Metabolomics as a Hypothesis-Generating Functional Genomics Tool for the Annotation of Arabidopsis thaliana Genes of "Unknown Function".

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.

A mutation in the LPAT1 gene suppresses the sensitivity of fab1 plants to low temperature.

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant grows as well as wild type at 22 degrees C, but after transfer to 2 degrees C fab1 plants cannot maintain photosynthetic function and die after 5 to 7 weeks at 2 degrees C. A fab1 suppressor line, S7, was isolated in a screen that identified mutants that remained alive after 16 weeks at 2 degrees C and were able to flower and produce seed after return to 22 degrees C. Relative to wild type, S7 plants had reduced levels of 16:3 fatty acid in leaf galactolipids, indicating reduced synthesis of chloroplast glycerolipids by the prokaryotic pathway of lipid metabolism. The suppressor mutation was identified, by map-based and candidate-gene approaches, as a hypomorphic allele of lysophosphatidic acid acyltransferase1 (lpat1), lpat1-3. LPAT1 encodes the enzyme that catalyzes the second reaction in the prokaryotic pathway. Several lines of evidence indicate that damage and death of fab1 plants at 2 degrees C may be a result of the increased proportion of phosphatidylglycerol (PG) in fab1 that are high-melting-point molecular species (containing only 16:0, 18:0, and 16:1,Delta3-trans fatty acids). Consistent with this proposal, the lpat1-3 mutation strongly affects the fatty acid composition of PG. The proportion of high-melting-point molecular species in PG is reduced from 48.2% in fab1 to 10.7% in fab1 lpat1-3 (S7), a value close to the 7.6% found in wild type.

PlantMetabolomics.org: a web portal for plant metabolomics experiments.

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.

Increasing UV-B induces biphasic leaf cell expansion in Phaseolus vulgaris, suggesting multiple mechanisms for controlling plant growth.

Leaf expansion, comprising cell division and cell enlargement, is controlled by light quality and quantity. The role of UV-B irradiance on leaf cell enlargement has not been determined. We studied the effect of a wide range of UV-B irradiances on the cell-enlargement-driven expansion of Phaseolus vulgaris L., cv. Contender (bush bean) leaf discs. Our growth method allowed separation of the cell enlargement phase of leaf expansion from the cell division phase. In two series of experiments with different types of UV-B screening filters, the effect of increasing levels of UV-B on the area of excised P. vulgaris leaf discs was investigated. One set of experiments utilized polyester (UV-B-absorbing) and cellulose acetate (UV-B-transmitting) filters. The other set utilized UV-B-absorbing and UV-B-transmitting acrylic filters. Regardless of which type of filter was used for screening, high (above summer solstice) levels of supplemental UV-B inhibited cell enlargement in a linear, dose-dependent manner, resulting in smaller leaf discs than treatment with UV-B-absorbing filters. Conversely, low levels of supplemental UV-B enhanced cell enlargement in a linear, dose-dependent manner, resulting in larger leaf discs than did treatment with UV-B-absorbing filters. The results suggest a biphasic response to UV-B, and that there is an optimum UV-B level that results in maximum leaf expansion by cell enlargement.

A suppressor of fab1 challenges hypotheses on the role of thylakoid unsaturation in photosynthetic function.

Leaf membrane lipids of the Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis 1 (fab1) mutant contain a 35% to 40% increase in the predominant saturated fatty acid 16:0, relative to wild type. This increase in membrane saturation is associated with loss of photosynthetic function and death of mutant plants at low temperatures. We have initiated a suppressor screen for mutations that allow survival of fab1 plants at 2 degrees C. Five suppressor mutants identified in this screen all rescued the collapse of photosynthetic function observed in fab1 plants. While fab1 plants died after 5 to 7 weeks at 2 degrees C, the suppressors remained viable after 16 weeks in the cold, as judged by their ability to resume growth following a return to 22 degrees C and to subsequently produce viable seed. Three of the suppressors had changes in leaf fatty acid composition when compared to fab1, indicating that one mechanism of suppression may involve compensating changes in thylakoid lipid composition. Surprisingly, the suppressor phenotype in one line, S31, was associated with a further substantial increase in lipid saturation. The overall leaf fatty acid composition of S31 plants contained 31% 16:0 compared with 23% in fab1 and 17% in wild type. Biochemical and genetic analysis showed that S31 plants contain a new allele of fatty acid desaturation 5 (fad5), fad5-2, and are therefore partially deficient in activity of the chloroplast 16:0 Delta7 desaturase. A double mutant produced by crossing fab1 to the original fad5-1 allele also remained alive at 2 degrees C, indicating that the fad5-2 mutation is the suppressor in the S31 (fab1 fad5-2) line. Based on the biophysical characteristics of saturated and unsaturated fatty acids, the increased 16:0 in fab1 fad5-2 plants would be expected to exacerbate, rather than ameliorate, low-temperature damage. We propose instead that a change in shape of the major thylakoid lipid, monogalactosyldiacylglycerol, mediated by the fad5-2 mutation, may compensate for changes in lipid structure resulting from the original fab1 mutation. Our identification of mutants that suppress the low-temperature phenotype of fab1 provides new tools to understand the relationship between thylakoid lipid structure and photosynthetic function.