Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max).

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 microM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots.

Recognition of Peroxisomes by Immunofluorescence in Transformed and Untransformed Tobacco Cells.

We report the visualization of peroxisomes in tobacco (Nicotiana tabacum) leaves using fluorescently labeled antibodies to glycolate oxidase. In transgenic tobacco leaves the expression of isocitrate lyase was also visualized. In dual probing experiments both enzymes were shown to be present together in all peroxisomes in transgenic tobacco leaves.

Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes.

The cDNA encoding castor bean endosperm isocitrate lyase (ICL) was expressed under the control of the promoter of the small subunit of pea ribulose bisphosphate carboxylase in transformed tobacco. ICL protein was detected using anti-ICL antibodies on immunoblots of total leaf protein extracts. Nycodenz density gradient separation of the extracts from the transgenic tobacco leaves showed ICL co-fractionated with hydroxypyruvate reductase, a peroxisomal matrix marker protein, and away from lactate dehydrogenase, a cytosolic marker protein. Immunoelectron microscopy of ultrathin leaf sections demonstrated the location of ICL within the matrix of the leaf peroxisomes of the transgenic plants. In vitro transcribed and translated ICL was also imported into leaf peroxisomes isolated from germinating sunflower seeds. The in vivo and in vitro import of this protein into leaf peroxisomes provides strong support for the notion that the import machinery of glyoxysomes and peroxisomes is very similar.

Physical map and properties of a 90-MDa plasmid of Azospirillum brasilense Sp7.

Homology was previously detected between the DNA restriction fragments containing Rhizobium meliloti nodulation genes and the 90-MDa plasmid, p90, of Azospirillum brasilense Sp7. Two DNA loci from Sp7 genome that complement mutations in the exopolysaccharide synthesis genes, exoB and exoC, of R. meliloti were also shown to be present on the plasmid. A more detailed characterization of the plasmid was undertaken to establish its physical map and to localize the nod homologies and other specific regions. Six loci were mapped, the region homologous to the nodulation genes, nodPQ, of R. meliloti, the exoB and exoC mutation-correcting loci, a locus for Ap resistance, a bla homology region different from the Ap resistance locus, and a region necessary for the maintenance of p90 as an independent replicon. Mobilization into Agrobacterium tumefaciens of p90-Tn5-Mob was obtained at a frequency of 10(-4), with the plasmid helper pJB3JI. Self-transfer of p90 was not demonstrated. Fragments of p90 hybridized with a plasmid of 90 MDa present in most A. brasilense and some A. lipoferum strains, suggesting a plasmid family in Azospirillum.

Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis.

Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K. W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol., 1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the wild type strain. Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size. Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis. Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location. This is the first report on plasmid localization of genes in Azospirillum.