In this section we highlight work (protocols, publications, databases) contributing to the achievement of specific project deliverables. An updated list of all publications acknowledging the support of European Commission to the AGRON-OMICS project is available on this page.
WP1 - Leaf Growth
Pilot leaf growth experiment
The purpose of this experiment was to compare leaf growth of 3 Arabidopsis genotypes (Ws, Ler, Col-4) grown under the same target environmental conditions in the laboratories of most participating partners, and to assess the biological and inter-laboratory variability. Based on the statistical analysis of leaf growth and several morphometric parameters, it was concluded that it is difficult to reach the same phenotype at different locations even if a very precise and common protocol is followed, including the use of identical material shipped from a central location (seed batch, soil, nutrient solution, pots). For this reason, the consortium has identified one core phenotyping platform (LEPSE, Montpellier) for all experiments involving the analysis of the same set of samples in different molecular profiling platforms.
Automated plant phenotyping platform
An automated plant phenotyping facility was set up and is operational at LEPSE since 2003 [Granier et al (2006) New Phytol 169(3):623-35 PMID:16411964]. It includes a controlled growth-chamber equipped with an automaton. A new platform has been set up at LEPSE in the frame of the AGRON-OMICS project. It enables the production of large quantity of high quality leaf material for molecular profiling experiments (WP3) and the recording of detailled growth parameters for series of Arabidopsis mutants, in the framework of the project consortium and in collaboration with different partners.
WP2 - Genetic Diversity
Candidate regions from growth QTL mapping experiments
Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations. Lisec, J., Meyer, R.C., Steinfath, M., Redestig, H., Becher, M., Witucka-Wall, H., Fiehn, O., Törjék, O, Selbig, J., Altmann, T., and Willmitzer, L. (2007) The Plant J PMID: 18047556
Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24 were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P < or = 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24-67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.
Growth regulatory genes from mutant screens
Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana Yunhai Li, Leiying Zheng, Fiona Corke, Caroline Smith, and Michael W. Bevan (2008) Genes Dev. 22 1331-1336 PMID: 18483219
Although the size of an organism is a defining feature, little is known about the mechanisms that set the final size of organs and whole organisms. Here we describe Arabidopsis DA1, encoding a predicted ubiquitin receptor, which sets final seed and organ size by restricting the period of cell proliferation. The mutant protein encoded by the da1-1 allele has a negative activity toward DA1 and a DA1-related (DAR) protein, and overexpression of a da1-1 cDNA dramatically increases seed and organ size of wild-type plants, identifying this small gene family as important regulators of seed and organ size in plants.
Lessons from a search for leaf mutants in Arabidopsis thaliana. Perez-Perez JM, Candela H, Robles P, Quesada V, Ponce MR, Micol JL.Int J Dev Biol. 2008 Oct 28. [Epub ahead of print] PMID: 19247929
Large-scale exploratory approaches to understanding gene function laid the foundations for the -omics era. Based on modern technologies for the structural and functional characterization of genomes, these curiosity-driven approaches allow systematic accumulation of vast amounts of data, enabling subsequent hypothesis-driven research. Some years before the dawn of genomics, exploratory approaches were already furthering our understanding of gene function in the form of saturation mutagenesis experiments aimed at the identification of all genes that mutate to a given phenotype. Forward genetic approaches, conducted on experimental organisms such as Drosophila melanogaster and Caenorhabditis elegans, have led to the isolation of mutants affected in specific developmental processes, whose cellular and molecular characterization has unraveled the underlying genetic mechanisms of animal development. To shed light on the making of plant leaves, in 1993 we initiated an attempt to identify as many viable and fertile mutants with abnormal leaf morphology as possible, using the Arabidopsis thaliana model organism. We identified 25 fast-neutron- and 153 ethyl-methane sulfonate-induced mutations, which fell into eight and 94 complementation groups, respectively. We also studied 115 publicly available mutant lines isolated by previous authors, which fell into 37 complementation groups. Although we did not reach saturation of the Arabidopsis thaliana genome, the broad spectrum of leaf morphological alterations identified is facilitating the dissection of specific leaf developmental processes. In a complementary approach, we also analyzed leaf architecture in natural accessions and two populations of recombinant inbred lines. Using a high-throughput gene mapping method, we have already cloned 25 of the genes identified by mutation, in some cases in collaboration with other groups. The products of these genes participate in various developmental processes, such as polar cell expansion, transduction of hormonal signals, gene regulation, plastid biogenesis, and chromatin remodeling, among others. The range of phenotypes and processes identified reveal the complexity of leaf ontogeny and will help explain the diversity of leaf morphology in nature.
