A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

Background  

The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.     

Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0)

Background  

We previously developed a high-throughput system called 'Ice-Cap' for growing Arabidopsis seedlings in a 96-well format and rapidly collecting tissue for subsequent DNA extraction and genotyping. While the originally described Ice-Cap method is an effective tool for high-throughput genotyping, one shortcoming of the first version of Ice-Cap is that optimal seedling growth is highly dependent on specific environmental conditions. Here we describe several technical improvements to the Ice-Cap method that make it much more robust and provide a detailed protocol for implementing the method.     

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.     

SSHscreen and SSHdb,generic software for microarray based gene discovery: application to the stress response in cowpea

Background  

Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process.     

Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

Background  

Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.     

UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Background  

Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids.     

<Emphasis Type="Italic">De novo</Emphasis> assembly of potential linear artificial chromosome constructs capped with expansive telomeric repeats

Background  

Artificial chromosomes (ACs) are a promising next-generation vector for genetic engineering. The most common methods for developing AC constructs are to clone and combine centromeric DNA and telomeric DNA fragments into a single large DNA construct. The AC constructs developed from such methods will contain very short telomeric DNA fragments because telomeric repeats can not be stably maintained in Escherichia coli.     

Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

Background  

We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.     

A novel fluorescent pH probe for expression in plants

Background  

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.     

Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants

Background  

We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.     

Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

Background  

The isolation of green fluorescent protein (GFP) and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types.     

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background  

Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.     

Development of expression vectors based on pepino mosaic virus

Background  

Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.     

High-throughput retrotransposon-based fluorescent markers: improved information content and allele discrimination

Background  

Dense genetic maps, together with the efficiency and accuracy of their construction, are integral to genetic studies and marker assisted selection for plant breeding. High-throughput multiplex markers that are robust and reproducible can contribute to both efficiency and accuracy. Multiplex markers are often dominant and so have low information content, this coupled with the pressure to find alternatives to radio-labelling, has led us to adapt the SSAP (sequence specific amplified polymorphism) marker method from a ³³P labelling procedure to fluorescently tagged markers analysed from an automated ABI 3730 xl platform. This method is illustrated for multiplexed SSAP markers based on retrotransposon insertions of pea and is applicable for the rapid and efficient generation of markers from genomes where repetitive element sequence information is available for primer design. We cross-reference SSAP markers previously generated using the ³³P manual PAGE system to fluorescent peaks, and use these high-throughput fluorescent SSAP markers for further genetic studies in Pisum.     

Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of <Emphasis Type="Italic">in vivo</Emphasis> protein phosphorylation sites

Background  

Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy.     

Genome-wide identification of microsatellites in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) using FIASCO and phpSSRMiner

Background  

Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.     

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.     

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.