Catálogo de publicaciones - libros

Suspension cultures offer a continuous and homogeneous source of cells growing under strictly controlled conditions, and are ideally suited to study molecular processes at the cellular level. cell suspension cultures are widely used to study cell structure, cell cycle and cell growth in higher plants. Despite the fact that tobacco protein identification is currently hampered by the lack of public genome sequence data, tobacco suspension cells are valuable as a model system to investigate protein dynamics in the context of cellular development. This chapter offers an overview of technical approaches developed for the separation, identification and interaction mapping of the cellular tobacco proteome. A number of proteomics applications used to study molecular regulation of cell cycle, cellular development and response to pathogen attacks are discussed.

In this chapter, we will focus on the contribution of proteomics to the identification and determination of the structure and function of CWPs as well as discussing new perspectives in this area. The great variety of proteins found in the plant cell wall is described. Some families, such as glycoside hydrolases, proteases, lectins, and inhibitors of cell wall modifying enzymes, are discussed in detail. Examples of the use of proteomic techniques to elucidate the structure of various cell wall proteins, especially with post-translational modifications such as -glycosylations, proline hydroxylation and -glycosylations, addition of GPI anchors, and phosphorylation, are given. Finally, the emerging understanding of the functions of cell wall proteins is discussed, as well as proposals for future research.

Proteins residing in the plasma membrane have key functions in transport, signal transduction, vesicle trafficking and many other important processes. To better understand these processes it is necessary to reveal the identity of plasma membrane proteins and to monitor modifications and regulation of their expression. This chapter is an overview of the methods used in plant plasma membrane proteomic studies and the results obtained so far. It focuses on studies using mass spectrometry for identification and includes aspects of plasma membrane fractionation, extraction and washing treatments, assessment of purity, separation methods for plasma membrane proteins and choice of techniques for protein cleavage. Finally, the results of plasma membrane proteomic studies are compared and problems with contaminating proteins are discussed.

The post-genomic era of biology has seen a significant shift in focus, from the genes themselves to the proteins they encode. Recent large-scale studies on the proteomes of chloroplasts and others types of plastid have provided significant new insights into the biogenesis, evolution, and functions of these organelles, and have raised some interesting questions. Many of the proteins that define several important sub-organellar compartments (including the envelope and thylakoid membrane systems, the stroma and plastoglobules) have been identified, and this information has been used to make in silico predictions about the entire complement of proteins in each case. Proteomics has revealed that a relatively large number of proteins inside chloroplasts do not possess canonical targeting information (such proteins lack transit peptides for engagement of the general import machinery), and this has led to the elucidation of novel and unusual pathways of chloroplast protein traffic. For example, it is now clear that some Arabidopsis proteins pass through the endoplasmic reticulum and Golgi en route to the chloroplast, and that these proteins may become glycosylated along the way. Comparative studies have been used to characterise organellar proteome changes in response to various environmental cues or genetic perturbations, whilst other approaches have shed light on the oligomerisation and covalent modification of plastidic proteins.

Plant mitochondria function in respiratory oxidation of organic acids and the transfer of electrons to oxygen via the respiratory electron transport chain, but also participate in wider catabolic and biosynthetic activities vital to plant growth and development. Over 2,000 different proteins are likely to be present in plant mitochondria and many have as yet undefined roles. A fuller systems understanding of the role of mitochondria will require continuing advancements in the isolation of high purity mitochondrial samples and an array of proteomic approaches to categorise their protein composition. In parallel to experimental analysis, prediction programs continue to be useful to search plant genome sequence data to identify putative mitochondrial proteins that are missing due to the physical and chemical limitations of current protein separation and analysis techniques. Studies on post-translational modifications (PTMs) of the primary sequence of mitochondrial proteins are providing important information on putative regulation and damage within the organelle but most such PTMs still need to be investigated in detail to define their biological significance. The interactions between proteins, and between proteins and ligands, define the large-scale structure of the mitochondrial proteome and we are beginning to undercover the dynamic interactions of this protein set, which defines the respiratory apparatus in plants.

