Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

Reverse-genetic analysis of the two biotin-containing subunit genes of the heteromeric acetyl-coenzyme A carboxylase in Arabidopsis indicates a unidirectional functional redundancy.

The heteromeric acetyl-coenzyme A carboxylase catalyzes the first and committed reaction of de novo fatty acid biosynthesis in plastids. This enzyme is composed of four subunits: biotin carboxyl-carrier protein (BCCP), biotin carboxylase, α-carboxyltransferase, and ß-carboxyltransferase. With the exception of BCCP, single-copy genes encode these subunits in Arabidopsis (Arabidopsis thaliana). Reverse-genetic approaches were used to individually investigate the physiological significance of the two paralogous BCCP-coding genes, CAC1A (At5g16390, codes for BCCP1) and CAC1B (At5g15530, codes for BCCP2). Transfer DNA insertional alleles that completely eliminate the accumulation of BCCP2 have no perceptible effect on plant growth, development, and fatty acid accumulation. In contrast, transfer DNA insertional null allele of the CAC1A gene is embryo lethal and deleteriously affects pollen development and germination. During seed development the effect of the cac1a null allele first becomes apparent at 3-d after flowering, when the synchronous development of the endosperm and embryo is disrupted. Characterization of CAC1A antisense plants showed that reducing BCCP1 accumulation to 35% of wild-type levels, decreases fatty acid accumulation and severely affects normal vegetative plant growth. Detailed expression analysis by a suite of approaches including in situ RNA hybridization, promoter:reporter transgene expression, and quantitative western blotting reveal that the expression of CAC1B is limited to a subset of the CAC1A-expressing tissues, and CAC1B expression levels are only about one-fifth of CAC1A expression levels. Therefore, a likely explanation for the observed unidirectional redundancy between these two paralogous genes is that whereas the BCCP1 protein can compensate for the lack of BCCP2, the absence of BCCP1 cannot be tolerated as BCCP2 levels are not sufficient to support heteromeric acetyl-coenzyme A carboxylase activity at a level that is required for normal growth and development.

AtRabD2b and AtRabD2c have overlapping functions in pollen development and pollen tube growth.

BACKGROUND: Rab GTPases are important regulators of endomembrane trafficking, regulating exocytosis, endocytosis and membrane recycling. Many Rab-like proteins exist in plants, but only a subset have been functionally characterized. RESULTS: Here we report that AtRabD2b and AtRabD2c play important roles in pollen development, germination and tube elongation. AtrabD2b and AtrabD2c single mutants have no obvious morphological changes compared with wild-type plants across a variety of growth conditions. An AtrabD2b/2c double mutant is also indistinguishable from wild-type plants during vegetative growth; however its siliques are shorter than those in wild-type plants. Compared with wild-type plants, AtrabD2b/2c mutants produce deformed pollen with swollen and branched pollen tube tips. The shorter siliques in the AtrabD2b/2c double mutant were found to be primarily due to the pollen defects. AtRabD2b and AtRabD2c have different but overlapping expression patterns, and they are both highly expressed in pollen. Both AtRabD2b and AtRabD2c protein localize to Golgi bodies. CONCLUSIONS: These findings support a partially redundant role for AtRabD2b and AtRabD2c in vesicle trafficking during pollen tube growth that cannot be fulfilled by the remaining AtRabD family members.

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.

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.

Genome wide co-expression among the starch debranching enzyme genes AtISA1, AtISA2, and AtISA3 in Arabidopsis thaliana.

Each of four starch debranching enzymes (DBE) is distinct and highly conserved across the plant kingdom; however, the specific functions of these proteins in carbohydrate metabolism are not well understood. DBEs function in both biosynthesis and degradation of starch, and two have been shown to function as multimers in various quarternary structures that can contain one or more DBE proteins, i.e. ISA1 homomultimers and ISA1/ISA2 heteromultimers. This study characterizes potential functional relationships between the three isoamylase-type DBE proteins (ISA) of Arabidopsis using a comprehensive bioinformatics analysis and promoter fusion approach to determine tissue-, subcellular-, and temporal specificity of gene expression. The results reveal complementary sets of expression patterns, in particular that AtISA1 (known to be involved in starch biosynthesis) and AtISA2 (a non-catalytic polypeptide) are co-expressed in some conditions in the absence of AtISA3 (known to be involved in starch degradation), whereas in other conditions AtISA2 is co-expressed with AtISA3 in the absence of AtISA1 (AtISA2 and AtISA3, but not AtISA1, are co-expressed specially in root columella cells and leaf hydathodes). Thus, AtISA2 may function in starch degradation, in addition to its role in starch biosynthesis. AtISA3 and several other potential regulatory genes, starch metabolic genes, and transcription factors, are specifically induced during cold acclimation; these transcription factors are candidates for involvement of cold-induced changes in starch metabolism. Finally, bioinformatics analysis using MetaOmGraph (http://www.metnetdb.org/MetNet_MetaOmGraph.htm) identifies Arabidopsis genes of unknown function that might be involved in starch metabolism in the cold.

Megagametophyte abnormalities of near-isogenic female partial-sterile soybean mutants ( Glycine max; Leguminosae).

Megagametogenesis of soybean, Glycine max (L.) Merr., cultivars Clark and Clark k2, and F1 hybrid of Clark (female parent) crossed with Clark k2 (male parent) were studied using stereo light microscopy and confocal scanning laser microscopy. Reproductive development in Clark and Clark k2 plants was compared to F1 hybrid plants. In mature pods, 6.4% of the ovules of Clark, 8.1% of the ovules of Clark k2, and 41.4% of the ovules of F1 hybrid plants were aborted. This female partial sterility was due to incomplete megagametophyte development: undeveloped polar nuclei-or developed but not in a position for fertilization; increased megagametophyte wall thickness; abnormal shape and/or premature degeneration of synergids and intact synergids throughout the life of the ovule; egg cell not well-developed or absent; and megagametophyte remaining uninucleate. Each of these abnormalities contributed to either lack of double fertilization or early megagametophyte abortion.