Current protocols in plant biology最新文献

The cytoskeleton is key to many essential processes in a plant cell, e.g., growth, division, and defense. Contrary to what “skeleton” implies, the cytoskeleton is highly dynamic, and is able to re-organize itself continuously. The advent of live-cell microscopy and the development of genetically encoded fluorophores enabled detailed observation of the organization and dynamics of the cytoskeleton. Despite the biological importance of the cytoskeletal dynamics, quantitative analyses remain laborious endeavors that only a handful of research teams regularly conduct. With this protocol, we provide a standardized step-by-step guide to analyze the dynamics of microtubules. We provide example data and code for post-processing in Fiji that enables researchers to modify and adapt the routine to their needs. More such tools are needed to quantitatively assess the cytoskeleton and thus to better understand cell biology. © 2019 by John Wiley & Sons, Inc.

Plant microRNAs (miRNAs) are ∼20- to 24-nucleotide small RNAs that post-transcriptionally regulate gene expression of mRNA targets. Here, we present a workflow to characterize the miRNA transcriptome of a non-model plant, focusing on miRNAs and targets that are differentially expressed under one experimental treatment. We cover RNA-seq experimental design to create paired small RNA and mRNA libraries and perform quality control of raw data, de novo mRNA transcriptome assembly and annotation, miRNA prediction, differential expression, target identification, and functional enrichment analysis. Additionally, we include validation of differential expression and miRNA-induced target cleavage using qRT-PCR and modified RNA ligase–mediated 5′ rapid amplification of cDNA ends, respectively. Our procedure relies on freely available software and web resources. It is intended for users that lack programming skills but can navigate a command-line interface. To enable an understanding of formatting requirements and anticipated results, we provide sample RNA-seq data and key input/output files for each stage. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Brachypodium distachyon is an excellent model system for the grasses and has been adopted as a research organism by many laboratories around the world. It has all of the biological traits required for a model system, including small stature, short life cycle, small genome, simple growth requirements, and a close relationship to major crop plants (cereals). In addition, numerous resources have been developed for working with this species, including genome sequences for many lines, sequenced mutant collections, and a large, freely available germplasm collection. Fortunately, among grasses B. distachyon is one of the most easily transformed species, an absolute necessity for a model system. Agrobacterium-mediated transformation is the preferred method to transform plants because it usually results in simple insertions of target DNA. In this article, we describe a method for Agrobacterium-mediated transformation of the inbred B. distachyon lines Bd21 and Bd21-3. Embryogenic callus induced from immature embryos is co-cultivated with Agrobacterium tumefaciens strain AGL1 or Agrobacterium rhizogenes strain 18r12v. Hygromycin and paromomycin are used as selective agents, with comparable transformation efficiencies (defined as the percentage of co-cultivated callus that produce transgenic plants) of 40% to 70%. It takes 20 to 30 weeks to obtain T₁ seeds starting from the initial step of dissecting out immature embryos. This protocol has been shown to be efficient and facile in several studies that resulted in the creation of over 22,000 T-DNA mutants. © 2019 by John Wiley & Sons, Inc.

Use of chemically inducible systems for transgene expression is a crucial requirement for modern plant biology research, as it allows (1) expression of transgenes that compromise plant viability or fertility when constitutively expressed and (2) spatiotemporal control of transgene expression levels. We describe the stringently regulated and highly responsive dexamethasone-inducible gene expression system pOp6/LhGR, which comprises the chimeric transcription activator LhGR and the corresponding pOp6 promoter. Upon induction, the LhGR activator binds to the pOp6 promoter and induces expression of the target gene of interest. We provide detailed protocols for inducing transgene expression at different developmental stages and in different plant species and discuss dexamethasone stability and use of its analogs. We also introduce new, versatile, GATEWAY-compatible binary vectors that are now available for the pOp6/LhGR system. © 2019 by John Wiley & Sons, Inc.

Functionally characterizing plant membrane transport proteins is challenging. Typically, heterologous systems are used to study them. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. These features facilitate almost background-free characterization of putative plant membrane transporters. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL-LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non-plant transporters with putative import function. © 2019 by John Wiley & Sons, Inc.

Gene-centered yeast one-hybrid (Y1H) screens using arrayed genome-wide transcription factor (TF) clone collections provide a simple and effective strategy to identify TF-promoter interactions using a DNA fragment as bait. In an effort to improve the assay we recently developed a Y1H system that uses a cell surface Gaussia luciferase reporter (gLUC59). Compared to other available methods, this luciferase-based strategy requires a shorter processing time, enhances the throughput and improves result analysis of gene-centered Y1H screens. Here, we described the procedure to perform high-throughput screens using this novel strategy, which involves a protocol for mating two haploid yeast strains carrying an arrayed TF clone collection and a promoter::gLUC59 reporter, respectively, and a protocol for analyzing gLUC59 activity in the resulting diploid cells. © 2019 by John Wiley & Sons, Inc.

Flavonoids are a class of specialized metabolites found in many different plant species. They protect against UV radiation, scavenge reactive oxygen species, are involved in plant defense responses, and are associated with plant-microorganism interactions. They have also been reported to possess health-promoting effects including anti-inflammatory, antioxidant, anticancer activity, and antihypertensive effects. Flavonoids encompass >10,000 structures where the types and amounts depend on the plant species, developmental stage, organ, and growth conditions. The diversity of flavonoid structures represents a significant challenge in the analysis of plant flavonoids. Many analytical techniques have been developed to detect and quantify flavonoids, and the most productive of these techniques use liquid chromatography (LC) coupled to mass spectrometry (MS) to analyze flavonoids due to the excellent combination of selectivity and sensitivity of MS. In addition, mass spectral libraries have been constructed to further aid flavonoid identification. Here, the use of ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) in plant flavonoid analyses, with an emphasis on sample extraction, flavonoid separation, and MS detection, is described. © 2018 by John Wiley & Sons, Inc.

In this unit, we describe a high-throughput absolute quantification protocol for 16 protein-bound amino acids (PBAAs) that combines a microscale protein hydrolysis step and an absolute quantification step using multiple reaction monitoring—based liquid chromatography–tandem mass spectrometry detection. The approach facilitates analysis of a few hundred samples per week by using a 96-well-plate extraction setup and avoiding use of additives. Importantly, the method uses only ∼3 mg of tissue per sample and includes 12 heavy-amino-acid internal standards to enable quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues. © 2018 by John Wiley & Sons, Inc.

Several plant growth systems are available to enhance the observation of the root system (e.g., hydroponic and aeroponic plant growth systems, use of transparent soils, etc.). This article describes the use of the ultrasound aeroponic system (USAS) to treat and to enhance access to the root systems of various model plant and crop species (e.g., Arabidopsis thaliana, Medicago truncatula, soybean, etc.). This system is also compatible with short-term (hr) and long-term (days/weeks) biotic and abiotic treatments of plants. Upon treatment, the ease of access to the plant root system facilitates phenotyping (e.g., analysis of root architecture, establishment of root light spectrum using remote sensing technology), microscopic, molecular, and biochemical experiments. In addition, to facilitate functional genomic studies, we combined the use of the USAS with the hairy root transformation system to grow and observe transgenic roots on composite legume plants. © 2018 by John Wiley & Sons, Inc.