Timothy Brears

Overexpression of Cytosolic Glutamine Synthetase. Relation to Nitrogen, Light, and Photorespiration

In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO2 photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO2 compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.

Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco

Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.

Regulation of Genes for Enzymes Along a Common Nitrogen Metabolic Pathway

We have characterized the multigene families encoding glutamine synthetase (GS) and asparagine synthetase (AS) in Pisum sativum. The isolated GS and AS genes have been used as probes to study the expression of individual members of these gene families during various aspects of plant development. These studies have shown that chloroplast GS2 and cytosolic GS are encoded by homologous nuclear genes which are differentially expressed in vivo (18,19). The nuclear gene for chloroplast GS2 is regulated by light, phytochrome, and photorespiration (4). In contrast, two nearly identical genes for cytosolic GS (GS3A and GS3B) are expressed at highest levels in developmental contexts where large amounts of nitrogen are mobilized in plants (22). Analysis of transgenic tobacco plants containing the pea GS promoters fused to a GUS reporter gene has shown that the genes for chloroplast GS2 and cytosolic GS3A are expressed in distinct cell types (5). These transgenic experiments have demonstrated that the chloroplast GS2 and cytosolic GS3A isoforms serve distinct, non-overlapping roles in plant nitrogen metabolism. Parallel studies on the gene family for plant AS have shown that peas contain two AS genes (AS1 and AS2) (20). The AS1 gene shows a dramatic dark-induced expression, which reflects the role of asparagine as the preferred nitrogen transport compound in dark-grown plants (20). Both AS1 and AS2 are expressed coordinately with genes for cytosolic GS during germination and nitrogen-fixation (20). Our combined studies on the gene families for GS and AS should uncover the molecular basis for the coordinate regulation of genes for enzymes along a common nitrogen metabolic pathway in plants.

The Molecular Biology of Amino Acid Biosynthesis in Plants

All living organisms have a fundamental requirement for nitrogen as a component of amino acids, proteins and nucleic acids. It is not surprising, therefore, that there are broad similarities in nitrogen metabolism in plants, animals and microorganisms, and that many enzymes involved in the biochemistry of nitrogen metabolism are shared by all groups of organisms. Indeed several of the plant genes which encode enzymes involved in nitrogen metabolism have been cloned by virtue of their sequence homology to their counterparts in microorganisms and mammals. However, the physiology of nitrogen metabolism is clearly very different in plants; one example in which this is particularly apparent is the biochemistry associated with photorespiration. In recent years the cloning of some of the genes involved in nitrogen metabolism has enabled cell-specific expression and metabolic regulation type experiments to be undertaken, which in turn directly address questions relating to metabolism and physiology in plants.