Regulation of gene expression during plant cell differentiation

At the cellular level, the means whereby plants attain their final form is achieved by the controlled integration of the processes of cell division and cell expansion. In the development of the seed into the adult plant, these integrated processes function to define anatomical sites called meristems out of which all cells of the plant are subsequently derived. In order to characterize the molecular mechanisms active at the meristems, a copy DNA (cDNA) expression library was constructed in bacteriophage lambda gt 11 using messenger RNA (mRNA) isolated from a homogeneous population of tobacco (Nicotiana tabacum L. cv xanthi) cells that had been induced to undergo rapid proliferation as a cell suspension culture. A heterologous antibody specifically raised against a protein abundantly accumulated during proliferative, non-differentiated growth in Sorghum was employed to select two different, non-overlapping cDNA recombinants. The recombinants were characterized through Southern and Northern analysis and DNA sequencing. Northern analysis indicated that the two recombinants cDNAs (named c2 and c10) are complementary to mRNA molecules of identical size which are subject to an identical form of developmental regulation in tobacco plants. The mRNAs are present in tissues that are actively involved in cell division (callus, cell suspensions, germinating seeds and tissues from early stages of seed development) but are absent from differentiated tissues (flowers, roots, expanded leaves and later stages of seed development). High steady-state levels of these mRNAs are induced in excised leaves under conditions that promote the induction of callus but not in intact leaves treated with 2,4-D. The mRNAs are present at high levels in green callus and cell suspensions suggesting that expression is not affected by light or photosynthesis. They are also present in heterologous tissues (N. sylvestris callus and suspension culture cells). The amino acid sequence derived from one cDNA sequence bears similarity to the DNA binding domains of zinc-finger proteins and activator domains of transcription factors involved in development. Synthetic peptides derived from the cDNAs were employed for the production of specific antibodies, which were subsequently employed for analysis of Western blots of proteins extracted from plant tissues. Affinity-purified antibody directed against a peptide comprising a zinc finger specifically identified a callus associated protein of identical molecular weight as the one recognized by the heterologous serum. Analysis of genomic digests indicated that the cognate gene(s) were present at a very low copy number in tobacco as well as in two other dicotyledonous species (N. sylvestris and Petunia hybrida). Tobacco genomic clones complementary to both cDNA were isolated, mapped and characterized