Protocol: A high-throughput DNA extraction system suitable for conifers

Background  

High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers.     

A high-throughput method for isolation of salicylic acid metabolic mutants

Background  

Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.     

Proposal for field sampling of plants and processing in the lab for environmental metabolic fingerprinting

Background  

Samples for plant metabolic fingerprinting are prepared generally by metabolism quenching, grinding of plant material and extraction of metabolites in solvents. Further concentration and derivatisation steps follow in dependence of the sample nature and the available analytical platform. For plant material sampled in the field, several methods are not applicable, such as, e.g., collection in liquid nitrogen. Therefore, a protocol was established for sample pre-treatment, grinding, extraction and storage, which can be used for analysis of field-collected plant material, which is further processed in the laboratory. Ribwort plantain (Plantago lanceolata L., Plantaginaceae) was used as model plant. The quality criteria for method suitability were high reproducibility, extraction efficiency and handling comfort of each subsequent processing step.     

Metabolic profiling of laser microdissected vascular bundles of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents.     

A rapid method of fruit cell isolation for cell size and shape measurements

Background  

Cell size is a structural component of fleshy fruit, contributing to important traits such as fruit size and texture. There are currently a number of methods for measuring cell size; most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes or chemicals to release single cells. Neither of these approaches is ideal for assaying large fruit numbers as both require a considerable time to prepare the tissue, with current methods of cell wall digestions taking 24 to 48 hours. Additionally, sectioning can lead to a measurement of a plane that does not represent the widest point of the cell.     

Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

Background  

Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment.     

A convenient method for simultaneous quantification of multiple phytohormones and metabolites: application in study of rice-bacterium interaction

Background  

Simultaneous analysis of multiple functional-related phytohormones and their metabolites will improve our understanding of interactions among different hormones in the same biologic process.     

High-contrast three-dimensional imaging of the Arabidopsis leaf enables the analysis of cell dimensions in the epidermis and mesophyll

Background  

Despite the wide spread application of confocal and multiphoton laser scanning microscopy in plant biology, leaf phenotype assessment still relies on two-dimensional imaging with a limited appreciation of the cells' structural context and an inherent inaccuracy of cell measurements. Here, a successful procedure for the three-dimensional imaging and analysis of plant leaves is presented.     

A database of PCR primers for the chloroplast genomes of higher plants

Background  

Chloroplast genomes evolve slowly and many primers for PCR amplification and analysis of chloroplast sequences can be used across a wide array of genera. In some cases 'universal' primers have been designed for the purpose of working across species boundaries. However, the essential information on these primer sequences is scattered throughout the literature.     

Rapid analysis of seed size in <Emphasis Type="Italic">Arabidopsis</Emphasis> for mutant and QTL discovery

Background  

Arabidopsis thaliana is a useful model organism for deciphering the genetic determinants of seed size; however the small size of its seeds makes measurements difficult. Bulk seed weights are often used as an indicator of average seed size, but details of individual seed is obscured. Analysis of seed images is possible but issues arise from variations in seed pigmentation and shadowing making analysis laborious. We therefore investigated the use of a consumer level scanner to facilitate seed size measurements in conjunction with open source image-processing software.     

pH-sensitivity of YFP provides an intracellular indicator of programmed cell death

Background  

Programmed cell death (PCD) is an essential process for the life cycle of all multicellular organisms. In higher plants however, relatively little is known about the cascade of genes and signalling molecules responsible for the initiation and execution of PCD. To aid with the discovery and analysis of plant PCD regulators, we have designed a novel cell death assay based on low cytosolic pH as a marker of PCD.     

Application of GC-MS for the detection of lipophilic compounds in diverse plant tissues

Background  

The concept of metabolite profiling has been around for decades and technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. However, studies are generally confined to polar compounds alone. Here we describe a simple method for lipophilic compounds analysis in various plant tissues.     

A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars

Background  

Microsatellites are popular molecular markers in many plant species due to their stable and highly polymorphic nature. A number of analysis methods have been described but analyses of these markers are typically performed on cumbersome polyacrylamide gels or more conveniently by capillary electrophoresis on automated sequencers. However post-PCR handling steps are still required. High resolution melting can now combine detailed sequence analysis with the closed-tube benefits of real-time PCR and is described here as a novel way to verify the identity of plant varieties such as grapevine and olive.     

Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

Background  

Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana.     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.