T-DNA TAGGING REVEALS A NOVEL CDNA TRIGGERING CYTOKININ-INDEPENDENT AND AUXIN-INDEPENDENT PROTOPLAST DIVISION

Activation T-DNA tagging was used to generate four cytokinin-independe nt (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lin es displayed similar phenotypes: reduced apical dominance, poorly deve loped roots, delayed growth and flowering, and male and female sterili ty. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both c ytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1 was used to isolate an active cDNA, cyi1a, that was able to trigger cytok inin-and auxin-independent protoplast division. Northern analysis show s that the transcript corresponding to cyi1a accumulates to high level s in the untransformed protoplasts shortly before the onset of cell di vision, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activ ity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.