Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein-protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r(2) = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1; 1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

We introduce a rational approach for associating genes with plant traits by combined use of a genome-scale functional network and targeted reverse genetic screening. We present a probabilistic network (AraNet) of functional associations among 19,647 (73%) genes of the reference flowering plant Arabidopsis thaliana. AraNet associations are predictive for diverse biological pathways, and outperform predictions derived only from literature-based protein interactions, achieving 21% precision for 55% of genes. AraNet prioritizes genes for limited-scale functional screening, resulting in a hit-rate tenfold greater than screens of random insertional mutants, when applied to early seedling development as a test case. By interrogating network neighborhoods, we identify AT1G80710 (now DROUGHT SENSITIVE 1; DRS1) and AT3G05090 (now LATERAL ROOT STIMULATOR 1; LRS1) as regulators of drought sensitivity and lateral root development, respectively. AraNet (http://www.functionalnet.org/aranet/) provides a resource for plant gene function identification and genetic dissection of plant traits.

PlantMetabolomics.org (PM) is a web portal and database for exploring, visualizing, and downloading plant metabolomics data. Widespread public access to well-annotated metabolomics datasets is essential for establishing metabolomics as a functional genomics tool. PM integrates metabolomics data generated from different analytical platforms from multiple laboratories along with the key visualization tools such as ratio and error plots. Visualization tools can quickly show how one condition compares to another and which analytical platforms show the largest changes. The database tries to capture a complete annotation of the experiment metadata along with the metabolite abundance databased on the evolving Metabolomics Standards Initiative. PM can be used as a platform for deriving hypotheses by enabling metabolomic comparisons between genetically unique Arabidopsis (Arabidopsis thaliana) populations subjected to different environmental conditions. Each metabolite is linked to relevant experimental data and information from various annotation databases. The portal also provides detailed protocols and tutorials on conducting plant metabolomics experiments to promote metabolomics in the community. PM currently houses Arabidopsis metabolomics data generated by a consortium of laboratories utilizing metabolomics to help elucidate the functions of uncharacterized genes. PM is publicly available at http://www.plantmetabolomics.org.

Metabolic networks reconstructed from sequenced genomes or transcriptomes can help visualize and analyze large-scale experimental data, predict metabolic phenotypes, discover enzymes, engineer metabolic pathways, and study metabolic pathway evolution. We developed a general approach for reconstructing metabolic pathway complements of plant genomes. Two new reference databases were created and added to the core of the infrastructure: a comprehensive, all-plant reference pathway database, PlantCyc, and a reference enzyme sequence database, RESD, for annotating metabolic functions of protein sequences. PlantCyc (version 3.0) includes 714 metabolic pathways and 2,619 reactions from over 300 species. RESD (version 1.0) contains 14,187 literature-supported enzyme sequences from across all kingdoms. We used RESD, PlantCyc, and MetaCyc (an all-species reference metabolic pathway database), in conjunction with the pathway prediction software Pathway Tools, to reconstruct a metabolic pathway database, PoplarCyc, from the recently sequenced genome of Populus trichocarpa. PoplarCyc (version 1.0) contains 321 pathways with 1,807 assigned enzymes. Comparing PoplarCyc (version 1.0) with AraCyc (version 6.0, Arabidopsis [Arabidopsis thaliana]) showed comparable numbers of pathways distributed across all domains of metabolism in both databases, except for a higher number of AraCyc pathways in secondary metabolism and a 1.5-fold increase in carbohydrate metabolic enzymes in PoplarCyc. Here, we introduce these new resources and demonstrate the feasibility of using them to identify candidate enzymes for specific pathways and to analyze metabolite profiling data through concrete examples. These resources can be searched by text or BLAST, browsed, and downloaded from our project Web site (http://plantcyc.org).

AraNet is a functional gene network for the reference plant Arabidopsis and has been constructed in order to identify new genes associated with plant traits. It is highly predictive for diverse biological pathways and can be used to prioritize genes for functional screens. Moreover, AraNet provides a web-based tool with which plant biologists can efficiently discover novel functions of Arabidopsis genes ( http://www.functionalnet.org/aranet/). This protocol explains how to conduct network-based prediction of gene functions using AraNet and how to interpret the prediction results. Functional discovery in plant biology is facilitated by combining candidate prioritization by AraNet with focused experimental tests.

Metabolomics is the methodology that identifies and measures global pools of small molecules (of less than about 1,000 Da) of a biological sample, which are collectively called the metabolome. Metabolomics can therefore reveal the metabolic outcome of a genetic or environmental perturbation of a metabolic regulatory network, and thus provide insights into the structure and regulation of that network. Because of the chemical complexity of the metabolome and limitations associated with individual analytical platforms for determining the metabolome, it is currently difficult to capture the complete metabolome of an organism or tissue, which is in contrast to genomics and transcriptomics. This paper describes the analysis of Arabidopsis metabolomics data sets acquired by a consortium that includes five analytical laboratories, bioinformaticists, and biostatisticians, which aims to develop and validate metabolomics as a hypothesis-generating functional genomics tool. The consortium is determining the metabolomes of Arabidopsis T-DNA mutant stocks, grown in standardized controlled environment optimized to minimize environmental impacts on the metabolomes. Metabolomics data were generated with seven analytical platforms, and the combined data is being provided to the research community to formulate initial hypotheses about genes of unknown function (GUFs). A public database (www.PlantMetabolomics.org) has been developed to provide the scientific community with access to the data along with tools to allow for its interactive analysis. Exemplary datasets are discussed to validate the approach, which illustrate how initial hypotheses can be generated from the consortium-produced metabolomics data, integrated with prior knowledge to provide a testable hypothesis concerning the functionality of GUFs.

High-throughput data are a double-edged sword; for the benefit of large amount of data, there is an associated cost of noise. To increase reliability and scalability of high-throughput protein interaction data generation, we tested the efficacy of classification to enrich potential protein protein interactions. We applied this method to identify interactions among Arabidopsis membrane proteins enriched in transporters. We validated our method with multiple retests. Classification improved the quality of the ensuing interaction network and was effective in reducing the search space and increasing true positive rate. The final network of 541 interactions among 239 proteins (of which 179 are transporters) is the first protein interaction network enriched in membrane transporters reported for any organism. This network has similar topological attributes to other published protein interaction networks. It also extends and fills gaps in currently available biological networks in plants and allows building a number of hypotheses about processes and mechanisms involving signal-transduction and transport systems.

Residing beneath the phenotypic landscape of a plant are intricate and dynamic networks of genes and proteins. As evolution operates on phenotypes, we expect its forces to shape somehow these underlying molecular networks. In this review, we discuss progress being made to elucidate the nature of these forces and their impact on the composition and structure of molecular networks. We also outline current limitations and open questions facing the broader field of plant network analysis.

Physiological responses, developmental programs, and cellular functions rely on complex networks of interactions at different levels and scales. Systems biology brings together high-throughput biochemical, genetic, and molecular approaches to generate omics data that can be analyzed and used in mathematical and computational models toward uncovering these networks on a global scale. Various approaches, including transcriptomics, proteomics, interactomics, and metabolomics, have been employed to obtain these data on the cellular, tissue, organ, and whole-plant level. We summarize progress on gene regulatory, cofunction, protein interaction, and metabolic networks. We also illustrate the main approaches that have been used to obtain these networks, with specific examples from Arabidopsis thaliana, and describe the pros and cons of each approach.