Basil J. Nikolau

Quality control for plant metabolomics: reporting MSI-compliant studies.

The Metabolomics Standards Initiative (MSI) has recently released documents describing minimum parameters for reporting metabolomics experiments, in order to validate metabolomic studies and to facilitate data exchange. The reporting parameters encompassed by MSI include the biological study design, sample preparation, data acquisition, data processing, data analysis and interpretation relative to the biological hypotheses being evaluated. Herein we exemplify how such metadata can be reported by using a small case study - the metabolite profiling by GC-TOF mass spectrometry of Arabidopsis thaliana leaves from a knockout allele of the gene At1g08510 in the Wassilewskija ecotype. Pitfalls in quality control are highlighted that can invalidate results even if MSI reporting standards are fulfilled, including reliable compound identification and integration of unknown metabolites. Standardized data processing methods are proposed for consistent data storage and dissemination via databases.

A proposed framework for the description of plant metabolomics experiments and their results

The study of the metabolite complement of biological samples, known as metabolomics, is creating large amounts of data, and support for handling these data sets is required to facilitate meaningful analyses that will answer biological questions. We present a data model for plant metabolomics known as ArMet (architecture for metabolomics). It encompasses the entire experimental time line from experiment definition and description of biological source material, through sample growth and preparation to the results of chemical analysis. Such formal data descriptions, which specify the full experimental context, enable principled comparison of data sets, allow proper interpretation of experimental results, permit the repetition of experiments and provide a basis for the design of systems for data storage and transmission. The current design and example implementations are freely available (http://www.armet.org/). We seek to advance discussion and community adoption of a standard for metabolomics, which would promote principled collection, storage and transmission of experiment data.

The metabolomics standards initiative (MSI)

In 2005, the Metabolomics Standards Initiative has been formed. An outline and general introduction is provided to inform about the history, structure, working plan and intentions of this initiative. Comments on any of the suggested minimal reporting standards are welcome to be sent to the open email list Msi-workgroups-feedback@lists.sourceforge.net

The Metabolomics Standards Initiative

In 2005, the Metabolomics Standards Initiative has been formed. An outline and general introduction is provided to inform about the history, structure, working plan and intentions of this initiative. Comments on any of the suggested minimal reporting standards are welcome to be sent to the open email list Msi-workgroups-feedback@lists.sourceforge.net

Plant biotin-containing carboxylases

Biotin-containing proteins are found in all forms of life, and they catalyze carboxylation, decarboxylation, or transcarboxylation reactions that are central to metabolism. In plants, five biotin-containing proteins have been characterized. Of these, four are catalysts, namely the two structurally distinct acetyl-CoA carboxylases (heteromeric and homomeric), 3-methylcrotonyl-CoA carboxylase and geranoyl-CoA carboxylase. In addition, plants contain a noncatalytic biotin protein that accumulates in seeds and is thought to play a role in storing biotin. Acetyl-CoA carboxylases generate two pools of malonyl-CoA, one in plastids that is the precursor for de novo fatty acid biosynthesis and the other in the cytosol that is the precursor for fatty acid elongation and a large number of secondary metabolites. 3-Methylcrotonyl-CoA carboxylase catalyzes a reaction in the mitochondrial pathway for leucine catabolism. The exact metabolic function of geranoyl-CoA carboxylase is as yet unknown, but it may be involved in isoprenoid metabolism. This minireview summarizes the recent developments in our understanding of the structure, regulation, and metabolic functions of these proteins in plants.

Platform biochemicals for a biorenewable chemical industry

The chemical industry is currently reliant on a historically inexpensive, petroleum-based carbon feedstock that generates a small collection of platform chemicals from which highly efficient chemical conversions lead to the manufacture of a large variety of chemical products. Recently, a number of factors have coalesced to provide the impetus to explore alternative renewable sources of carbon. Here we discuss the potential impact on the chemical industry of shifting from non-renewable carbon sources to renewable carbon sources. This change to the manufacture of chemicals from biological carbon sources will provide an opportunity for the biological research community to contribute fundamental knowledge concerning carbon metabolism and its regulation. We discuss whether fundamental biological research into metabolic processes at a holistic level, made possible by completed genome sequences and integrated with detailed structural understanding of biocatalysts, can change the chemical industry from being dependent on fossil-carbon feedstocks to using biorenewable feedstocks. We illustrate this potential by discussing the prospect of building a platform technology based upon a concept of combinatorial biosynthesis, which would explore the enzymological flexibilities of polyketide biosynthesis.

