Eve Syrkin Wurtele

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.

MINING MEDLINE: ABSTRACTS, SENTENCES, OR PHRASES?

A growing body of works address automated mining of biochemical knowledge from digital repositories of scientific literature, such as MEDLINE. Some of these works use abstracts as the unit of text from which to extract facts. Others use sentences for this purpose, while still others use phrases. Here we compare abstracts, sentences, and phrases in MEDLINE using the standard information retrieval performance measures of recall, precision, and effectiveness, for the task of mining interactions among biochemical terms based on term co-occurrence. Results show statistically significant differences that can impact the choice of text unit.

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.

Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein.

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH2-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5′ signal sequence, an open reading frame of 718 nucleotides, a 3′ AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.

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.

Direct profiling and imaging of plant metabolites in intact tissues by using colloidal graphite-assisted laser desorption ionization mass spectrometry

Laser desorption/ionization (LDI)-based imaging mass spectrometry (MS) has been applied to several biological systems to obtain information about both the identities of the major chemical species and their localization. Colloidal graphite-assisted LDI (GALDI) MS imaging was introduced for the imaging of small molecules such as phospholipids, cerebrosides, oligosaccharides, flavonoids, and other secondary metabolites with high spatial homogeneity due to finely dispersed particles. Mass profiles and images of Arabidopsis thaliana have been recorded directly from various plant surfaces and cross sections. The main targeted metabolites were flavonoids and cuticular waxes, both of which are important in many aspects of functional genomics, proteomics, and metabolomics. The mass spectral profiles revealed tissue-specific accumulation of flavonoids in flowers and petals. In addition, many other location-specific ions were observed. The location and the degree of light-induced accumulation of flavonoids in stem sections were successfully probed by GALDI MS.

Plants contain multiple biotin enzymes: Discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom☆

Acetyl-CoA carboxylase is the sole biotin enzyme previously reported in plants. Western analysis with 125I-streptavidin of proteins extracted from carrot somatic embryos visualized six biotin-containing polypeptides, the relative molecular masses of which are 210,000, 140,000, 73,000, 50,000, 39,000, and 34,000. This multiplicity of the biotin-containing polypeptides can be partly explained by the discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and pyruvate carboxylase in extracts of somatic carrot embryos, biotin enzymes previously unknown in the plant kingdom. These biotin enzymes seem to be widely distributed in the plant kingdom.

Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically ‘perfected’ enzyme from a non-enzymatic ancestor over a defined period of plant evolution.

Characterization of a Gene That Is Expressed Early in Somatic Embryogenesis of Daucus carota

The EMB-1 mRNA of carrot (Daucus carota) was isolated as an embryo abundant cDNA clone (T.H. Ulrich, E.S. Wurtele, B.J. Nikolau [1990] Nucleic Acids Res 18: 2826). Northern analyses of RNA isolated from embryos, cultured cells, and a variety of vegetative organs indicate that the EMB-1 mRNA specifically accumulates in embryos, beginning at the early stages of embryo development. In situ hybridization with both zygotic and somatic embryos show that the EMB-1 mRNA begins to accumulate at low levels throughout globular embryos. Accumulation of EMB-1 mRNA increases and becomes more localized as embryos mature; in torpedo embryos, EMB-1 mRNA preferentially accumulates in the meristematic regions, particularly the procambium. The similarity in distribution of EMB-1 mRNA in both zygotic and somatic embryos indicates that much of the spatial pattern of expression of the emb-1 gene is dependent on the developmental program of the carrot embryo and does not require maternal or endosperm factors. The EMB-1 protein (relative molecular weight 9910) is a very hydrophilic protein that is a member of a class of highly conserved proteins (typified also by the Em protein of wheat and the Lea D 19 protein of cotton) that may be ubiquitous among angiosperm embryos but whose functions are as yet unknown. The carrot genome appears to contain one or two copies of the emb-1 gene. A 1313-base pair DNA fragment of the carrot genome containing the emb-1 gene was isolated and sequenced. The gene is interrupted by a single intron of 99 base pairs. Primer extension experiments identify two EMB-1 mRNAs, differing by 6 bases at their 5[prime] ends that are transcribed from this gene.