New insights into peripherin expression in cochlear neurons.

Peripherin is an intermediate filament protein that is expressed in peripheral and enteric neurons. In the cochlear nervous system, peripherin expression has been extensively used as a differentiation marker by preferentially labeling the type II neuronal population at adulthood, but yet without knowing its function. Since the expression of peripherin has been associated in time with the process of axonal extension and during regeneration of nerve fibers in other systems, it was of interest to determine whether peripherin expression in cochlear neurons was a static phenotypic trait or rather prone to modifications following nerve injury. In the present study, we first compared the expression pattern of peripherin and beta III-tubulin from late embryonic stages to the adult in rat cochlea. The staining for both proteins was seen before birth within all cochlear neurons. By birth, and for 2 or 3 days, peripherin expression was gradually restricted to the type II neuronal population and their projections. In contrast, from postnatal day (P) 10 onwards, while the expression of beta III-tubulin was still found in projections of all cochlear neurons, only the type I population had beta III-tubulin immunoreactivity in their cell bodies. We next investigated the expression of peripherin in axotomized cochlear neurons using an organotypic explant model. Peripherin expression was surprisingly re-expressed in a vast majority of neurons after axotomy. In parallel, the expression and localization of beta III-tubulin and peripherin in dissociated cultures of cochlear neurons were studied. Both proteins were distributed along the entire neuronal length but exhibited complementary distribution, especially within the projections. Moreover, peripherin immunoreactivity was still abundant in the growth cone, whereas that of beta III-tubulin was decreasing at this compartment. Our findings are consistent with a model in which peripherin plays an important structural role in cochlear neurons and their projections during both development and regenerative processes and which is compatible with the assumption that frequently developmentally regulated factors are reactivated during neuronal regeneration.

Enzymic potential for fructose 6-phosphate phosphorylation by guard cells and by palisade cells in leaves of the broad bean Vicia faba L.

Guard cells and palisade cells were dissected from freeze-dried leaflets of the broad bean, Vicia faba L. Individual cell samples (6-12 ng) were assayed for ATP-dependent and pyrophosphate-dependent phosphofructokinases. The assay indicator, NADH loss, was monitored in real time in oil droplets with a computer-driven microfluorometer. On a protein basis, both activities were 10-fold higher in guard cells than in palisade cells, indicating (i) elevated carbon metabolism in guard cells to meet demands for energy and carbon skeletons required during stomatal opening, and (ii) parallel glycolytic pathways in guard cells, one responsive to the potent regulatory metabolite fructose 2,6-bisphosphate and the other not. Future work will be devoted to clarifying the roles of the cytosolic and chloroplastic compartments in guard cells.

Malate content of picoliter samples of Raphanus sativus cytoplasm.

Malate, which plays many essential roles in plant metabolism, is a potent in vitro inhibitor of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC). Because PEPC activity leads to malate biosynthesis, malate is assumed to attenuate its own synthesis in situ. To test this hypothesis, we measured directly the malate content of picoliter samples of Raphanus root-hair cytoplasm using quantitative histochemical techniques. We also obtained an estimate for malate accumulation in these cells. These values were compared with the PEPC activity of individual root hairs (less than 2 ng). The results indicate that high cytoplasmic malate concentration does not severely inhibit PEPC in situ. We suggest that the focus for studies on the regulation of organic anion accumulation be on the interactive effects of malate and other PEPC effectors.

Effect of GA(4 + 7) on the initiation and development of the inflorescence bud of evergreen azalea.

Apical buds of evergreen azalea (Rhododendron sp.) were treated with GA(4 + 7) at different stages of development. Treatment of vegetative buds stimulated shoot growth, slightly delayed both flower initiation and development, but increased the number of flower primordia. Treatment at the time of floral transition induced bud abortion at an early stage of the reproductive development. Treatment of inflorescence buds which contained at least one complete flower substituted for chilling in overcoming dormancy and prevented inflorescence bud abortion.

Elevation in the Sucrose Content of the Shoot Apical Meristem of Sinapis alba at Floral Evocation.

Nanogram tissue samples from apical meristems of Sinapis alba were assayed for sucrose, total soluble hexosyl equivalents ( identical with glucose and fructose plus hexoses from sucrose hydrolysis), and total soluble glucosyl equivalents ( identical with glucose plus glucose from sucrose hydrolysis). On dry weight basis, sucrose concentration increased by more than 50% within 10 hours after the start of either a long photoperiod or a short photoperiod displaced by 10 hours in the 24-hour cycle (;displaced short day'). (These treatments induce flower initiation) Glucose and fructose concentrations were close to zero in vegetative meristems and remained low compared to sucrose in meristems of induced plants. Within a single meristem, the peripheral and the central zones had similar concentrations of sucrose. Our results indicate that an early physiological event in floral transition is the accumulation of sucrose in the meristem.

