Plants require nitrogen during their vegetative and reproductive growth phases. Nitrogen is made available to the plant through soil mineralization, the application of nitrogen fertilizer, or both. It has been estimated, however, that between 50 and 70 percent of nitrogen applied to crops is lost from the plant-soil system [Peoples, M. B. et al., “Minimizing Gaseous Losses of Nitrogen,” In Nitrogen Fertilizer in the Environment (Bacon, P. E., ed.) Marcel Dekker, pp. 565-606 (1995)]. Nitrogen is one of the most expensive plant nutrients to supply, nitrogen fertilizer is not always available at a reasonable cost, and excessive application of nitrogen fertilizer can result in environmental challenges. Corn is an example of an agronomically important plant that often requires nitrogen fertilizers to perform at its genetic potential.
Native ICDH can exist in the mitochondria, chloroplast and cytosol, with each having a different physiological impact although the catalytic action may be similar. In general, ICDH1 is found in the cytosol and ICDH2 is found in the chloroplast.
For co-factor reducing power, ICDH can use either nicotinamde adenine dinucleotide (NAD+) or nicotinamde adenine dinucleotide phosphate (NADP+), depending on which metabolic pathway it is active. Some publications indicate that the main function of ICDH may be to generate reducing power (NADH, NADPH) for other metabolic reactions, for example, in the β-oxidation of unsaturated fatty acids. Other theories include the suggestion that the reaction product, 2-oxyglutarate (OG), could be used to support amino acid synthesis via the GOGAT cycle (Hodges, M. Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation. J. Exp. Botany (2002), 53, 905). In addition, the over-expression of the ICDH enzyme in a stack with another gene or genes may allow the effective utilization of the additional carbon skeletons. A previous study of transgenic tobacco plants that overexpressed a mitochondrial icdh gene was focused on redox pathways and did not mention nor evaluate any possible impact on nitrogen utilization (Gray, G., Villarimo, A., Whitehead, C., McIntosh, L. Transgenic Tobacco (Nicotiana tabacum L.) Plants with Increased Expression Levels of Mitochondrial NADP+-dependent Isocitrate Dehydrogenase: Evidence Implicating this Enzyme in the Redox Activation of the Alternative Oxidase, Plant and Cell Physiology 2004; 45, 1413-1425).
Regulation of NAD- and NADP-dependent isocitrate dehydrogenases (NAD-ICDH, EC 1.1.1.41 and NADP_ICDH, EC 1.1.1.42) is complex due to expression, substrates, compartments and post-translational regulation. While it is unclear which ICDH version generates OG for amino acids, any such OG would have to be in, or enter, the chloroplast where nitrogen is assimilated into amino acids. The literature suggests that plant cytostolic versions of ICDH are homodimers with subunits of approximately 47 kD. Mitochondrial ICDH is suspected to have more subunits. Bacterial versions of ICDH may be monomeric and have been considered to overcome the typical regulation of expression and function that occurs with plant ICDH in plants, that is, phosphorylation may inactivate the homodimer.
Cytostolic NADP-specific ICDH catalyzes the conversion of citrate to oxoglutarate. One strategy is to design a construct containing a gene encoding a monomeric prokaryotic-type isocitrate dehydrogenase gene (icdh), and to direct overexpression of ICDH in the cytoplasm of plants. The expressed ICDH enzyme will enhance the plant's ability to utilize available nitrogen via an enhanced flow of carbon into the nitrogen assimilatory mechanism. Here, we describe the overexpression and characterization of asynthetic icdh gene based on selection from among bacterial icdh sequences and optimized for expression in corn, and the stacking of a icdh genes with other transgenes.