Extending SILAC to proteomics of plant cell lines

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) is a widespread method for metabolic labeling of cells and tissues in quantitative proteomics; however, incomplete incorporation of the label has so far restricted its wider use in plants. Here, we argue that differential labeling by two different versions of the labeled amino acids renders SILAC fully applicable to dark-grown plant cell lines. By comparing Arabidopsis thaliana cell cultures labeled with two versions of heavy Lys (Lys-4 and Lys-8), we show that this simple modification of the SILAC protocol enables similar quantitation accuracy, precision, and reproducibility as conventional SILAC in animal cells.     

Ionic channels in plant cell membranes

The use of patch clamp methods for identifying ion-specific channels and other transport structures in plant cell membranes is described. Methodology, basic concepts that underlie data analysis, and applications of this powerful technique are emphasized.     

Quantitative plant proteomics

Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.     

Large-scale plant proteomics

Large-scale and high throughput approaches increasingly play an essential role in the study of biological systems, which are per se highly complex. Therefore, they need to be examined by these extensive methods to receive information about the large genomic and proteomic networks. In plant biology, this purpose has a strong support through the accessability of the complete genome sequence of the model plant Arabidopsis thaliana. This brief review intends to focus on the basics and the state-of-the-art of these high-throughput technologies and their application to plant proteomics. It describes protein microarrays, the use of antibodies, 2-DE and MS methods and the yeast two hybrid system, which are emerging as the major technologies for plant proteomics.     

Turgor pressure sensing in plant cell membranes

Experimental evidence is reviewed which shows that the cell membrane is compressible by both mechanical and electrical forces. Calculations are given which show that significant changes in the thickness of cell membranes can occur as a result of (a) direct compression due to the turgor pressure; (b) indirect effects due to the stretching of the cell wall; and (c) the stresses induced by the electric field in the membrane.     

Differential proteomics of plant development

In this mini-review, recent advances in plant developmental proteomics are summarized. The growing interest in plant proteomics continually produces large numbers of developmental studies on plant cell division, elongation, differentiation, and formation of various organs. The brief overview of changes in proteome profiles emphasizes the participation of stress-related proteins in all developmental processes, which substantially changes the view on functional classification of these proteins. Next, it is noteworthy that proteomics helped to recognize some metabolic and housekeeping proteins as important signaling inducers of developmental pathways. Further, cell division and elongation are dependent on proteins involved in membrane trafficking and cytoskeleton dynamics. These protein groups are less prevalently represented in studies concerning cell differentiation and organ formation, which do not target primarily cell division. The synthesis of new proteins, generally observed during developmental processes, is followed by active protein folding. In this respect, disulfide isomerase was found to be commonly up-regulated during several developmental processes. The future progress in plant proteomics requires new and/or complementary approaches including cell fractionation, specific chemical treatments, molecular cloning and subcellular localization of proteins combined with more sensitive methods for protein detection and identification.     

Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes

Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.     

植物蛋白质组学研究进展

植物结构蛋白质组学研究目的在不断变化。早期旨在比较不同基因型和株系,估测系统发育距离。随后是用蛋白N端Edman测序法或氨基酸分析法鉴定蛋白。如今,应用双向凝胶电泳和质谱法,已能联手成功地解决蛋白分离和鉴定问题。这将有助于对不同突变系与野生型作比较和对不同的表达基因鉴定,应用数据库和作图软件有可能获得功能蛋白构象。     

Focus on plant proteomics.

Proteomics covers the systematic analysis of proteins expressed by a genome, from the identification of their primary amino-acid sequence to the determination of their relative amounts, their state of modification and association with other proteins or molecules of different types. Tremendous progress has been made in this field in the past few years, especially in plant biology, mostly due to major developments of mass spectrometry dedicated to protein analyses and advanced information technology. The aim of this special issue of Plant Physiology and Biochemistry devoted to Plant Proteomics is not to present a comprehensive coverage of this rapidly expanding field but to focus on the representation of some key aspects to illustrate the importance of proteomics in plant functional genomics.     

Modification-specific proteomics in plant biology

Post-translational modifications (PTMs) are involved in the regulation of a wide range of biological processes, and affect e.g. protein structure, activity and stability. Several hundred PTMs have been described in the literature, but relatively few have been studied using mass spectrometry and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM, but also to determine the functional relevance in the context of regulation, response to abiotic stress etc. Protein phosphorylation is the only PTM that has been studied extensively at the proteome wide level in plants using mass spectrometry based methods.     

Induced changes in permeability of plant cell membranes to water

The half-time for THO equilibration was three times longer for a living carrot (Daucus carota L.) cylinder than for a dead one. Furthermore, the energy of activation of THO flux was more than twice as high for the living cylinder. Passage through living membranes thus constitutes a rate-limiting step for THO flux in carrot tissue.     

Translational plant proteomics: a perspective

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics.     

植物蛋白质组学的研究进展

植物蛋白质组学的研究取得了重要进展。全面介绍了植物器官蛋白质组、植物组织蛋白质组及植物亚细胞蛋白质组的研究进展。