Changes in adenine-nucleotide content in the apical bud of Sinapis alba L. during floral transition.

The total adenylate pool of the apical buds of vegetative plants of Sinapis alba L. continuously grown in short days fluctuates over a 24-h cycle with the minimum occurring at the end of the dark period. In the buds of plants induced to flower by a single long-day treatment, total adenylate pool increases above the control level 16 h after the start of the long day, resulting mainly from a rise in ATP and ADP contents. This occurs 6 h after the increase in the soluble carbohydrate content previously shown to occur in the apical buds of plants induced to flower (Bodson 1977, Planta 135, 19-23). A transient rise of the energy charge occurs 22 h after the start of the inductive long day.

Promotion of Flowering in Brassica campestris L. cv Ceres by Sucrose.

Flower initiation of the quantitative long-day plant Brassica campestris cv Ceres was earlier and at a lower final leaf number when sucrose was added to the medium in which plants were grown in sterile culture. The optimal concentration of sucrose was 40 to 80 millimolar. This flower-promoting effect of sucrose was not osmotic, as mannitol, sodium chloride, and polyethylene glycol were not effective at equal osmotic potentials.Seedlings grown heterotrophically after treatment with 4-chloro-5-(dimethylamino)-2-phenyl-3-(2H)-pyridazinone to prevent chlorophyll accumulation were also induced to form flower primordia earlier as the sucrose concentration in the medium was increased up to 80 millimolar. Inclusion of 4 millimolar sodium nitrate in the culture medium of green plants did not reduce the flower-promoting effects of sucrose but delayed initiation in plants grown without added sucrose.Removal of CO(2) during a single main or supplementary light period, or both, greatly reduced flower initiation. It is concluded that sucrose may be an important controlling factor determining floral initiation in Brassica.

Flowering in Xanthium strumarium: INITIATION AND DEVELOPMENT OF FEMALE INFLORESCENCE AND SEX EXPRESSION.

Vegetative plants of Xanthium strumarium L. grown in long days were induced to flower by exposure to one or several 16-hour dark periods. The distribution of male and female inflorescences on the flowering shoot was described, and a scoring system was designed to assess the development of the female inflorescences. The time of movement of the floral stimulus out of the induced leaf and the timing of action of high temperature were shown to be similar for both the apical male and lateral female inflorescences.Strong photoperiodic induction of the plants favored female sex expression, while maleness was enhanced by exogenous gibberellic acid. The problem of the control of sex expression in Xanthium is discussed in relation to the distribution pattern of male and female inflorescences on the flowering shoot and to the state of the meristem at the time of the arrival of the floral stimulus.

Cytokinin as a Possible Component of the Floral Stimulus in Sinapis alba.

Results of previous investigations indicated that one of the early and essential events occurring in the apical meristem of Sinapis alba L. during the transition to flowering is the release to mitosis of the G(2) nuclei; the trigger to mitosis is generated in the leaves and its movement out of the leaves begins around 16 hours after the start of the inductive treatment. The mitotic wave in the meristem culminates 10 hours later.In this paper, it is shown that a single application of a cytokinin (benzyladenine or zeatin) at concentrations ranging from 1 to 20 mug/ml directly to the apical bud of vegetative plants, at a time corresponding to the time of movement of the mitotic trigger in induced plants, produces a mitotic wave which is very similar to that found in induced plants. It is thus proposed that the mitotic component of the floral stimulus in Sinapis is a cytokinin. As the cytokinins are completely unable to induce flowering, it appears that there is a multicomponent floral stimulus in this species.

Changes in the carbohydrate content of the leaf and the apical bud of Sinapis during transition to flowering.

Vegetative plants of Sinapis alba L. were induced to flower by a single long day of 20 h or by a single short day of 8 h starting at an unusual time of the 24-h cycle ("displaced short day"). The soluble sugar and starch contents of the just-expanded leaf and the apical bud were measured at various times after the start of each of these two photoinductive treatments. Associated with the induction of flowering there were temporary increases in the soluble sugar and starch contents of the leaf and of the bud. These increases were apparent 14 h after the start of the long day and 12 h after the start of the displaced short day. The starch content of the bud increased later. These results indicate that an increase of the soluble sugar content of the bud is required for its transition from the vegetative to the reproductive condition.