Use of mass spectrometry for imaging metabolites in plants

We discuss and illustrate recent advances that have been made to image the distribution of metabolites among cells and tissues of plants using different mass spectrometry technologies. These technologies include matrix-assisted laser desorption ionization, desorption electrospray ionization, and secondary ion mass spectrometry. These are relatively new technological applications of mass spectrometry and they are providing highly spatially resolved data concerning the cellular distribution of metabolites. We discuss the advantages and limitations of each of these mass spectrometric methods, and provide a description of the technical barriers that are currently limiting the technology to the level of single-cell resolution. However, we anticipate that advances in the next few years will increase the resolving power of the technology to provide unprecedented data on the distribution of metabolites at the subcellular level, which will increase our ability to decipher new knowledge concerning the spatial organization of metabolic processes in plants.

Reverse Genetic Characterization of Cytosolic Acetyl-CoA Generation by ATP-Citrate Lyase in Arabidopsis

Acetyl-CoA provides organisms with the chemical flexibility to biosynthesize a plethora of natural products that constitute much of the structural and functional diversity in nature. Recent studies have characterized a novel ATP-citrate lyase (ACL) in the cytosol of Arabidopsis thaliana. In this study, we report the use of antisense RNA technology to generate a series of Arabidopsis lines with a range of ACL activity. Plants with even moderately reduced ACL activity have a complex, bonsai phenotype, with miniaturized organs, smaller cells, aberrant plastid morphology, reduced cuticular wax deposition, and hyperaccumulation of starch, anthocyanin, and stress-related mRNAs in vegetative tissue. The degree of this phenotype correlates with the level of reduction in ACL activity. These data indicate that ACL is required for normal growth and development and that no other source of acetyl-CoA can compensate for ACL-derived acetyl-CoA. Exogenous malonate, which feeds into the carboxylation pathway of acetyl-CoA metabolism, chemically complements the morphological and chemical alterations associated with reduced ACL expression, indicating that the observed metabolic alterations are related to the carboxylation pathway of cytosolic acetyl-CoA metabolism. The observations that limiting the expression of the cytosolic enzyme ACL reduces the accumulation of cytosolic acetyl-CoA–derived metabolites and that these deficiencies can be alleviated by exogenous malonate indicate that ACL is a nonredundant source of cytosolic acetyl-CoA.

Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis

Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A4B4 configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by theACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of theACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.

High-Spatial and High-Mass Resolution Imaging of Surface Metabolites of Arabidopsis thaliana by Laser Desorption-Ionization Mass Spectrometry Using Colloidal Silver

High-spatial resolution and high-mass resolution techniques are developed and adopted for the mass spectrometric imaging of epicuticular lipids on the surface of Arabidopsis thaliana. Single cell level spatial resolution of approximately 12 mum was achieved by reducing the laser beam size by using an optical fiber with 25 mum core diameter in a vacuum matrix-assisted laser desorption ionization-linear ion trap (vMALDI-LTQ) mass spectrometer and improved matrix application using an oscillating capillary nebulizer. Fine chemical images of a whole flower were visualized in this high spatial resolution showing substructure of an anther and single pollen grains at the stigma and anthers. The LTQ-Orbitrap with a MALDI ion source was adopted to achieve MS imaging in high mass resolution. Specifically, isobaric silver ion adducts of C29 alkane (m/z 515.3741) and C28 aldehyde (m/z 515.3377), indistinguishable in low-resolution LTQ, can now be clearly distinguished and their chemical images could be separately constructed. In the application to roots, the high spatial resolution allowed molecular MS imaging of secondary roots and the high mass resolution allowed direct identification of lipid metabolites on root surfaces.