Igor C. Oliveira

Glutamate-receptor genes in plants.

In animal brains, ionotropic glutamate receptors (GluRs) function as glutamate-activated ion channels in rapid synaptic transmission. We have now discovered that genes encoding putative ionotropic GluRs exist in plants, and we present preliminary evidence for their involvement in light-signal transduction. It may be that signalling between cells by excitatory amino acids in animal brains evolved from a primitive signalling mechanism that existed before the divergence of plants and animals. Our findings also help to explain why neuroactive compounds made by plants work on receptors in human brains.

Use of Arabidopsis Mutants and Genes To Study Amide Amino Acid Biosynthesis

Studies of enzymes involved in nitrogen assimilation in higher plants have an impact on both basic and applied plant research. First, basic research in this area should uncover the mechanisms by which plants regulate genes involved in a metabolic pathway. Second, because nitrogen is a rate-limiting element in plant growth (Hageman and Lambert, 1988), it may be possible to increase the yield or improve the quality of crop plants by the molecular or genetic manipulation of genes involved in nitrogen assimilation. Research on nitrogen assimilation into amino acids has been complicated by the fact that some of these reactions are catalyzed by multiple isoenzymes located in distinct subcellular compartments. With traditional biochemical approaches, it has been impossible to sort out the function of each isoenzyme in plant nitrogen metabolism. The discovery that genes for chloroplastic and cytosolic isoenzymes of glutamine synthetase (GS) are expressed in distinct cell types (Edwards et al., 1990; Carvalhoet al., 1992; Kamachi et al., 1992)suggeststhat traditional biochemical studies, which begin with tissue disruption, artificially mix isoenzymes that may not coexist in the same cell type in vivo. Thus, in vitro biochemical methods commonly used to define the rate-limiting enzyme in a pathway in unicellular microorganisms may lead to erroneous interpretations when employed to study plant metabolic pathways. An alternative way to define the in vivo function of a particular isoenzyme or to define a rate-limiting enzyme in a pathway is by mutant analysis, as shown by studies of Escherichia coli and yeast. Plant mutants defective in particular isoenzymes of GS or ferredoxin-dependent glutamate synthase (Fd-GOGAT) have been identified in screens for photorespiratory mutants in Arabidopsis and barley (Somerville and Ogren, 1980,1982; Wallsgrove et al., 1987). More recently, Arabidopsis mutants with alterations in the activity of additional enzymes of nitrogen assimilation have been identified using a screening method that does not depend on a growth phenotype (Schultz and Coruzzi, 1995). The in vivo role of the mutated isoenzyme

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.

CCAAT Box Enhancer Binding Protein (C/EBP-) Stimulates B Element-mediated Transcription in Transfected Cells

A construct comprising three tandemly repeated copies of the κB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-κBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-κB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-κBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein α (C/EBP-α) (but not C/EBP-β or C/EBP-). Stimulation by C/EBP-α required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-κBCAT was seen after co-transfection with C/EBP-α in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-κB proteins, we co-transfected C/EBP-α with NF-κB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP-α reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a κB probe revealed that the addition of recombinant C/EBP-α protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-κB proteins had no effect. Our findings show that C/EBP-α may cause activation of some κB element-containing genes lacking C/EBP binding sites.

Recent Progress in the Elucidation of Interferon-Gamma Actions: Molecular Biology and Biological Functions

It has been known for a long time that the two major classes of interferons, IFN-alpha/beta and IFN-gamma, are similar in many of their actions despite the fact that they are structurally unrelated and bind to separate receptors. Recent studies revealed overlapping, common components in IFN-alpha/beta and IFN-gamma signaling. The second chain of the IFN-gamma receptor was recently identified. The generation of mice with targeted disruptions of the genes for IFN-gamma or the IFN-gamma receptor is helping to understand the essential functions of IFN-gamma in host defenses. Other studies have shown that the transcription factor IRF-1 is essential for the IFN-gamma-mediated activation of the gene for the inducible nitric oxide synthase.