Current protocols in plant biology最新文献

Cover: In Perez de Souza et al. (https://doi.org/10.1002/cppb.20100), Initial data quality check using principal component analysis (PCA). (A) before and (B) after outlier removal.

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors.

Basic Protocol 1: Metabolic profiling by gas chromatography–mass spectrometry (GC-MS)

Support Protocol 1: Preparation of freeze-dried cassava material

Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS

Support Protocol 3: Preparation of retention-time standard mixture

Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography–mass spectrometry (IC-MS)

Support Protocol 4: Preparation of standards and recovery experimental procedure

Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids

Alternate Protocol: Determination of soluble sugars and starch

Basic Protocol 4: Determination of anions

Basic Protocol 5: Determination of elements

Basic Protocol 6: Determination of total protein

Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites

Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol

Basic Protocol 9: Determination of starch quality

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein–small molecule interactions in biological systems remains unknown. PROtein–metabolite interactions using size separation (PROMIS) is a method for studying protein–small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography–mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule–protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors.

Basic Protocol 1: Preparation of native cell lysate from plant material

Support Protocol: Bradford assay to determine protein concentration

Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography

Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol

Basic Protocol 4: Metabolomics analysis

Basic Protocol 5: Proteomics analysis

Metabolomics has grown into one of the major approaches for systems biology studies, in part driven by developments in mass spectrometry (MS), providing high sensitivity and coverage of the metabolome at high throughput. Untargeted metabolomics allows for the investigation of metabolic phenotypes involving several hundreds to thousands of metabolites. In this approach, all signals in a mass chromatogram are processed in an unbiased way, allowing for a deeper investigation of metabolic phenotypes, but also resulting in significantly more complex data processing and post-processing steps. In this article, we discuss all the intricacies involved in extracting and analyzing metabolites by chromatography coupled to MS, as well as the processing and analysis of such datasets. © 2019 The Authors.

Basic Protocol 1: Metabolite extraction for LC-MS

Alternate Protocol: Methyl tert-butyl ether (MTBE) extraction for multiple mass spectrometry platforms (GC-polar, LC-polar, LC-lipid)

Basic Protocol 2: LC-MS analysis

Support Protocol 1: GC-MS derivatization and analysis

Support Protocol 2: Lipid analysis

Basic Protocol 3: LC-MS data processing

Basic Protocol 4: Data analysis

Basic Protocol 5: Metabolite annotation

Support Protocol 3: Molecular networking using MetNet

Support Protocol 4: Co-injection of authentic standards

Enzyme-enzyme interactions can be discovered by affinity purification mass spectrometry (AP-MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP-MS. The success of AP-MS depends on the levels and stability of the bait protein, the stability of the protein-protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in-gel-digestion AP-MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics-based in-solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP-MS experiment constitutes a single sample for LC-MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold-change abundance (FC-A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co-sublocalization of “bait” and “prey,” we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC-A values of at least four within-biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme-enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors.

Basic Protocol 1: Gene cloning to the destination vectors

Alternate Protocol: In-Fusion or Gibson gene cloning protocol

Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf

Basic Protocol 3: Affinity purification of protein complexes

Basic Protocol 4: On-bead trypsin/LysC digestion and C18 column peptide desalting and concentration

Basic Protocol 5: Data analysis and quality control

Cover: In Boschiero et al. (https://doi.org/10.1002/cppb.20098), Workflow for Basic Protocol 1. (A) Pipeline overview of SSP-coding gene discovery with MAKER and SPADA tools. (B) Transcriptome workflow for file generation of transcript evidence to be used by MAKER including alignment, assemble, and GFF3 file process. (C) Genome annotation workflow using MAKER software. See 220098.

Hundreds to thousands of small secreted peptides (SSPs) are encoded in plant genomes but have been overlooked, and most remain unannotated and unstudied. Despite their low profile, they have been found to confer dramatic effects on growth and development of plants. With the growing appreciation of their significance, the development of appropriate methods to identify and functionally assess the myriad SSPs encoded in plant genomes has become critical. Here, we provide protocols for the computational and physiological analysis of SSPs in plant genomes. We first describe our methodology successfully used for genome-wide identification and annotation of SSP-coding genes in the model legume Medicago truncatula, which can be readily adapted for other plant species. We then provide protocols for the functional analysis of SSPs using various synthetic peptide screens. Considerations for the design and handling of peptides are included. © 2019 by John Wiley & Sons, Inc.

Insertional mutant libraries of microorganisms can be applied in negative depletion screens to decipher gene functions. Because of underrepresentation in colonized tissue, one major bottleneck is analysis of species that colonize hosts. To overcome this, we developed insertion pool sequencing (iPool-Seq). iPool-Seq allows direct analysis of colonized tissue due to high specificity for insertional mutant cassettes. Here, we describe detailed protocols for infection as well as genomic DNA extraction to study the interaction between the corn smut fungus Ustilago maydis and its host maize. In addition, we provide protocols for library preparation and bioinformatic data analysis that are applicable to any host-microbe interaction system. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cover: In Chen et al. (https://doi.org/10.1002/cppb.20088), Flow chart for Agrobacterium-mediated transformation of B. distachyon. See e20088.

The cell wall is an intricate mesh largely composed of polysaccharides that vary in structure and abundance. Apart from cellulose biosynthesis, the assembly of matrix polysaccharides such as pectin and hemicellulose occur in the Golgi apparatus before being transported via vesicles to the cell wall. Matrix polysaccharides are biosynthesized from activated precursors or nucleotide sugars. The composition and assembly of the cell wall is an important aspect in plant development and plant biomass utilization. The application of anion-exchange chromatography to determine the monosaccharide composition of the insoluble matrix polysaccharides enables a complete profile of all major sugars in the cell wall from a single run. While porous carbon graphite chromatography and tandem mass spectrometry delivers a sensitive and robust nucleotide sugar profile from plant extracts. Here we describe detailed methodology to quantify nucleotide sugars within the cell and profile the non-cellulosic monosaccharide composition of the cell wall. © 2019 by John Wiley & Sons, Inc.