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Recombinant phage X177.4 contains a gene for j3 phaseolin, a major storage glycoprotein of French bean seed. A 3.8-kilobase Bgl II-Bam}Il fragment containing the entire 1700-base-pair coding region, together with 863 base pairs of 5' and 1226 base pairs of 3' flanking sequence, was inserted into the A66 Ti plasmid of Agrobacterium tumefaciens and used to transform tobacco. The level of phaseolin in the seeds of plants regenerated from cloned tissue was 1000-fold higher than in other tissues. The molecular weight of the phaseolin RNA transcript in tobacco seeds was identical to that found in bean seeds. The phaseolin protein in tobacco seed was glycosylated and appeared to undergo removal of the signal peptide. However, a large proportion of the phaseolin was cleaved into discrete peptides. These same peptides were formed as phaseolin was degraded during tobacco seed germination. The phaseolin gene appeared to be inserted as a single copy, and the proportion of phaseolin per genome copy in tobacco seeds (up to 3% of the total embryo proteins) resembled that in the bean seeds (40% of total seed protein, expressed from about 14 copies per diploid genome). Furthermore, the transplanted gene was turned on during tobacco seed development, and its protein product, phaseolin, was localized in the embryonic tissues. Finally, the phaseolin gene was inherited as a Mendelian dominant trait in tobacco.The transfer of foreign genetic information to broad-leafed plants by means (4) and maintained on Murashige and Skoog medium (MS) without hormone supplement. The tissue was cloned by the feeder plate method (9) and then placed in liquid culture to induce shoot-stem elongation (10). The shoots were grafted (11) onto 6-to 8-week-old N. tabacum var. Xanthi plants and grown at 22°C with a 16-hr photoperiod. Flowers were self-pollinated, and the seeds were allowed to mature.Quantitative and Qualitative Protein Assays. Proteins were extracted and quantified as described (8). Phaseolin was quantitated in tissues by dot-immunobinding assay (12). Protein patterns were analyzed after fractionation on a 13% polyacrylamide gel (13) or by two-dimensional gel electrophoresis (14) followed by electrophoretic immunoblot analysis with polyclonal antiserum to phaseolin (15). Antigenantibody complexes were visualized by treating the filters with 251I-labeled Staphylococcus protein A, followed by autoradiography.For immunodetection of concanavalin A-bound proteins, tissue extracts were incubated with concanavalin ASepharose beads followed by elution of the bound fraction with 1% NaDodSO4. The bound and unbound fractions were subjected to NaDodSO4/polyacrylamide gel electrophoresis followed by immunoblot analysis as described earlier.Isolation of RNA and Blot-Hybridization Analysis. Total RNA was isolated from leaves as described (16). RNA from developing seeds was prepared by isolation of polysomes followed by phenol extraction of the polysome pellet (17 3320The publication costs of this article were defrayed in part by page charge pay...

Zeins, the seed storage proteins of maize, are a group of alcoholsoluble polypeptides of different molecular masses that share a similar amino acid composition but vary i n their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal i s to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whelher this protein would be stable i n nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, p-phaseolin, in nonseed tissues and to determine the basis for i t s stability/instability. We have introduced the 15-kD zein and bean p-phaseolin-coding sequences behind the 35s cauliflower mosaic virus promoter into tobacco (Nicofiana fabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein i s stable not only i n seeds but in all nonseed tissues tested, whereas the P-phaseolin protein accumulated only i n mid-and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the p-phaseolin protein i n nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention i n the ER or to the proteaseresistant nature of the protein.Seed storage proteins constitute a potentially useful class of proteins for the improvement of forage crops if they can be made to accumulate in leaves. It is a fairly simple genetic engineering feat to introduce a seed protein-coding sequence behind a strong constitutive promoter into transgenic plants and ensure high rates of synthesis of the corresponding protein. However, stability of the protein in an alien environment is still not clearly defined and has to be treated on a case by case basis.Seed proteins are synthesized during seed development and accumulate in protein bodies. These proteins are then utilized by the emerging seedling during germination. The major seed protein in dicotyledonous plants are the saltsoluble globulins, which are stored in vacuole-derived protein bodies. Most monocot seed storage proteins are '

