This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.

The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink--source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.

Transgenic tobacco lines expressing Arath-CYCD2 or Arath-CYCD3 genes under a cauliflower mosaic virus 35S promoter are modified in the timing of their development, but not in the phenotype of their vegetative organs. They display an increased rate of leaf initiation, which is shown to be associated with distinct changes in the structural organization of their shoot apical meristem (SAM). Constitutive expression of Arath-CYCD2 leads to a progressive modification of the SAM structural organization with predominant periclinal divisions in the L3 layer and to the loss of the classical cytophysiological zonation, the central zone being reduced to the central cells of the L1 and L2 layers. These changes reveal a particular sensitivity of the corpus cells (L3) to Arath-CYCD2 over-expression and suggest a role for CYCD2 in controlling the planes of cell division in these cells. The SAM structural modifications in the Arath-CYCD3 over-expressing lines are less drastic; only an increased cell number together with a reduced cell size, particularly in the L1 layer, characterizes the peripheral zones. This could be related to the shortening of the G1-phase duration that renders cell growth incomplete between successive mitoses. Cell proliferation continues beyond the SAM in the developing internodes and confers a delayed senescence to Arath-CYCD3 over-expressing juvenile tissues. In addition, the ploidy levels of mature stem tissues in both types of transgenic lines are unaffected, suggesting that the studied G1 to S cell-cycle genes have no effect on the extent of endoreduplication in tobacco stem tissues.

Lentil root statocytes show a strict structural polarity of their organelles with respect to the g vector. These cells are involved in the perception of gravity and are responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles and could also be involved in the transduction of the gravitropic signal. A pre-embedding immunogold silver technique was carried out with a monoclonal antibody in order to study the distribution of actin cytoskeleton in the statocytes at the electron microscopic level. Some areas were never labelled (cell wall, vacuole, nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm always close to, or on the nuclear and amyloplast envelopes and the tonoplast. Associations of 2 to 6 dots in file were observed, but these short files were not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 micron. This work demonstrated that each statocyte organelle was enmeshed in an actin web of short filaments arranged in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of immunogold labelling. The diffuse fluorescence of the cytoplasm could be explained by the fact that the meshes of the web should be narrow. The vicinity of actin and of the amyloplasts envelope could account for the movement of these organelles that was observed in spatial microgravity.