Oakes et al., Bio/Technology; vol. 9, pp. 982-986 (Oct. 1991). "Gene Manipulation Plant Improvement." Stadler Genet. Symp. 16th, pp. 577-603 (1984). Herrera-Estrella et al., “Photoregulation of Nuclear Gene Expression." Abstracts of papers Presented at the 1984 meeting on Molecular Biology of the Photosynthetic Appa ratus, May 9-13, 1984. Douglas et al., Proc. Natl. Acad. Sci., vol. 81, pp.3983-3987 (1984). Hurt et al., FEBS, vol. 178. No. 2, pp. 306-310 (1984). Kalderon et al., Nature, vol. 311. pp. 33-38 (1984). Cheung et al., Proc. Natl. Acad. Sci., vol. 85, pp. 391-395 (1988). Smeekens et al., TIBS, vol. 15, No. 73-76 (1990). Horsch et al., Science vol. 223, p. 496 (1984). Lewin, R., Science, vol. 237, p. 1570 (1987). Reecket al., Cell, vol. 50, p. 667 (1987). Schell and Van Montagu, "Regulated Expression of Foreign Gene of Plants". Molecular Developmental Biology, pp. 3-13 (1986). Schreier et al., EMBO.J., vol. 4, No. 1, pp. 25-32 (1985). Cocking et al. Nature, vol. 293, No. 5830, pp. 265-270 (1981). Broglie et al. 1984, Science 224:838-843. Broglie et al. 1983, Bio/Technology 1:55-61. Henera-Estrella et al., 1984, Nature 310:115-120. DeBlocket al. 1984, EMBO J3(8) 1681-1689. Ellis, R. 1984, Chloroplast Biogenesis, Cambridge Univer sity Press: Cambridge, England, pp. 1-9. Hirschberg et al. 1983, Science 222:1346-1349. Goodman et al. 1987. Science 236:48-54. Murai et al. 1983, Science 222:476-482. Shields, R. 1984, Nature 310:98. Shields, S. 1985, Nature 317:668. Joos, H. et al. (1983) Cell 32:1057-1067, Genetic Avalgeioq T-DNA transcriptions in kopaleine cebemgalls.

ABSTRACT The common nodulation genes nodA, B, and C are highly conserved between different Rhizobium species and are required for the nodulation of legumes and non-legumes. The expression of these genes in Rhizobium meliloti is under both positive and negative control. Monospecific polyclonal antibodies were used to localize the NodA and NodB proteins in the cytosol of R. meliloti. These proteins are involved in the generation of small heat-stable compounds that stimulate the mitosis of different plant protoplasts. Our experiments suggest that the NodC transmembrane protein is not involved in the synthesis of these factors. Gene fusion experiments were used to define the membrane-anchor domain which is necessary for the insertion of the NodC protein into the membrane. A highly hydrophobic transmembrane-anchor domain was found near the carboxyl terminus, separating a large extracellular domain which contains a cystein-rich cluster from a short putative intracellular domain. The domain structure of the dime...

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding Δ1-pyrroline-5-carboxylate synthase (P5CS), the first enzyme of the proline biosynthetic pathway, were isolated from a λZap-cDNA library constructed from salt stressed Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open reading frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and proB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% identity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thaliana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS-1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT-PCR approach. A rapid increase in the steady-state transcript level of both genes in roots was observed by RT-PCR upon exposure of hydroponically grown 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducible genes, yet a higher response was observed for MsP5CS-2.

Lipochitooligosaccharides (LCOs) are plant growth regulators that promote at subfemtomolar concentrations cell division in tobacco protoplasts. In response to LCO treatment, tobacco cells release a second growth factor that fully mediates the growth-promoting activities of the initial extracellular LCO stimulus. This diffusible growth factor was isolated from the protoplasts' culture filtrate and shown to be a peptide. We report that the LCO-induced mitogen released by tobacco cells and a synthetic heptadecapeptide derived from region 2 of the tobacco homolog of the early nodulin gene ENOD40 are antigenically related and qualitatively indistinguishable in their ability to stimulate cell division.

