Abstract：
:Receptor-like protein kinases (RLKs) are a large and important group of plant proteins involved in numerous aspects of development and stress response. Within this family, homo-oligermization of receptors followed by autophosphorylation of the intracellular protein kinase domain appears to be a widespread mechanism to regulate protein kinase activity. In vitro studies of several RLKs have identified autophosphorylation sites involved in regulation of catalytic activity and signaling in vivo. Recent work has established that multiple RLKs are biochemically active when expressed in E. coli and readily autophosphorylate prior to purification or subsequent manipulation. This observation has led us to develop a simplified method for assaying RLK phosphorylation status as an indirect measure of intrinsic autophosphorylation activity. The method involves expressing a recombinant RLK protein kinase domain in E. coli, followed by SDS-PAGE of boiled cell lysate, and sequential staining with the phosphoprotein stain Pro-Q Diamond and a colloidal Coomassie total protein stain. We show this method can be used to measure and quantify in vitro autophosphorylation levels of recombinant wildtype and mutant versions of the Arabidopsis RLK HAESA, as well as to detect transphosphorylation activity of recombinant HAESA against a protein kinase inactive version of itself. Our method has several advantages over traditional protein kinase assays. It does not require protein purification, transfer, blotting, or radioactive reagents. It allows for rapid and quantitative assessment of autophosphorylation levels and should have general utility in the study of any autophosphorylating protein kinase expressed in E. coli.
journal_name
Plant Methodsjournal_title
Plant methodsauthors
Taylor I,Seitz K,Bennewitz S,Walker JCdoi
10.1186/1746-4811-9-22subject
Has Abstractpub_date
2013-06-26 00:00:00pages
22issue
1issn
1746-4811pii
1746-4811-9-22journal_volume
9pub_type
杂志文章相关文献
Plant Methods文献大全abstract：: Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-5-3
更新日期：2009-02-27 00:00:00
abstract：Background:Over the last years, several plant science labs have started to employ fluctuating growth light conditions to simulate natural light regimes more closely. Many plant mutants reveal quantifiable effects under fluctuating light despite being indistinguishable from wild-type plants under standard constant light...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-019-0546-1
更新日期：2019-12-23 00:00:00
abstract：:Current methods of in-house plant phenotyping are providing a powerful new tool for plant biology studies. The self-constructed and commercial platforms established in the last few years, employ non-destructive methods and measurements on a large and high-throughput scale. The platforms offer to certain extent, automa...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-015-0072-8
更新日期：2015-04-17 00:00:00
abstract：BACKGROUND:Nectar reabsorption is a widely known phenomenon, related to the strategy of resource-recovery and also to maintain the nectar homeostasis at the nectary. The method currently performed to demonstrate nectar being reabsorbed involves the use of radioactive tracers applied to the nectary. Although this method...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-9-20
更新日期：2013-06-19 00:00:00
abstract：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 gen...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-3-1
更新日期：2007-01-08 00:00:00
abstract：Background:Low pollen viability may limit grapevine yield under certain conditions, causing relevant economic losses to grape-growers. It is usually evaluated by the quantification of the number of viable and non-viable pollen grains that are present in a sample after an adequate pollen grain staining procedure. Althou...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-017-0267-2
更新日期：2018-01-09 00:00:00
abstract：BACKGROUND:Genetic studies on the molecular mechanisms of the regulation of root growth require the characterisation of a specific root phenotype to be linked with a certain genotype. Such studies using classical labour-intensive methods are severely hindered due to the technical limitations that are associated with th...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-016-0135-5
更新日期：2016-06-24 00:00:00
abstract：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 ...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-4-8
更新日期：2008-05-19 00:00:00
abstract：BACKGROUND:Pollen tubes extend rapidly when pollen grains are incubated in defined media. Tube extension requires many critical functions of plant cells including molecular signaling, cytoskeleton remodeling, secretion, and cell wall synthesis. Consequently, pollen tube growth has been established as a way to conduct p...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-10-3
更新日期：2014-01-23 00:00:00
abstract：Background:Plasmopara obducens is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of Impatiens that causes high crop loss. Currently, there are no available methods for the microscopic detection of P. obducens from leaves of impatiens, which may be contributing to the spread of the...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-018-0362-z
更新日期：2018-10-24 00:00:00
abstract：Background:Image processing techniques have been widely used in the analysis of leaf characteristics. Earlier techniques for processing digital RGB color images of plant leaves had several drawbacks, such as inadequate de-noising, and adopting normal-probability statistical estimation models which have few parameters a...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-020-0561-2
更新日期：2020-02-26 00:00:00
abstract：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. RESULTS:A method was developed for simultaneous quantification of multiple phytohormones, abscisic acid, indole-3-ace...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-8-2
更新日期：2012-01-15 00:00:00
abstract：:Chloroplasts play a great role for sustained wellbeing of life on the planet. They have the power and raw materials that can be used as sophisticated biological factories. They are rich in energy as they have lots of pigment-protein complexes capable of collecting sunlight, in sugar produced by photosynthesis and in m...
