Systems Biology Group
Traditional approaches for understanding complex biological systems have employed reductionist methods that have focused on individual components to comprehend the complexity observed. A systems biology approach assumes that every component can influence a system and thus attempts to integrate information from a multitude of experiments from a diverse range of conditions to understand complex biological systems. The large number of experiments required to satisfy this approach have become possible in recent years with the advanced development of microarrays, sequencers and mass spectrometers. The Systems Biology Group within the Feedstocks Division at The Joint BioEnergy Institute is employing integrative strategies to further our fundamental understanding of the plant cell wall.
Subcellular Partitioning of Plant Cell Wall Biosynthesis
The plant cell wall is comprised of a range of complex sugar polymers including cellulose, hemicellulose and pectin. These compounds collectively provide the structure and rigidity to the plant cell wall. The details of precisely how this complex sugar matrix is constructed are currently poorly understood. The Golgi apparatus is an organelle within the plant cell that produces a large proportion of these matrix polysaccharides (namely hemicellulose and pectin) and secretes them to the cell wall for incorporation.
Plant Golgi Proteomics
Project Lead: Dr Harriet Parsons
In order to better understand the role of this organelle in cell wall biosynthesis we are attempting to characterize this organelle using proteomics. Our current knowledge of the protein constituent that comprise the plant Golgi is relatively poor when compared to other subcellular components within the cell. While several factors have contributed to this shortfall, the major problem has been problems associated with its isolation. We are using an orthogonal approach which employs traditional density centrifugation followed by charge based separation of the organelle on a Free Flow Electrophoresis system.
Figure 1. Strategy for isolation of plant Golgi using density centrifugation and the FFE
Plant Cystosolic Proteome
Project Lead: Dr Jun Ito
The biosynthesis of the cell wall involves the coordinated regulation of hundreds of processes in several compartments within the plant cell. Matrix polysaccharides are synthesized within the Golgi apparatus from nucleotide sugars and secreted to the cell wall for incorporation. Carbon metabolism plays an important role in the supply of nucleotide sugars for the biosynthesis of both cellulose and hemicellulose. The production of the majority of these substrates occurs within the cytosol. We have now completed an extensive proteomic survey of the Arabidopsis cytosol and revealed its principal role in the regulation of protein synthesis and degradation. This in-depth proteomic analysis identified many of the enzymes involved in sugar metabolism and significantly all cytosolic localized enzymes involved in the biosynthesis of principal nucleotide sugars involved in matrix polysaccharide biosynthesis.
Figure 2. Functional breakdown of the ca. 1300 proteins identified in the cytosol by mass spectrometry.
Nucleotide Sugar Transporters
Project Lead: Dr Katy Christiansen
The supply of cytosolic synthesized nucleotide sugars for matrix polysaccharide biosynthesis is regulated by nucleotide sugar transporters residing in the Golgi membranes. Comparative bioinformatics profiling has identified over 50 putative nucleotide sugar transporters, although very few have been functionally characterized. Given the extensive number of candidates, it seems probable when each nucleotide sugar has a distinct transporter. Our proteomic characterization of the Golgi apparatus identified a number of putative nucleotide sugar transporters from a range of phylogenic subfamilies. We have developed a high-throughput screen that employs a yeast endomembrane vesicular mutant and heterologus expression of nucleotide sugars to functionally characterize the Arabidopsis nucleotide sugar transporter family. The technique has been validated with the GDP-Man transporter and is being used to screen substrate specificity for unknown transporters.
Figure 3. Validation of the yeast transporter screen with the detection of GDP-Man in yeast vesicles by LC-MS/MS
Mining the Plant Proteome
Project Leads: Dr Andrew Carroll and Dr Hiren Joshi
The past decade has witnessed an explosion in plant functional genomics data, with the field of proteomics being no exception. Recently, JBEI led a consortium to develop an aggregation portal (http://gator.masc-proteomics.org/) to summarize disparate proteomics data from a variety of online repositories at a single online gateway. The creation of the aggregation portal provided access to an unprecedented amount of data and the integration and analysis of these data have provided a bioinformatics driven technique to target protein modifications in the model plant Arabidopsis. The majority of pre-matched spectral data available through the MASCP Gator represents unmodified regions of the protein, these spectral ‘tracks’ can be employed to ascertain modified regions within the protein. In conjunction with mass spectrometry compatible peptide modeling, non-synonymous single nucleotide polymorphisms (nsSNPs) in Arabidopsis natural variants and conserved identity in cross species orthologs it is possible to create a systematic technique to target modified regions within proteins. An initial survey of previously published protein glycosylation from Arabidopsis indicates that the technique can identify areas where glycosylation sites have been previously mapped.
Figure 4. Screenshot of the MASCP Gator developed to summarize proteomics data in the model plant Arabidopsis.
Profiling the Plant Cell Wall
Project Lead: Dr A. Michelle Smith-Moritz
Traditional biochemical approaches have been extensively utilized to identify and characterize genes involved in cell wall biosynthesis. Thus new techniques are required to enable differential analysis of cell walls to uncover new genetic reasons for differences. We have recently developed an unbiased procedure to analyze plant cell wall mutant lines employing spectroscopy. Whole dried plant material was analyzed by Fourier Transform Near Infrared Spectroscopy (FT-NIR), spectral data were then subjected to data compression by Principle Components Analysis (PCA) and an unbiased outlier determination procedure involving Mahalanobis distance is applied. Experimental variations in monosaccharide composition have enabled the development of a partial least squares (PLS) regression technique to model the composition of major sugars (Ara, Glc, Gal, Xyl) from rice samples analyzed by FT-NIR.
Figure 5. Experimental determination of xylose from plant samples (blue *) compared to composition prediction by PLS (red x). circle and square are average values for all samples.