David and Goliath: what can the tiny weed Arabidopsis teach us to improve biomass production in crops? Gonzalez N, Beemster GT, Inzé D. Curr Opin Plant Biol. 2008 Dec 30. PMID: 19119056
In the next decades, the world market for plant-derived products is expected to expand exponentially. Not only do we rely on plants to feed the growing world population, but plants will also play a pivotal role in providing a significant part of our increasing energy demands. Whereas in the 1960s the green revolution contributed to increase plant productivity, it is expected that biotechnological advances will further boost biomass production and plant yield. To do this effectively, it will be necessary to understand how the molecular machinery that determines yield parameters operates. Although of no direct economic significance, the model plant Arabidopsis can be used to find genes and regulatory networks controlling biomass production, which, in turn, can be applied for further growth improvement in other species including cereals.
WP3 - Molecular Profiling
First draft of the Arbidopsis Peptide Atlas
The Arabidopsis Peptide Atlas assembles high-quality genome-wide expression data at the proteome level. This information is important for two reasons: first, identified peptides provide expression evidence for predicted gene models and as such allow verifying or disproving gene prediction; and second, this strategy generates for each identified protein a list of detectable peptides. The detection of specific peptides from a protein is essential for the design of targeted quantitative proteomics experiments. These can be used in selected reaction monitoring (SRM) for absolute protein quantification. Peptides that unambiguously identify a protein in complex proteome samples are called "proteotypic" peptides and can be considered "biomarkers" for the presence and the quantity of a protein in the sample. Using this approach, the absolute quantities of proteins in entire pathways can be determined, making this strategy very attractive for systems biology approaches.
Genome-Scale Proteomics Reveals Arabidopsis thaliana Gene Models and Proteome Dynamics Baerenfaller K, Grossmann J, Grobei MA, Hull R, Hirsch-Hoffmann M, Yalovsky S, Zimmermann P, Grossniklaus U, Gruissem W, Baginsky S. (2008) Science 320: 938-941 PMID: 18436743
We have assembled a proteome map for Arabidopsis thaliana from high-density, organ-specific proteome catalogs that we generated for different organs, developmental stages, and undifferentiated cultured cells. We matched 86,456 unique peptides to 13,029 proteins and provide expression evidence for 57 gene models that are not represented in the TAIR7 protein database. Analysis of the proteome identified organ-specific biomarkers and allowed us to compile an organ-specific set of proteotypic peptides for 4105 proteins to facilitate targeted quantitative proteomics surveys. Quantitative information for the identified proteins was used to establish correlations between transcript and protein accumulation in different plant organs. The Arabidopsis proteome map provides information about genome activity and proteome assembly and is available as a resource for plant systems biology.
Contrasting fluorescent tags to be used in leaves
Building blocks for plant gene assembly. Karimi, M., Bleys, A., Vanderhaeghen, R. and Hilson, P. (2007) Plant Physiology 145:1183-1191 PMID: 17965171
The MultiSite Gateway cloning system, based on site-specific recombination, enables the assembly of multiple DNA fragments in predefined order, orientation, and frame register. To streamline the construction of recombinant genes for functional analysis in plants, we have built a collection of 36 reference Gateway entry clones carrying promoters, terminators, and reporter genes, as well as elements of the LhG4/LhGR two-component system. This collection obeys simple engineering rules. The genetic elements (parts) are designed in a standard format. They are interchangeable, fully documented, and can be combined at will according to the desired output. We also took advantage of the MultiSite Gateway recombination sites to create vectors in which two or three genes can be cloned simultaneously in separate expression cassettes. To illustrate the flexibility of these core resources for the construction of a wide variety of plant transformation vectors, we generated various transgenes encoding fluorescent proteins and tested their activity in plant cells. The structure and sequence of all described plasmids are accessible online at http://www.psb.ugent.be/gateway/. All accessions can be requested via the same Web site.
