Marek Mutwil

PlaNet: Combined Sequence and Expression Comparisons across Plant Networks Derived from Seven Species

Genes that are similarly expressed, or coexpressed, are often involved in related biological processes. Such coexpressed relationships also appear to be conserved across species. The PlaNet platform enables comparative analysis of genome-wide coexpression networks across seven plant species, thus enabling prediction of gene function and elucidation of the identity of functional homologs. The model organism Arabidopsis thaliana is readily used in basic research due to resource availability and relative speed of data acquisition. A major goal is to transfer acquired knowledge from Arabidopsis to crop species. However, the identification of functional equivalents of well-characterized Arabidopsis genes in other plants is a nontrivial task. It is well documented that transcriptionally coordinated genes tend to be functionally related and that such relationships may be conserved across different species and even kingdoms. To exploit such relationships, we constructed whole-genome coexpression networks for Arabidopsis and six important plant crop species. The interactive networks, clustered using the HCCA algorithm, are provided under the banner PlaNet (http://aranet.mpimp-golm.mpg.de). We implemented a comparative network algorithm that estimates similarities between network structures. Thus, the platform can be used to swiftly infer similar coexpressed network vicinities within and across species and can predict the identity of functional homologs. We exemplify this using the PSA-D and chalcone synthase-related gene networks. Finally, we assessed how ontology terms are transcriptionally connected in the seven species and provide the corresponding MapMan term coexpression networks. The data support the contention that this platform will considerably improve transfer of knowledge generated in Arabidopsis to valuable crop species.

GeneCAT--novel webtools that combine BLAST and co-expression analyses.

The gene co-expression analysis toolbox (GeneCAT) introduces several novel microarray data analyzing tools. First, the multigene co-expression analysis, combined with co-expressed gene networks, provides a more powerful data mining technique than standard, single-gene co-expression analysis. Second, the high-throughput Map-O-Matic tool matches co-expression pattern of multiple query genes to genes present in user-defined subdatabases, and can therefore be used for gene mapping in forward genetic screens. Third, Rosetta combines co-expression analysis with BLAST and can be used to find ‘true’ gene orthologs in the plant model organisms Arabidopsis thaliana and Hordeum vulgare (Barley). GeneCAT is equipped with expression data for the model plant A. thaliana, and first to introduce co-expression mining tools for the monocot Barley. GeneCAT is available at http://genecat.mpg.de

Cellulose synthesis: a complex complex.

Cellulose is the worlds most abundant biopolymer and a key structural component of the plant cell wall. Cellulose is comprised of hydrogen-bonded beta-1,4-linked glucan chains that are synthesized at the plasma membrane by large cellulose synthase (CESA) complexes. Recent advances in visualization of fluorescently labelled complexes have facilitated exploration of regulatory modes of cellulose production. For example, several herbicides, such as isoxaben and 2,6-dichlorobenzonitrile that inhibit cellulose production appear to affect different aspects of synthesis. Dual-labelling of cytoskeletal components and CESAs has revealed dynamic feedback regulation between cellulose synthesis and microtubule orientation and organization. In addition, fluorescently tagged CESA2 subunits may substitute for another subunit, CESA6, which suggests both plasticity and specificity for one of the components of the CESA complex.

Functional Analysis of the Cellulose Synthase-Like Genes CSLD1, CSLD2, and CSLD4 in Tip-Growing Arabidopsis Cells

A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase superfamily in Arabidopsis (Arabidopsis thaliana), CELLULOSE SYNTHASE-LIKE D1 (CSLD1), CSLD2, and CSLD4. CSLD2 is required for normal root hair growth but has a different role from that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3, and CSLD2 mutants produce root hairs with a range of abnormalities, with many root hairs rupturing late in development. Remarkably, though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semiquantitative reverse transcription-polymerase chain reaction, and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures, and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3, suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission, and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes, although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesize a polysaccharide that has a specialized structural role in the cell walls of tip-growing cells.

Towards revealing the functions of all genes in plants

The great recent progress made in identifying the molecular parts lists of organisms revealed the paucity of our understanding of what most of the parts do. In this review, we introduce computational and statistical approaches and omics data used for inferring gene function in plants, with an emphasis on network-based inference. We also discuss caveats associated with network-based function predictions such as performance assessment, annotation propagation, the guilt-by-association concept, and the meaning of hubs. Finally, we note the current limitations and possible future directions such as the need for gold standard data from several species, unified access to data and tools, quantitative comparison of data and tool quality, and high-throughput experimental validation platforms for systematic gene function elucidation in plants.

