We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast acel (activating gopper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the 3-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 ,M CuS04 to the nutrient solution or after application of 0.5 ,uM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal ofcopper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu 2؉ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.

No abstract

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P2 isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N2 fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P2 but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P, isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P1 was three orders of magnitude lower than for AAT-P2. Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P2.MAbs' have proven to be useful diagnostic reagents with many applications in the study of tissue of legumes. AAT-P2 is nodule specific and induced during rhizobial infection of roots concomitant with the onset of N2 fixation and is associated with the proplastid fraction (1). Both enzymes have been separated, purified, and characterized enzymically (13). This is the first report of monoclonal antibodies prepared against plant AAT, although there is a recent report of a polyclonal antibody against an AAT isoform from Panicum maximum (1 1). Polyclonal antibodies have been produced against animal enzymes (17) to study the evolution rates of vertebrate aminotransferases, but this was not extended to plants (17). MAbs have been produced against porcine mitochondrial AAT (20). This paper reports the production and characterization ofa range ofMAbs generated against AAT-P2 MATERIALS AND METHODS Plant Growth and Rhizobium InoculationLupin seeds (Lupinus angustifolius [L] cv Uniharvest) were purchased from Dalgety N.Z. Ltd. Seeds were surface-sterilized, germinated on agar, and transferred to sterile pumice troughs before inoculation with Rhizobium lupini NZP2257. Plant/Rhizobium cultures were grown either in glass houses during summer (day length 2 12 h) or in a controlled environment growth cabinet with a day length of 12 h and a day/ night temperature regime of 24°C/2 1°C (14). Purfication of AAT-P2Nodules (18-21 d old) were harvested and homogenized in two volumes of 50 mM Tris (pH 8.0) containing 0.4 M sucrose and 50 ,ug/mL pyridoxal phosphate. AAT-P, and AAT-P2 were separated and partially purified by ammonium sulfate fractionation, gel filtration on Sepharose CL-6B, and ion exchange chromatography on DEAE Sepharose essentially as described prev...