WP4 - Molecular Interactions
List of 500 genes known or suspected to be related to plant growth
One essential collective task that was initiated at the beginning of the project is to build a scaffold for genes and molecular modules that are involved in the control of growth and development of plant leaves. The concrete results of this effort that will continue for the duration of AGRON-OMICS activities are the corner stone of the project knowledge base. The majority of partner laboratories contributed to the compilation of an initial gene list of over 1000 genes of which about 900 are unique entries. This list represented the starting point of the work performed in this deliverable consisting in selecting a second sub-list of about 500 genes entering the cloning of open reading frames (ORFs) work pipeline. As a result, more than 400 genes with ORFs not represented in the SSP or ATOME ORFeomes were selected for capture via the Gateway recombinational cloning system. The ORF entry clones will be publicly available shortly.
Protocols for analyzing protein-protein interactions in planta
Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD (2008) Plant J. 2008 Jul 4 PMID: 18643979
The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co-localization or bi-molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium- to high-throughput analyses of fusion proteins harboring fluorescent tags in a whole-plant cellular context.
Boosting tandem affinity purification of plant protein complexes. Van Leene J, Witters E, Inzé D, De Jaeger G. (2008) Trends Plant Sci. 2008 Sep 2. PMID: 18771946
Protein-interaction mapping based on the tandem affinity purification (TAP) approach has been successfully established for several systems, such as yeast and mammalian cells. However, relatively few protein complex purifications have been reported for plants. Here, we highlight solutions for the pitfalls and propose a major breakthrough in the quest for a better TAP tag in plants.
WP6 Data Management and Integration
The Arabidopsis Reactome knowledgebase website.
Arabidopsis Reactome: A Foundation Knowledgebase for Plant Systems Biology. Tsesmetzis N, Couchman M, Higgins J, Smith A, Doonan JH, Seifert GJ, Schmidt EE, Vastrik I, Birney E, Guanming Wu, D'Eustachio P, Stein LD, Morris RJ, Bevan MW, Walsh SV (2008) Plant Cell 10.1105/tpc.108.057976 PMID: 18591350
New ways of capturing and representing biological knowledge are needed to enable individual researchers to remain abreast of relevant discoveries and to permit computational approaches for interpreting the large volumes of diverse data generated by modern biological research. Here, we describe a promising approach that expands the term "reaction" to represent biological processes. We show how users can represent a wide variety of biological processes in plants in terms of the concept of a reaction and assemble the information obtained from the model plant Arabidopsis thaliana into an online knowledgebase called Arabidopsis Reactome. Its curated and imported pathways currently cover 8% of the Arabidopsis proteome. Arabidopsis Reactome events have also been electronically projected onto five other predicted plant proteomes. Such a system allows the visualization and interpretation of high-throughput data, hypothesis formulation in systems biology, and is a useful learning resource. The Arabidopsis Reactome project (www.arabidopsisreactome.org) is open access, open source, and open to contributions.
Establishment of a robust statistical method for combining transciptomics and proteomics data
Transciptomics and proteomics data are very different in nature: different coverage, sensitivities, and dynamic range. The direct comparison of both data types requires a statistical adjustment and evaluation of biases occasioned by technology or any of the above-mentioned parameters. A normalization and data correction method needs to be developed to achieve more precise data integration between the transcriptome and the proteome. For an application of original methodology, see Baerenfaller et al (2008) Science 320: 938-941 PMID: 18436743.
WP8 Knowledge and Technology Transfer
WP9 Training and Mobility
The material for the workshops organized within this workpackage is available here.
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