Cold Spring Harbor protocols最新文献_第3页

Ethyl Methanesulfonate Treatment of Maize Pollen for Development of Segregating Mutant Populations or Targeted Mutagenesis. 玉米花粉甲基磺酸乙酯处理对分离突变群体发育或定向诱变的影响。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108651

Rajdeep S Khangura, Norman B Best, Brian P Dilkes

{"title":"Ethyl Methanesulfonate Treatment of Maize Pollen for Development of Segregating Mutant Populations or Targeted Mutagenesis.","authors":"Rajdeep S Khangura, Norman B Best, Brian P Dilkes","doi":"10.1101/pdb.prot108651","DOIUrl":"https://doi.org/10.1101/pdb.prot108651","url":null,"abstract":"In maize, abundant pollen production and easy controlled pollination permit the direct mutagenesis of pollen to produce populations of independent mutant lines. Pollen can be treated with alkylating agents, such as ethyl methanesulfonate (EMS), to induce point mutations. The ease of applying and decontaminating this mutagen after the mutagenesis application and the advantages provided by the mutation spectra for subsequent bioinformatic analysis make EMS an attractive mutagen. We provide a maize pollen mutagenesis protocol with a list of critical supplies, a step-by-step procedure, and troubleshooting tips. Pollen is freshly collected and suspended in an emulsion of EMS and paraffin oil. The slurry of pollen, oil, and EMS is then directly placed on prepared maize silks to perform pollinations. Mutations result during embryogenesis due to replication-dependent mispairing at alkylated residues contributed by sperm nuclei. Thus, each seed bears an independent set of mutations. These progenies can be analyzed directly, as is the case in targeted mutagenesis experiments or the exploration of dominant genetic variation. Alternatively, the progenies of self-pollinated plants can be screened in the next generation to discover novel recessive mutations. In addition to the dose of EMS and contact time, the genetic background of maize can significantly influence outcomes, and some optimization of dose and contact time may be required for a genetic background and specific use case. Although we outline good practices for safe handling of EMS and waste, researchers should consult their local safety officers to ensure safe handling, decontamination, and disposal of EMS, which is toxic.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215148","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

Optimized Methods for Applying and Assessing Heat, Drought, and Nutrient Stress of Maize Seedlings in Controlled Environment Experiments. 在受控环境实验中应用和评估玉米幼苗热、旱和养分胁迫的优化方法。

Cold Spring Harbor protocols Pub Date : 2025-06-02 DOI: 10.1101/pdb.top108467

Alejandra Quiñones, Leonardo W Lima, Katherine M Murphy, Anna L Casto, Malia A Gehan, Cory D Hirsch

{"title":"Optimized Methods for Applying and Assessing Heat, Drought, and Nutrient Stress of Maize Seedlings in Controlled Environment Experiments.","authors":"Alejandra Quiñones, Leonardo W Lima, Katherine M Murphy, Anna L Casto, Malia A Gehan, Cory D Hirsch","doi":"10.1101/pdb.top108467","DOIUrl":"10.1101/pdb.top108467","url":null,"abstract":"Maize (Zea mays), also known as corn, is an important crop that plays a crucial role in global agriculture. The economic uses of maize are numerous, including for food, feed, fiber, and fuel. It has had a significant historical importance in research as well, with important discoveries made in maize regarding plant domestication, transposons, heterosis, genomics, and epigenetics. Unfortunately, environmental stresses cause substantial yield loss to maize crops each year. Yield losses are predicted to increase in future climate scenarios, posing a threat to food security and other sectors of the global economy. Developing efficient methods to study maize abiotic stress responses is a crucial step toward a more resilient and productive agricultural system. This review describes the importance of and methods for studying the effects of heat, drought, and nutrient deficiency on early developmental stages of maize grown in controlled environments. Studying the early effects of environmental stressors in controlled environments allows researchers to work with a variety of environmental conditions with low environmental variance, which can inform future field-based research. We highlight the current knowledge of physiological responses of maize to heat, drought, and nutrient stress; remaining knowledge gaps and challenges; and information on how standardized protocols can address these issues.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108467"},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459804","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

Maize Abiotic Stress Treatments in Controlled Environments. 受控环境中的玉米非生物胁迫处理。

Cold Spring Harbor protocols Pub Date : 2025-06-02 DOI: 10.1101/pdb.prot108620

Katherine M Murphy, Anna L Casto, Leonardo Chavez, Leonardo W Lima, Alejandra Quiñones, Malia A Gehan, Cory D Hirsch

{"title":"Maize Abiotic Stress Treatments in Controlled Environments.","authors":"Katherine M Murphy, Anna L Casto, Leonardo Chavez, Leonardo W Lima, Alejandra Quiñones, Malia A Gehan, Cory D Hirsch","doi":"10.1101/pdb.prot108620","DOIUrl":"10.1101/pdb.prot108620","url":null,"abstract":"Maize (Zea mays) is one of the world's most important crops, providing food for humans and livestock and serving as a bioenergy source. Climate change and the resulting abiotic stressors in the field reduce crop yields, threatening food security and the global economy. Water deficit (i.e., drought), heat, and insufficient nutrients (e.g., nitrogen and phosphorus) are major environmental stressors that affect maize yields, and impact growth and development at all stages of the plant life cycle. Understanding the biological processes underlying these responses in maize has the potential to increase yields in the face of abiotic stress. Optimizing individual or combined abiotic stress treatments in controlled environments reduces potential noise in data collection that can be present under less controlled growth conditions. Here, we describe methods and conditions for controlled abiotic stress treatments and associated controls during early vegetative growth of maize, conducted in greenhouses or growth chambers. This includes the environmental conditions, equipment, soil preparation, and intensity and duration of heat, drought, nitrogen deficiency, and phosphorous deficiency. Controlled experiments at early growth stages are informative for future in-field studies that require greater labor and inputs, saving researchers time and growing space, and thus research funds, before testing plants across later stages of development. We suggest that stress treatments be severe enough to result in a measurable phenotype, but not so severe that all plants die prior to sample collection. This protocol is designed to set important standards for replicable research in maize.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108620"},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459803","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