The nucleolus is a prominent sub-nuclear structure found in all eukaryotes. It is where the ribosomal RNA genes are transcribed and ribosomes are synthesised. However, much evidence has now accumulated that the nucleolus is involved in many other nuclear processes. Nucleoli are of moderate protein complexity, comprising a few hundred proteins, and can be isolated for proteomic analysis. In this chapter we describe the purification and analysis of plant nucleoli by proteomic methods and summarise the current results. We also discuss more specific tagging methods that have been used to analyse individual protein complexes, as well as methods for analysing post-translational modifications of nucleolar proteins. Finally we discuss the assessment of the reliability of such proteomic data, and the presentation and curation of this type of data.

Germinated pollen grains form pollen tubes that accomplish rapid polarized growth within female gametophytic tissues in order to deliver the sperm cell to the ovule. This process is essential for successful plant fertilization in vivo. Pollen tubes are considered to be an ideal model system to study tip growth. A more comprehensive understanding of proteins involved in pollen development, germination and tube elongation is important not only for unraveling the intricate machinery of sexual reproduction in flowering plants, but has considerable potential for the improvement of crop plant production. Integrated proteomic, genomic and cell biological studies offer powerful tools for dissection of the fascinating mechanisms of pollen development and tip-growth regulation in higher plants. Here, we provide a brief overview of current advances in pollen and pollen tube proteomics.

Plant diseases caused by fungi and oomycetes are considered to represent a severe limiting factor to food production, with major economic impact. Therefore, it is of general concern to elucidate the details of fungal plant pathogenesis, including the molecular mechanisms of disease and the nature of the host cellular response. In recent years, proteomic analyses have emerged as a powerful approach to study effector proteins that derive from phytopathogens as well as defence-related proteins that are induced in infected plants to overcome disease. Proteomics now represents a valuable complement to genomic approaches. This chapter reviews recent advances in proteomic research, focussing on both the above-mentioned aspects, i.e. (1) the molecular mechanisms by which fungal plant pathogens establish infection, and (2) the defence-related strategies of plants to counteract or resist such challenges. Types or classes of proteins involved in these processes that have been investigated with proteomic or related methods are presented here. These include extracellular and cytoplasmic effector proteins, pathogen-associated molecular patterns, and inducible defence-related plant proteins such as pathogenesis-related proteins as well as phosphorylated proteins involved in disease signalling. Additional aspects of the responses to disease of plants, concerning specificity, systemic signal transduction and systemic acquired resistance, are also addressed.

Symbiotic nitrogen fixation, the primary pathway by which inorganic nitrogen is made available for living organisms, requires complex communication between the bacterial microsymbiont and the host plant, beginning in the soil and ending at nodule senescence. The symbiosis takes the form of a highly complex structure referred to as a nodule, which appears as a tumor-like growth on the roots of certain leguminous plants. Both partners exchange signals and change metabolically and morphologically in response to their fellow symbiont. These changes by necessity must be coordinated and complementary. Proteomic analysis has revealed extensive changes in the proteomes of each organism during symbiosis.

Since proteins are key effectors of plant responses to environmental cues, including recognition, signalling, transport and defence reactions, much interest has focussed on characterising proteins involved in the establishment and functioning of arbuscular mycorrhizal (AM) symbiosis. The recent development of high-throughput techniques in the model species is providing, step-by-step, a large-scale analysis of symbiosis-related proteins. Depending on the symbiotic stage and the abundance of mycorrhizal material, different proteomic strategies, which can be combined with other large-scale approaches (transcriptomic and metabolomic), can be used and are presented in this chapter. In addition to providing an update on AM related-proteomics research, our aim is to highlight proteomic strategies available to study AM symbiosis. Finally, further developments that can be expected from the most recent technical improvements in plant proteomics are discussed for this particular research area.