the most critical. At the molecular level, Drosophila nociception shares many conserved features with vertebrate nociception, making it a useful organism for medically relevant research in this area. Here, we review three larval assays for nociceptive behavior using mechanical stimuli, optogenetic activation, and the naturalistic stimuli of parasitoid wasp attacks. Together, the assays described have been successfully used by many laboratories in studies of the molecular, cellular, and circuit mechanisms of nociception. In addition, the simple nature of the assays we describe can be useful in teaching laboratories for undergraduate students.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108172"},"PeriodicalIF":0.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11787404/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878474","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