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A tremendous amount of DNA sequence information is now available to scientists and engineers. These DNA sequences provide the foundation for studying how the genome of an organism is functioning and they are particularly useful for metabolic engineers interested in manipulating plants for the production of chemicals and enzymes. Functional genomics relies on high-throughput techniques for measuring the mRNA (the transcriptome), protein (the proteome), and metabolite (the metabolome) components of plants as well as their organs and tissues. Microarray technologies, recent advances in protein mass spectrometry, and high-throughput metabolite analyses are beginning to provide detailed information on the total mRNA, protein, and metabolite components of plants. This knowledge will allow scientists to monitor changes in proteins and metabolites in plants. Ultimately, it may allow them to discover new metabolic pathways and to model metabolic and regulatory networks in plants.

A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.

The four component proteins of the glycine decarboxylase multienzyme complex (the P-, H-, T-, and L-proteins) comprise over one-third of the soluble proteins in mitochondria isolated from the leaves of C3 plants. Together with serine hydroxymethyltransferase, glycine decarboxylase converts glycine to serine and is the site of photorespiratory CO2 and NH3 release. The component proteins of the complex are encoded on nuclear genes with N-terminal presequences that target them to the mitochondria. The isolated complex readily dissociates into its component proteins and reassociates into the intact complex in vitro. Because of the intimate association between photosynthesis and photorespiration, the proteins of the complex are present at higher levels in leaves in the light. The expression of these genes is controlled at the transcriptional level and the kinetics of expression are closely related to those of the small subunit of Rubisco. Deletion analysis of fusions between the promoter of the H-protein of the complex and the reporter gene beta-glucuronidase in transgenic tobacco has identified a region responsible for the tissue specificity and light dependence of gene expression. Gel shift experiments show that a nuclear protein in leaves binds to this region. Glycine decarboxylase has proven to be an excellent system for studying problems in plant biochemistry ranging from protein-protein interactions to control of gene expression.

Monoclonal antibodies specific for the dicarboxylate transporter of pea mitochondria were prepared and used to identify this substrate carrier. The hybridoma library was derived from mice that had been immunized with total mitochondrial membranes. The monoclonal antibodies specific for the dicarboxylate transporter were selected by screening hybridoma supernatants for their ability to inhibit malate- and succinate-dependent oxalacetate reduction by osmotically shocked pea leaf mitochondria. Three monoclonal antibodies were shown to be specific for the dicarboxylate transporter by their ability to 1) inhibit malate and succinate metabolism without affecting alpha-ketoglutarate, citrate, pyruvate, glycine, glutamate, or aspartate metabolism by mitochondria and 2) inhibit malate uptake by a partially purified transporter fraction reconstituted into asolectin vesicles. The dicarboxylate transporter was identified by Western blotting and immunoprecipitation and had an apparent molecular mass of 26,000 Da. The techniques described should prove useful for identifying a number of membrane proteins that can be assayed in situ but are difficult to assay following dissolution of the membrane.

Pea leaf mitochondria showed complex kinetics for malate metabolism. O2 uptake increased as malate concentration increased from 0 to 10 mM, reached a plateau between 10 and 20 mM malate, and then increased again up to 40 mM malate. Analysis of the products of malate oxidation by high-performance liquid chromatography revealed that the first phase of O2 uptake coincided with the synthesis of both pyruvate and oxalacetate (OAA) while the second phase of O2 uptake at higher malate levels usually occurred with a large increase in OAA formation. The biphasic response in O2 uptake and the changing ratios of pyruvate and OAA synthesis did not appear to be the direct result of the differing Km values of malate dehydrogenase and malic enzyme. Rather, they resulted from thermodynamic properties of these two malate oxidases and the kinetics of the two NADH dehydrogenases found in plant mitochondria. At low malate concentrations the rotenone-sensitive NADH dehydrogenase was active and could accept electrons from both malate oxidases. This NADH dehydrogenase became saturated at about 10 mM malate. At higher malate concentrations the rotenone-insensitive NADH dehydrogenase was increasingly important and its increased electron transport capacity was best exploited by malate dehydrogenase. At the higher malate concentrations an increasing portion of the electrons from malate reduce O2 through the alternative oxidase. Although this coincided with the second phase of malate-dependent O2 uptake it was not required for this phase to be seen.