journal_title：Plant methods
pub_type： 杂志文章,评审
doi：10.1186/s13007-017-0179-1
更新日期：2017-04-19 00:00:00
abstract：Background:To investigate potential effects of herbicide phytotoxic on crops, a major challenge is a lack of non-destructive and rapid methods to detect plant growth that could allow characterization of herbicide-resistant plants. In such a case, hyperspectral imaging can quickly obtain the spectrum for each pixel in t...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-020-00576-7
更新日期：2020-03-05 00:00:00
abstract：:The genomic sequences of many important Triticeae crop species are hard to assemble and analyse due to their large genome sizes, (in part) polyploid genomes and high repeat content. Recently, the draft genomes of barley and bread wheat were reported thanks to cost-efficient and fast NGS technologies. The genome of bar...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-9-35
更新日期：2013-09-06 00:00:00
abstract：Background:Functional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation. Thus, the research carried out on gene function in Pinus tabuliformis is typically performed by heterologous expression base...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-020-00594-5
更新日期：2020-04-10 00:00:00
abstract：Background:Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bo...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-019-0408-x
更新日期：2019-03-09 00:00:00
abstract：Background:Most bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; i...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-019-0465-1
更新日期：2019-07-31 00:00:00
abstract：Background:Plastome (plastid genome) sequences provide valuable information for understanding the phylogenetic relationships and evolutionary history of plants. Although the rapid development of high-throughput sequencing technology has led to an explosion of plastome sequences, annotation remains a significant bottlen...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-019-0435-7
更新日期：2019-05-21 00:00:00
abstract：BACKGROUND:Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent ...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-10-22
更新日期：2014-07-05 00:00:00
abstract：Background:Progress in high-throughput molecular methods accompanied by more complex experimental designs demands novel data visualisation solutions. To specifically answer the question which parts of the specifical biological system are responding in particular perturbation, integrative approach in which experimental ...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-018-0345-0
更新日期：2018-08-30 00:00:00
abstract：BACKGROUND:Plant metabolites are commonly functionally classified, as defense- or growth-related phytohormones, primary and specialized metabolites, and so forth. Analytical procedures for the quantifications of these metabolites are challenging because the metabolites can vary over several orders of magnitude in conce...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-016-0130-x
更新日期：2016-05-26 00:00:00
abstract：BACKGROUND:RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs). Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-3-13
更新日期：2007-11-25 00:00:00
abstract：BACKGROUND:Establishing transcriptional regulatory networks that include protein-protein and protein-DNA interactions has become a key component to better understanding many fundamental biological processes. Although a variety of techniques are available to expose protein-protein and protein-DNA interactions, unequivoc...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-4-25
更新日期：2008-10-27 00:00:00
abstract：BACKGROUND:Identifying cis-regulatory elements is critical in understanding the direct and indirect regulatory mechanisms of gene expression. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high throughput sequencing to identify regions of regulat...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-015-0087-1
更新日期：2015-09-04 00:00:00
abstract：:[This corrects the article DOI: 10.1186/s13007-017-0166-6.]. ...
journal_title：Plant methods
pub_type： 杂志文章,已发布勘误
doi：10.1186/s13007-017-0249-4
更新日期：2017-11-16 00:00:00
abstract：BACKGROUND:A multitude of different imaging systems are already available to image genetically altered RNA species; however, only a few of these techniques are actually suitable to visualize endogenous RNA. One possibility is to use fluorescently-labelled and hybridization-sensitive probes. In order to yield more infor...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/1746-4811-10-15
更新日期：2014-05-31 00:00:00
abstract：BACKGROUND:The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter wa...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-017-0191-5
更新日期：2017-05-22 00:00:00
abstract：Background:Strigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation. Simultaneously, root exuded strigolactones mediate rhizosphere signaling towards beneficial arbuscular mycorrhizal fungi, but also attract parasitic plants. The seed germination o...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-020-00669-3
更新日期：2020-09-17 00:00:00
abstract：Background:The cellular morphology of plant organs is strongly related to other physical properties such as shape, size, growth, mechanical properties or chemical composition. Cell morphology often vary depending on the type of tissue, or on the distance to a specific tissue. A common challenge in quantitative plant hi...
journal_title：Plant methods
pub_type： 杂志文章
doi：10.1186/s13007-020-00603-7
更新日期：2020-05-06 00:00:00
