Cold Spring Harbor protocols最新文献

Seed mutagenesis using alkylating chemical agents such as ethyl methanesulfonate (EMS) can generate somatic and germinal mutations in many plant species. In monoecious plants like maize, the sperm- and egg-producing reproductive germlines are derived from distinct cell lineages in the embryo. This separation results in independent mutations inherited via the egg and sperm lineages and prevents the recovery of recessive mutant phenotypes in diploid progeny after the first round of self-pollination. Thus, two generations of self-pollination are required to screen for recessive mutations when conducting seed mutagenesis. The additional time and manual self-pollination make this approach laborious. However, a high mutation rate and the ability to screen for somatic sectors in heterozygous mutant plants and other defined genetic backgrounds make seed mutagenesis an effective but underutilized mutagenesis tool for maize research. This protocol provides the directions and optimization steps to perform effective seed mutagenesis in maize. A high frequency of somatic mutations from seed mutagenesis can be achieved, but comes at the expense of poor and disordered growth, failure to form reproductive structures, and low or no seed production at high EMS concentrations or long contact times. In experiments where germinal mutations are a goal, an optimum dose of EMS is required in the first generation. Maize genetic backgrounds vary in their sensitivity to EMS, requiring some pilot testing in new genetic backgrounds. Researchers using this protocol can carry out seed mutagenesis safely and effectively to develop libraries of mutants or alleles for various experiments.

Maize is a globally important staple that is used as food for human and animal consumption, fuel, and other industrial applications. Pathogens affect all stages of the plant life cycle and every plant organ, and lead to significant yield losses. An integrated strategy incorporating cultural and chemical management practices, as well as development of resistant plant varieties, is needed to prevent yield losses due to plant diseases. Large numbers of breeding material must be screened to develop pathogen-resistant maize varieties. Inoculation methods must be high-throughput to accommodate the large screening experiments. Additionally, there needs to be an extensive understanding of the plant-pathogen interaction to use a targeted biotechnology-based approach, which takes advantage of knowledge of the system to engineer resistance. To evaluate germplasm for breeding and biotechnology approaches, inoculation methods must replicate natural infection, and disease severity must be rated consistently to accurately screen germplasm or gather data on pathogens of interest. Here, we review inoculation and rating methods for Gibberella ear rot, seedling blight caused by Globisporangium ultimum var. ultimum, and Goss's wilt that are efficient and high-throughput. We also introduce fluorescence microscopy techniques for leaf samples infected with Exserohilum turcicum, the causal agent of northern corn leaf blight. These pathogens all cause significant yield losses, and in particular, Gibberella ear rot is associated with the accumulation of harmful mycotoxins. Understanding how pathogens cause disease and how plants defend against attack is a major goal of maize pathology studies and critical for developing integrated management strategies.

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.

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.

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.

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.

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.

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.

Flavonoids represent a large class of phenolic specialized metabolites and play crucial roles in plant-environment interactions, including responses to biotic and abiotic factors. While the core flavonoid biosynthesis pathway is well known in several plant species, enzymes involved in modifying core flavonoid structures, furnishing them with distinct biological activities, continue to be identified. Anthocyanins, a specific type of flavonoid pigment, serve various functions, including attracting pollinators and seed-dispersing organisms when accumulated in flowers and seeds. Anthocyanins also accumulate in vegetative tissues of many plants, especially under unfavorable conditions. In this review, we present an overview of the diverse structures, various distributions, and multiple functions of flavonoids in plants.

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.