Measuring the Readily Releasable Synaptic Vesicle Pool at the Drosophila Larval Neuromuscular Junction. 测量果蝇幼虫神经肌肉接头处可随时释放的突触小泡库

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.prot108425

Tingting Wang, C Andrew Frank

{"title":"Measuring the Readily Releasable Synaptic Vesicle Pool at the Drosophila Larval Neuromuscular Junction.","authors":"Tingting Wang, C Andrew Frank","doi":"10.1101/pdb.prot108425","DOIUrl":"10.1101/pdb.prot108425","url":null,"abstract":"Presynaptic homeostatic potentiation (PHP) is a type of homeostatic regulation that stabilizes synaptic output under conditions where postsynaptic receptor function is impaired. PHP manifests as a significant increase in presynaptic neurotransmitter release, compensating for decreased postsynaptic receptor activity and thus maintaining stable excitation levels in postsynaptic cells. Presynaptic neurotransmitter release is calcium-dependent, initiated by calcium influx through voltage-gated calcium channels localized at the presynaptic active zones. This calcium influx triggers the fusion of vesicles from the readily releasable vesicle pool (RRP) that are ready for immediate release. Two key presynaptic cellular mechanisms are essential for PHP's induction and maintenance. First, a compensatory rise in the abundance of presynaptic calcium channels (and consequently, an increase in calcium influx) occurs when postsynaptic glutamate receptors are suppressed. Second, the RRP size enlarges during PHP. PHP is disrupted if either of these processes is impaired. This protocol outlines the use of the two-electrode voltage-clamp technique for assessing the RRP during PHP, induced either pharmacologically or genetically, at the Drosophila neuromuscular junction (NMJ). Electrophysiological recordings typically take place at the NMJ of muscle 6 in abdominal segments A2 and A3.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108425"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522016/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140848078","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

Measuring Presynaptic Calcium Influx at the Drosophila Larval Neuromuscular Junction. 测量果蝇幼虫神经肌肉接头处的突触前钙离子流入量

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.prot108426

Tingting Wang, C Andrew Frank

{"title":"Measuring Presynaptic Calcium Influx at the Drosophila Larval Neuromuscular Junction.","authors":"Tingting Wang, C Andrew Frank","doi":"10.1101/pdb.prot108426","DOIUrl":"10.1101/pdb.prot108426","url":null,"abstract":"Synaptic transmission plays a critical role in information processing and storage within the nervous system. The triggering of action potentials activates voltage-gated calcium channels at presynaptic active zones, facilitating the calcium-dependent release of synaptic vesicles. Homeostatic mechanisms are crucial in stabilizing synaptic function. At the Drosophila neuromuscular junction, a compensatory increase in presynaptic neurotransmitter release occurs when postsynaptic glutamate receptor function is pharmacologically or genetically impaired, thereby stabilizing synaptic output. This adaptation is known as presynaptic homeostatic potentiation (PHP). Recent advancements, including confocal and super-resolution imaging techniques, have demonstrated an increase in presynaptic calcium influx during both the rapid induction and long-term maintenance of PHP. These observations indicate that the abundance and structural organization of presynaptic calcium channels, along with various active zone components, undergo modifications following the suppression of postsynaptic glutamate receptors. Such findings underscore the critical roles of trafficking and stabilization of presynaptic calcium channels and active zone proteins in homeostatic plasticity. This protocol describes using calcium indicators and confocal imaging methods to measure single-action potential-evoked presynaptic calcium influx during PHP.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108426"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522019/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140862359","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 Total Anthocyanins, Determination of Anthocyanidin Core Structures, and Characterization of Specific Anthocyanins from Maize. 提取和定量总花青素、确定花青素核心结构以及鉴定玉米中的特定花青素。

Cold Spring Harbor protocols Pub Date : 2025-05-05 DOI: 10.1101/pdb.prot108577

Nan Jiang, Lina Gomez, Erich Grotewold

{"title":"Extraction and Quantification of Total Anthocyanins, Determination of Anthocyanidin Core Structures, and Characterization of Specific Anthocyanins from Maize.","authors":"Nan Jiang, Lina Gomez, Erich Grotewold","doi":"10.1101/pdb.prot108577","DOIUrl":"10.1101/pdb.prot108577","url":null,"abstract":"Anthocyanins are flavonoid pigments that accumulate in fruits and flowers that serve as attractants for pollinators and seed-dispersing organisms. Anthocyanins exhibit diverse chemical structures, characterized both by different anthocyanidin core structures and numerous chemical modifications of the anthocyanidin core. Here, we describe a protocol for the extraction and quantification of total anthocyanins, as well as for the characterization of anthocyanidin core structures and specific anthocyanins, using a spectrophotometer, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-two-dimensional mass spectrometry (UHPLC-MS/MS). The method involves anthocyanin extraction using acidic methanol, anthocyanin quantification using a spectrophotometer, determination of anthocyanidin core structure from hydrolyzed anthocyanin extracts using UHPLC-MS/MS, separation of different anthocyanins using HPLC, and characterization of specific anthocyanins using UHPLC-MS/MS. As an example, we describe how we have used this protocol to extract and quantify total anthocyanins from maize leaves, identify cyanidin as the core anthocyanidin structure, and characterize three specific anthocyanins that accumulate in maize leaves, each having a cyanidin core with decorations of a hexose group, and a malonyl or coumaroyl moiety.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108577"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142072224","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

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