Cold Spring Harbor protocols最新文献

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

Corrigendum: Agrobacterium-Mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants.

Cold Spring Harbor protocols Pub Date : 2025-03-20 DOI: 10.1101/pdb.corr108644

Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang

引用次数: 0

Agrobacterium-Mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants. 利用农杆菌介导的热带玉米幼苗叶轮外植体转化。

Cold Spring Harbor protocols Pub Date : 2025-03-20 DOI: 10.1101/pdb.prot108596

Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang

{"title":"Agrobacterium-Mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants.","authors":"Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang","doi":"10.1101/pdb.prot108596","DOIUrl":"10.1101/pdb.prot108596","url":null,"abstract":"Conventional maize transformation has largely relied on immature embryos as explants, and is thus often hampered by the limited access to high-quality immature embryos year-round. Here, we present a detailed protocol using seedling leaf whorls as alternative explants for tropical maize inbred transformation. This approach involves the use of a cassette that drives the expression of the morphogenic transcription factors (MTFs) Baby boom (Bbm) and Wuschel2 (Wus2), which have been shown to greatly enhance transformation efficiency. We outline here the steps required for the preparation of seedling leaf whorl explants and subsequent Agrobacterium infection, and describe the tissue culture regimen that results in transgenic plant regeneration. Because constitutive expression of Bbm and Wus2 prevents normal plant regeneration and the production of fertile plants, the cassette containing these genes must be excised. As such, we include the steps for the Cre/loxP-mediated excision of the MTF gene cassette. The protocol outlines a year-round, more affordable, and efficient approach for carrying out maize transformation for crop improvement.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281403","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

Building AuxInYeast Synthetic Biology Strains for Biochemical Characterization of Maize Auxin Hormone Signaling Components.

Cold Spring Harbor protocols Pub Date : 2025-03-12 DOI: 10.1101/pdb.prot108634

Román Ramos Báez, Amy Lanctot, Britney L Moss

{"title":"Building AuxInYeast Synthetic Biology Strains for Biochemical Characterization of Maize Auxin Hormone Signaling Components.","authors":"Román Ramos Báez, Amy Lanctot, Britney L Moss","doi":"10.1101/pdb.prot108634","DOIUrl":"https://doi.org/10.1101/pdb.prot108634","url":null,"abstract":"The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the plant auxin hormone signaling pathway. As a plant synthetic biology chassis, Saccharomyces cerevisiae yeast offers rapid growth, well-established genetic and biochemical tools, and core eukaryotic cellular machinery compatible with heterologous plant gene expression. The AuxInYeast system for maize consists of yeast cells containing the minimal necessary set of plant auxin signaling parts: a receptor (ZmTIR1/AFB), repressor (ZmIAA), corepressor (REL2), transcription factor (ZmARF), and auxin response cis-element (auxRE). In plants, auxin binding to a receptor:repressor complex triggers ubiquitination and degradation of the repressor, preventing it from binding to transcription factors on auxin response elements. Thus, auxin-induced repressor degradation allows for the activation of auxin-inducible transcriptional responses. Tagging various auxin signaling components with fluorescent protein reporters then enables quantitative measurement of signaling dynamics via high-throughput approaches such as flow cytometry. As these signaling proteins each belongs to large gene families, AuxInYeast users can build strains with defined components to study their behaviors in isolation or various combinations. Such strains enable researchers to dissect auxin sensitivity, the dynamics of auxin repressor degradation and transcriptional activation, and promoter architecture. It also allows a head-to-head comparison of maize components with orthologs from other plant species to test the evolutionary conservation of component interactions. This protocol describes the construction of such strains. Finally, this protocol and the AuxInYeast approach can also be adapted to assay other multicomponent maize biochemical pathways in yeast.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613786","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

Synthetic Biology Approaches to Study Maize Signaling Pathways.

Cold Spring Harbor protocols Pub Date : 2025-03-12 DOI: 10.1101/pdb.top108450

Amy Lanctot, Román Ramos Báez, Britney L Moss

{"title":"Synthetic Biology Approaches to Study Maize Signaling Pathways.","authors":"Amy Lanctot, Román Ramos Báez, Britney L Moss","doi":"10.1101/pdb.top108450","DOIUrl":"https://doi.org/10.1101/pdb.top108450","url":null,"abstract":"Synthetic biology approaches merge the tenets of engineering with established biological techniques to answer fundamental questions about living systems and to engineer biological forms and functions. Following the engineering principle of design-build-test-iterate, this review serves as a guide to applying synthetic principles and approaches in maize. We outline strategies for (1) choosing the optimal model organism to serve as a heterologous chassis for maize signaling pathways, (2) designing and building biological parts and devices to express pathway components, (3) choosing an analytical technique to measure pathway function, and (4) optimizing and troubleshooting the designed system. Auxin is a hormone that is essential for plant growth and development, regulating cellular proliferation and differentiation. Considering the importance of auxin for maize development in aerial and underground tissue, it was an obvious starting point for synthetic biology approaches. We use the maize nuclear auxin response recapitulated in yeast (AuxInYeast) system to showcase the power of heterologous expression approaches for testing fundamental attributes of the evolution, genetics, and biochemistry of signaling pathways that may be challenging to assay in planta. This approach involves co-expression of maize auxin signaling components in Saccharomyces cerevisiae coupled with fluorescence flow cytometry to quantify signaling activity. We and others have used this system to interrogate the dynamics of pathway signaling, interactions between paralogous components, and the adaptation of auxin signaling over large evolutionary distances. Thus, the AuxInYeast system is a fast, high-throughput, hypothesis-generating platform that can be readily adapted by the maize community to creatively answer questions about fundamental maize biology and to drive development of novel tools for breeding and plant engineering.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613787","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

Testing AuxInYeast Synthetic Biology Strains via Fluorescence Flow Cytometry.

Cold Spring Harbor protocols Pub Date : 2025-03-12 DOI: 10.1101/pdb.prot108635

Britney L Moss, Amy Lanctot, Román Ramos Báez

{"title":"Testing AuxInYeast Synthetic Biology Strains via Fluorescence Flow Cytometry.","authors":"Britney L Moss, Amy Lanctot, Román Ramos Báez","doi":"10.1101/pdb.prot108635","DOIUrl":"https://doi.org/10.1101/pdb.prot108635","url":null,"abstract":"Understanding how the auxin hormone signaling pathway components come together to orchestrate cellular responses is key to engineering the growth and development of maize. Although a variety of techniques exist to measure auxin activities in plants, many are time- and resource-intensive or do not easily allow for high-throughput quantitative measurement of component libraries. The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the auxin hormone signaling pathway from essentially any plant. AuxInYeast uses Saccharomyces cerevisiae yeast as a heterologous expression platform for auxin signaling pathway components with fluorescent tags that facilitate measurement of auxin perception, repression, and activation. This protocol describes how to use fluorescence flow cytometry for these AuxInYeast experiments. As a case study, we focus on AuxInYeast strains built to measure maize auxin perception (i.e., those that express receptors and fluorescently tagged repressors that degrade upon auxin exposure). This protocol describes two different types of cytometry assays. The Steady-State Assay measures the extent of auxin-induced repressor degradation at one or two time points across many AuxInYeast strains and is particularly useful for initial assessment of whether auxin-induced degradation occurs and for dose response assays. The Time-Course Assay is used to measure auxin-induced repressor degradation dynamics over 2-3 h in a smaller number of strains. It is most useful for assessing the range of degradation rates across sets of repressors or receptors, and to precisely determine the impact of mutations and natural variation on degradation rate.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613789","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