Toward the Storage Metabolome: Profiling the Barley Vacuole

While recent years have witnessed dramatic advances in our capacity to identify and quantify an ever-increasing number of plant metabolites, our understanding of how metabolism is spatially regulated is still far from complete. In an attempt to partially address this question, we studied the storage metabolome of the barley (Hordeum vulgare) vacuole. For this purpose, we used highly purified vacuoles isolated by silicon oil centrifugation and compared their metabolome with that found in the mesophyll protoplast from which they were derived. Using a combination of gas chromatography-mass spectrometry and Fourier transform-mass spectrometry, we were able to detect 59 (primary) metabolites for which we know the exact chemical structure and a further 200 (secondary) metabolites for which we have strong predicted chemical formulae. Taken together, these metabolites comprise amino acids, organic acids, sugars, sugar alcohols, shikimate pathway intermediates, vitamins, phenylpropanoids, and flavonoids. Of the 259 putative metabolites, some 12 were found exclusively in the vacuole and 34 were found exclusively in the protoplast, while 213 were common in both samples. When analyzed on a quantitative basis, however, there is even more variance, with more than 60 of these compounds being present above the detection limit of our protocols. The combined data were also analyzed with respect to the tonoplast proteome in an attempt to infer specificities of the transporter proteins embedded in this membrane. Following comparison with recent observations made using nonaqueous fractionation of Arabidopsis (Arabidopsis thaliana), we discuss these data in the context of current models of metabolic compartmentation in plants.

Large-Scale Co-Expression Approach to Dissect Secondary Cell Wall Formation Across Plant Species

Plant cell walls are complex composites largely consisting of carbohydrate-based polymers, and are generally divided into primary and secondary walls based on content and characteristics. Cellulose microfibrils constitute a major component of both primary and secondary cell walls and are synthesized at the plasma membrane by cellulose synthase (CESA) complexes. Several studies in Arabidopsis have demonstrated the power of co-expression analyses to identify new genes associated with secondary wall cellulose biosynthesis. However, across-species comparative co-expression analyses remain largely unexplored. Here, we compared co-expressed gene vicinity networks of primary and secondary wall CESAsin Arabidopsis, barley, rice, poplar, soybean, Medicago, and wheat, and identified gene families that are consistently co-regulated with cellulose biosynthesis. In addition to the expected polysaccharide acting enzymes, we also found many gene families associated with cytoskeleton, signaling, transcriptional regulation, oxidation, and protein degradation. Based on these analyses, we selected and biochemically analyzed T-DNA insertion lines corresponding to approximately twenty genes from gene families that re-occur in the co-expressed gene vicinity networks of secondary wall CESAs across the seven species. We developed a statistical pipeline using principal component analysis and optimal clustering based on silhouette width to analyze sugar profiles. One of the mutants, corresponding to a pinoresinol reductase gene, displayed disturbed xylem morphology and held lower levels of lignin molecules. We propose that this type of large-scale co-expression approach, coupled with statistical analysis of the cell wall contents, will be useful to facilitate rapid knowledge transfer across plant species.

Laying down the bricks: logistic aspects of cell wall biosynthesis

Plant cell wall polysaccharides are synthesised at the plasma membrane and in the Golgi apparatus. Current research efforts mainly try to address how these molecules are synthesised or modified. However, it is clear that polysaccharide synthesis in the two compartments needs to be carried out in a coordinated fashion, and that carbohydrates and proteins that are delivered from the Golgi to the cell surface have to undergo a range of modifications. Consequently, there appears to be a need for a fine-tuned system that coalesces signals from the wall, synthesis of carbohydrate-based molecules and vesicle shuttling. Several recent papers have scratched the surface for an initial understanding of these linked processes. For example, the impairment of the proton pumping activity in the trans-Golgi network, which is part of the cells trafficking system, results in growth defects, changes in Golgi stack morphology and cellulose deficiency. An increased understanding of how cell wall synthesis is coordinated with the secretory machinery may facilitate avenues for modulating cell wall contents and therefore overall plant biomass.

Elucidating gene function and function evolution through comparison of co-expression networks of plants.

The analysis of gene expression data has shown that transcriptionally coordinated (co-expressed) genes are often functionally related, enabling scientists to use expression data in gene function prediction. This Focused Review discusses our original paper (Large-scale co-expression approach to dissect secondary cell wall formation across plant species, Frontiers in Plant Science 2:23). In this paper we applied cross-species analysis to co-expression networks of genes involved in cellulose biosynthesis. We showed that the co-expression networks from different species are highly similar, indicating that whole biological pathways are conserved across species. This finding has two important implications. First, the analysis can transfer gene function annotation from well-studied plants, such as Arabidopsis, to other, uncharacterized plant species. As the analysis finds genes that have similar sequence and similar expression pattern across different organisms, functionally equivalent genes can be identified. Second, since co-expression analyses are often noisy, a comparative analysis should have higher performance, as parts of co-expression networks that are conserved are more likely to be functionally relevant. In this Focused Review, we outline the comparative analysis done in the original paper and comment on the recent advances and approaches that allow comparative analyses of co-function networks. We hypothesize that in comparison to simple co-expression analysis, comparative analysis would yield more accurate gene function predictions. Finally, by combining comparative analysis with genomic information of green plants, we propose a possible composition of cellulose biosynthesis machinery during earlier stages of plant evolution.

Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the corresponding proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of analyses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.