Extracellular Proteomes of Arabidopsis Thaliana and Brassica Napus Roots: Analysis and Comparison by MudPIT and LC-MS/MS

Abstract

An important principle of the functional organization of plant cells is the targeting of proteins to specific subcellular locations. The physical location of proteins within the apoplasm/ rhizosphere at the root-soil interface positions them to play a strategic role in plant response to biotic and abiotic stress. We previously demonstrated that roots of Triticum aestivum and Brassica napus exude a large suite of proteins to the apoplasm/rhizosphere [Basu et al. (1994) Plant Physiol 106:151-158; Basu et al. (1999) Physiol Plant 106:53-61]. This study is a first step to identify low abundance extracytosolic proteins from Arabidopsis thaliana and Brassica napus roots using recent advances in the field of proteomics. A total of 16 extracytosolic proteins were identified from B. napus using two-dimensional gel electrophoresis, tandem mass spectrometry (LC-MS/MS) and de novo sequencing. Another high-throughput proteomics approach, Multidimensional Protein Identification Technology (Mud PIT) was used to identify 52 extracytosolic proteins from A. thaliana. Signal peptide cleavage sites, the presence/absence of transmembrane domains and GPI modification were determined for these proteins. Functional classification grouped the extracellular proteins into different families including glycoside hydrolases, trypsin/ protease inhibitors, plastocyanin-like domains, copper-zinc superoxide dismutases, gamma-thioinins, thaumatins, ubiquitins, protease inhibitor/ seed storage/lipid transfer proteins, transcription factors, class III peroxidase, and plant basic secretory proteins (BSP). We have also developed an on-line, Extracytosolic Plant Proteins Database (EPPdb, http://eppdb.biology.ualberta.ca) to provide information about these extracytosolic proteins.

Extracellular Proteins in Pea Root Tip and Border Cell Exudates

“…In recent years the secretion of several specific endogenous proteins from the root cap, including a Gly-rich glycoprotein and ribosome inactivating proteins, has been documented (Matsuyama et al, 1999;Park et al, 2002). Basu et al (2006) used MudPIT and LC-MS/MS to document that 52 extracytosolic proteins are released from roots of Arabidopsis grown in liquid shake culture for 11 d. There was little overlap among the proteins identified in that study and those in the pea root cap secretome (Table II). This may reflect the divergence in species, collection methods, and seedling age, the possible presence of microbial activities, or the fact that root caps of Brassicacae species do not produce populations of living border cells .…”

Root Secretion of Defense-related Proteins Is Development-dependent and Correlated with Flowering Time

“…Root secretion of proteins has been previously studied by other researchers (24,30,31,59), but the possible functional relationship of these secreted proteins with plant development is poorly understood. Previous studies analyzed the root secretion of proteins only at a specific stage of the plant development or in response to particular stress conditions (30,31).…”

“…Previous studies analyzed the root secretion of proteins only at a specific stage of the plant development or in response to particular stress conditions (30,31). Although Basu et al (31) reported several extracytosolic proteins released from 10-day-old Arabidopsis roots, the temporal accumulation or possible regulation of secreted proteins or whether they are predisposed to be secreted in response to plant developmental changes were not clearly studied.…”

“…However, there is no information in the literature about whether this induction of expression of defense-related genes is a systemic response throughout the plant from leaves to roots and into the rhizosphere. Two-dimensional gel electrophoresis (2-DE) 4 following different mass spectrometry methods has proven to be a useful tool for identification of proteins in the root exudates of Arabidopsis, pea, and alfalfa (24,26,[30][31][32]. In this study, we conducted a systematic proteomic analysis of root exudates of the model plant Arabidopsis thaliana to better understand the variation of protein secretion by roots throughout plant development.…”

Comparative Analyses of Extracellular Matrix Proteome: An Under-Explored Area in Plant Research

“…Defining proteins that change in abundance, form, location or other activities may indicate the presence and functional significance of a protein. Whereas comparative ECM proteome research is quite advanced in animals (Zhu et al, 2007) and yeast (Kim et al, 2007) (Liepman et al, 2010;Basu et al, 2006;Bayer et al, 2006;Borderies et al, 2003;Chivasa et al, 2002;Feiz et al, 2006;Jamet et al 2008a), Medicago sativa (Soares et al, 2007;Watson et al, 2004), and crop plants for, e.g., Oryza sativa (Choudhary et al, 2010), Brassica napus (Basu et al, 2006) Zea mays (Zhu et al, 2006) and Cicer arietinum (Bhushan et al, 2006). Around 500 CWPs of Arabidopsis, representing about one third of its estimated cell wall proteome, have been described (Liepman et al, 2010) while 219, 143, 102, 58 CWPs were identified in rice, chickpea, maize and Brassica, respectively.…”