ENOD40 is expressed at an early stage in root nodule organogenesis in legumes. Identification of ENOD40 homologs in nonleguminous plants suggests that this gene may have a more general biological function. In vitro translation of soybean ENOD40 mRNA in wheat germ extracts revealed that the conserved nucleotide sequence at the 5 end (region I) encodes two peptides of 12 and 24 aa residues (peptides A and B). These peptides are synthesized de novo from very short, overlapping ORFs. Appropriate ORFs are present in all legume ENOD40s studied thus far. In this case small peptides are directly translated from polycistronic eukaryotic mRNA. The 24-aa peptide B was detected in nodules by Western blotting. Both peptides specifically bind to the same 93-kDa protein, which was affinity purified from soybean nodules. Using peptide mass fingerprinting, we identified this binding protein as nodulin 100, which is a subunit of sucrose synthase. Based on our data we suggest that ENOD40 peptides are involved in the control of sucrose use in nitrogen-fixing nodules.translation ͉ short open reading frames ͉ peptide signals T he nodule on the roots of legumes is an organ induced by rhizobia in which they are hosted intracellularly and where they find an ideal environment for symbiotic nitrogen fixation. The plant genes that are specifically induced by nodulation factor-secreting rhizobia during early stages of nodule development have been termed early nodulin (ENOD) genes. Among these genes, ENOD40 is one of the earliest nodulins and appears to play an important role in root nodule organogenesis. ENOD40 is induced by nodulation factors and the phytohormone cytokinin, and its expression precedes the first cortical cell division (1, 2). In mature nodules, the expression of ENOD40 has been detected in cells surrounding vascular bundles (3, 4). In addition, this gene also is expressed at low levels in stem and root cells (3). Recent work (5) has revealed ENOD40 homologs also in the monocotyledonous plants rice and maize. ENOD40 is therefore widespread in the plant kingdom, suggesting that it may have a general biological function.A remarkable feature of legume ENOD40 genes is that they contain only short ORFs. Therefore, it was initially proposed that this gene functions as an RNA (6, 7). All ENOD40 genes studied thus far contain two highly conserved regions. Recently, it was reported that the 5Ј located conserved region I of soybean (Glycine max) ENOD40 encodes a small peptide (8). This work claimed that the peptide renders tobacco cells insensitive to high concentrations of auxin. However, these data were obtained by counting tobacco cells undergoing division and could not be reproduced by using other proliferation assays (9). Moreover, a study with transgenic clover containing an auxin-responsive promoter-␤-glucuronidase (GUS) fusion failed to show the involvement of this peptide in perturbing auxin balance (10). Although genetic approaches using translational fusions were used to study ENOD40 expression (11,12), no direct biochemica...

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed been isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

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basis of action in plants is fragmentary. Plants may synthes-Cordeiro 1 , Els Prinsen 2 , Jeff Schell 1 and Richard ize cytokinins either directly by addition of isopentenylpyro-Walden 1,* phosphate to AMP by an adenylate:isopentenyltransferase 1 Max-Planck-Institut fü r Zü chtungsforschung, Carl-von-(cytokinin synthase) producing isopentenyladenosine 5Ј Linne-Weg 10, 50829 Kö ln, Germany, and phosphate ([9R-5ЈP]iP) which in turn serves as an inter-