Cyclic AMP is an important signalling molecule in prokaryotes and eukaryotes1, but its significance in higher plants has been generally doubted2 because they have low adenylyl cyclase activity and barely detectable amounts of cAMP3. Here we used activation T-DNA tagging to create tobacco cell lines that can proliferate in the absence of the phytohormone auxin in the culture media4,5. The sequence tagged in one line, axi 141, was used to isolate a complementary DNA encoding adenylyl cyclase, the first from a higher plant. Sequence analysis reveals that the tobacco adenylyl cyclase is probably soluble, contains characteristic leucine-rich repeats, and bears similarity with adenylyl cyclase from the yeast Schizosaccharomyces pombe. Expression of the cDNA in Escherichia coli results in an increase in endogenous cAMP levels, and in yeast its expression functionally complements the cry1 mutation. Tobacco protoplasts treated with cAMP, or the adenylyl cyclase activator forskolin, no longer require auxin to divide. This finding, together with the observation that the adenylyl cyclase inhibitor dideoxyadenosine inhibits cell proliferation in the presence of auxin, suggests that cAMP is involved in auxin-triggered cell division in higher plants.

In particular in Germany, many general rather negative conclusions were reached regarding the possible social and industrial impact of plant biotechnology. Presently many of these conclusions are in urgent need of reevaluation. Since the impact of plant biotechnology is presently reconsidered broadly, the same should be done with regard to „Biodiesel“.

In the rhizosphere, soil bacteria of the genus Rhizobium are induced to synthesize lipo-chitooligosaccharide (LCO) signal molecules that in turn trigger the formation of nodules on the roots of leguminous plants (Dénarié and Cullimore, 1993). The important role of LCOs as plant growth regulators has stimulated considerable interest in their synthesis. Therefore, we developed simplified procedures for the synthesis of LCOs which employ both an enzymatic and a chemical step (Röhrig et al., 1995 and 1996). We demonstrated that these synthetic LCO signals, which trigger nodule organogenesis in legumes, also promote cell division of tobacco protoplasts at very low concentrations in the absence of auxin and cytokinin. Furthermore, LCOs induce the expression of AXI1 (Röhrig et al., 1995), a gene implicated in auxin signaling (Hayashi et al., 1992). More recently, using the protoplast division assay and transient expression assays with a construct in which the AXI1 promoter was linked to the GUS reporter gene, we showed that the N-octadecenoylated monosaccharide glucosamine has all structural requirements for a biological active glycolipid. The assays also revealed that the N-acylated galactosamine epimer was inactive and specifically inhibited LCO action (Röhrig et al., 1996).

Intron sequences from monocotyledonous and dicotyledonous origin were used to abolish marker gene expression in prokaryotes (Escherichia coli and Agrobacterium tumefaciens) but permit expression in selected eukaryotic systems using the eukaryotic specific splicing mechanism. A 1014 bp maize Shrunken-1 (Sh 1) intron 1 flanked by exon1 and exon2 sequences was cloned into the N-terminal of the NPT II-coding region. Transient gene expression analysis revealed that the modified neomycin phosphotransferase II (NPT II) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, is expressed in barley protoplasts, but poorly expressed in tobacco protoplasts. In dicotyledonous cells AU-rich sequences are known to be important for efficient splicing and therefore an attempt was made to improve expression of the NPT II gene, containing the Sh 1 intron 1, in tobacco by increasing the AU content from 57% to 69%. Reverse transcriptase PCR analysis of RNA from transiently expressed NPT II transcripts from tobacco protoplasts revealed that despite the increase in AU-content, NPT II was still poorly expressed. Cryptic splice sites were identified as one possible cause for missplicing of the Sh1 intron 1 in dicots and poor levels of expression. Alternatively, cloning of the 198 bp intron 2 of the potato STLS 1 gene (81% AU) into the N-terminal part of the NPT II-coding region resulted in proper expression of NPT II in tobacco as well as in barley protoplasts and abolished marker gene expression in prokaryotes. The successful insertion of an intron into a selectable marker gene which completely abolishes gene expression in prokaryotes, without affecting expression of chimeric genes in monocotyledonous and dicotyledonous plant cells provides a suitable system to reduce the number of false-positives in transgenic plant production.