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

SummaryHigher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS 1 ) or in the chloroplast (GS 2 ). Glutamine synthetase 1 is regulated in different plants at the transcriptional level and there are some reports of regulation at the level of protein stability. Here we present data that clearly establish that GS 1 in plants is also regulated at the level of transcript turnover and at the translational level. Using a Glycine max (soybean) GS 1 transgene, with and without its 3¢ untranslated region (UTR), driven by the constitutive CaMV 35S promoter in Medicago sativa (alfalfa) and Nicotiana tabacum (tobacco), we show that the 3¢ UTR plays a major role in both transcript turnover and translation repression in both the leaves and the nodules. Our data suggest that the 3¢ UTR mediated turnover of the transcript is regulated by a nitrogen metabolite or carbon/nitrogen ratios. We also show that the 3¢ UTR of the gene for the soybean GS 1 confers post-transcriptional regulation on a reporter gene. Our dissection of post-transcriptional and translational levels of regulation of GS in plants shows that the situation in plants strongly resembles that in other organisms where GS is regulated at almost all levels. Multistep regulation of GS shows the high priority given by organisms to regulating and ensuring optimal control of nitrogen substrates and preventing overproduction of glutamine and drainage of the glutamate pool.

Glutamine synthetase (GS) is the key enzyme in ammonia assimOxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ⅐OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels.GS (EC 6.3.1.2) is a key enzyme in nitrogen metabolism. It catalyzes the biosynthesis of Gln from Glu, ATP, and ammonium. GS from bacteria consists of 12 identical subunits arranged in two hexamers stacked face to face; the side-to-side interface of a pair of subunits constitutes an active site containing two Mn 2ϩ ions (Yamashita et al., 1989). The two divalent metal ions in the active site are distinguished by their dissociation constants (Villafranca et al., 1985). Saturation of the high-affinity site, n 1 , in each subunit by Mn 2ϩ or Mg 2ϩ induces a conformational change, converting the enzyme from a catalytically inactive to a catalytically active conformation (Hunt and Ginsburg, 1980). The metal ion at the n 1 site also plays a catalytic role in the binding of Glu, whereas the second metal ion, n 2 , is involved in the binding of ATP (Hunt and Ginsburg, 1980; Liaw et al., 1993).In bacteria GS has been shown to be regulated by cumulative feedback inhibition, covalent modification, and repression/derepression (Stadtman, 1990). Although normally stable, bacterial GS is turned over when cells are starved for nitrogen (Fulks and Stadtman, 1985), suggesting that the intracellular level of GS in bacterial cells is also regulated by proteolysis. The degradation of GS in Escherichia coli and Klebsiella aerogenes appears to involve two steps: (a) the enzyme is inactivated by oxidative modification of a single His residue per subunit (Levine, 1983a; Rivett and Levine, 1990) and (b) the altered enzyme is then degraded by endogenous proteases that are capable of degrading the oxidized enzyme but exhibit little activity on native GS (Roseman and Levine, 1987;Stadtman and Berlett, 1997).In plants GS is an octamer and has a native molecular mass of approximately 320 to 380 kD (Stewart et al., 1980). Conservation in the amino acid sequence in the active site of GS across kingdoms suggests that plant GS is mechanistically similar to bacterial GS (Shatters and Kahn, 1989; Sanangelantoni et al., 1990). There...

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH 4 ϩ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS 1 ) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of ␤-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-␤-glucuronidase gene construct, suggesting that the transcript for the GS 1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS 1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS 1 transcripts when fed with high levels of NO 3 Ϫ . However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS 1 is regulated at the level of RNA stability and protein turnover.

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