Current protocols in plant biology最新文献

Plant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Plant growth–promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol of pathogens are eco-friendly alternatives to chemical usage and have less energy, environmental, and economic costs. PGPB isolation and evaluation are essentials steps for determining bacteria that are able to improve plant development and productivity. In this unit, we present protocols to isolate bacteria from soil and plant roots (“putative” diazotrophic and endospore-forming bacteria), as well to evaluate some of their beneficial characteristics for the promotion of plant growth (e.g., nitrogen fixation, production of indolic compounds and siderophores, phosphate solubilization, and 1-aminocyclopropane-1-carboxylate deaminase activity). © 2017 by John Wiley & Sons, Inc.

Medicago truncatula was chosen by the legume community, along with Lotus japonicus, as a model plant to study legume biology. Since then, numerous resources and tools have been developed for M. truncatula. These include, for example, its genome sequence, core ecotype collections, transformation/regeneration methods, extensive mutant collections, and a gene expression atlas. This review aims to describe the different genetic and genomic tools and resources currently available for M. truncatula. We also describe how these resources were generated and provide all the information necessary to access these resources and use them from a practical point of view. © 2017 by John Wiley & Sons, Inc.

Target identification remains a challenging step in plant chemical genomics approaches. Drug affinity responsive target stability (DARTS) represents a straightforward technique to identify small molecules’ protein targets and assist in the characterization of interactions between small molecules and putative targets identified by other methods. When a small molecule interacts with a protein, it has the potential to stabilize the protein's structure, resulting in a reduced susceptibility to protease action. During the DARTS procedure, protein extracts are treated with proteolytic enzymes, and only proteins that bind to the small molecule are protected from proteolysis. DARTS represents a protocol independent of the molecule's mechanism of action or chemical structure. Another advantage of DARTS is that it does not require additional modifications or tagging of the small molecule. The protocols outlined in this article describe in detail the DARTS technique applied to plant proteins and propose several detection procedures according to protein abundance. © 2017 by John Wiley & Sons, Inc.

Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium-mediated transformation. However, the large size of BiBACs and low-copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning experiments more difficult than other available binary vector systems. Therefore, a protocol that outlines preparation, modification, and transformation of high-molecular weight (HMW) constructs is advantageous for researchers looking to use BiBACs in plant genomics research. This unit does not cover the cloning of HMW DNA into BiBAC vectors. Researchers looking to clone HMW DNA into BiBACs can refer to Zhang et al. (2012; doi: 10.1038/nprot.2011.456). © 2017 by John Wiley & Sons, Inc.

The field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method that tests for the direct physical interaction of a candidate receptor protein and a peptide ligand. This protocol describes the generation of the ligand probe, transient expression of a receptor protein, enrichment of membrane-bound receptors, photo-crosslinking and click chemistry-mediated reporter addition, and detection of the receptor-ligand complex. Copper-based click chemistry confers several advantages, including the versatility to use almost any azide-containing reporter molecule for detection or visualization of the complex and enables addition of the reporter molecule after receptor-ligand binding which reduces the need for bulky ligand modifications that could interfere with the interaction. © 2017 by John Wiley & Sons, Inc.

Commercial maize hybrid production has corroborated the usefulness of producing inbred lines; however, the delivery of new lines has always been a major time constraint in breeding programs. Traditional methods for developing inbred lines typically require 6 to 10 generations of self-pollination to obtain sufficient homozygosity. To bypass the time and costs associated with the development of inbred lines, doubled haploid (DH) systems have been widely adopted in the commercial production of maize. Within just two generations, DH systems can create completely homozygous and homogeneous lines. A typical maize DH system, utilizing anthocyanin markers R1-nj or Pl1 for haploid selection, is described in this protocol. © 2017 by John Wiley & Sons, Inc.

We present a simple one-pot extraction protocol, which rapidly isolates hydrophilic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlined here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z-score values, which normalize the expression data into a format that can easily be cross-compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation. © 2017 by John Wiley & Sons, Inc.

There is a growing interest in the use of plant growth–promoting rhizobacteria to improve crop productivity as a partial substitute for nitrogen fertilizer. Bacteria-colonizing plants may be epiphytic or endophytic. This article describes reproducible protocols to quantify the level of colonization in each plant compartment. The protocols were developed using several cereal crops such as maize, rice, sorghum, and wheat. © 2017 by John Wiley & Sons, Inc.

To fully exploit the potential of plant microbiome alterations to improve plant health, reliable methods must be used to prepare and characterize microbiome samples. The power of culture-independent studies is that they allow the characterization of novel microbial community members, but only microbial members consistently represented between different research groups are likely to become broadly applicable treatments. The identification of plant microbiome members can be affected by several experimental stages, including design, sample preparation, nucleic acid extraction, sequencing, and analysis. The protocols described here therefore aim to highlight crucial steps that experimenters should consider before beginning a plant microbiome study. © 2017 by John Wiley & Sons, Inc.