Stamens and gibberellin in the regulation of corolla pigmentation and growth in Petunia hybrida

Removal of stamens, or even of only the anthers, at an early stage of corolla development, before the start of main anthocyanin production, inhibited both growth and pigmentation of attached corollas of Petunia. When only one or two stamens were removed from one side, the inhibition was restricted to the corolla side adjacent to the detached stamens. Application of gibberellic acid (GA₃) substituted for the stamens in its effect on both growth and pigmentation. In detached corollas, isolated at the early-green stage and grown in vitro in sucrose medium, GA₃ promoted growth and was essential for anthocyanin synthesis. A marked enhancement of anthocyanin production was observed 48 h before the increase in corolla growth rate. Corollas detached at later stages were able to continue their growth and pigmentation in sucrose without GA₃. When Paclobutrazol (-[(4-chlorophenyl)-ethyl]-(1,1-dimethylethyl)-H-1,2,4-triazol-1-ethanol), an inhibitor of gibberellin biosynthesis, was added to the growth medium of in-vitro-grown corollas, pigmentation was inhibited but there was no effect on corolla growth. Low levels of GA₃ counteracted the Paclobutrazol effect on pigmentation but did not affect growth. The above results indicate that the effect of GA₃ (and probably that of the stamens) on corolla growth is independent of its effect on pigmentation. Gibberellic acid and paclobutrazol had no effect on [¹⁴C]sucrose uptake by in-vitro-grown corollas. The activity of phenylalanine ammonialyase was correlated with the effect of stamens and GA₃ on pigmentation in corollas grown in vivo and in vitro.Abbreviations GA gibberellin - GA₃ gibberellic acid - PAC Paclobutrazol - PAL phenylalanine ammonia-lyase     

Biochemical differentiation in the tobacco flower probed with monoclonal antibodies

We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes cell-and tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch.Abbreviations ELISA enzyme-linked immunosorbent assay - Fg immunoglobulin - kDa kilodalton     

Comparison of de-novo flower-bud formation in a photoperiodic and a day-neutral tobacco

Regeneration of flower buds in thin tissue layers from pedicels of photoinduced short-day (SD) tobacco, Nicotiana tabacum L. cv. Maryland Mammoth, is described. Up to seven flower buds per explant were obtained in a medium containing Murashige and Skoog's macro- and microclements, 100 mg/l myoinositol, 0.1 mg/l thiamine-HCl, 6% glucose, 5 M N⁶-benzylaminopurine, and 0.5 M -naphthaleneacetic acid. Usually some vegetative buds were also formed in the pedicel thin tissue layers. Thin tissue layers from other positions in the induced SD tobacco regenerated vegetative buds only. A comparative study with a day-neutral (DN) tobacco, Samsun, showed that the capacity to form de-novo flower buds was more localized and less strongly determined in photoperiodic than in the DN tobacco. The differences between the photoperiodic and DN tobaccos in flower-bud regeneration capacity are thus quantitative and not qualitative. The basis for this quantitative difference is not known, but may depend on factors controlling production of floral stimulus (florigen) and competency of cells to respond to florigen, and-or stability of the determined state to form flower buds in vitro.Abbreviations BAP N⁶-benzylaminopurine - DN day-neutral - GA₃ gibberellic acid - LD long-day - MM Maryland Mammoth - NAA -naphthaleneacetic acid - SD short-day     

Control of the developmental pathway of tobacco pollen in vitro

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.     

Spermidine and flower-bud differentiation in thin-layer explants of tobacco

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.     

Modulation of the number of membrane-bound auxin-binding sites during the growth of batch-cultured tobacco cells

We studied the modulation of the number of membrane-bound naphthaleneacetic acid (NAA)-binding sites during the growth cycle of tobacco cells in batch culture. Both cell number and specific NAA-binding increased exponentially, but at different rates and for different periods. This caused a characteristic modulation of the number of binding sites per cell during the growth cycle: During the first day of the lag phase this number decreased; in the exponential phase it rose markedly, and in the stationary phase it was constant.Abbreviations MES 4-morpholinoethanesulphonic acid - NAA 1-naphthaleneacetic acid     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

Differential growth of georeacting maize roots

The growth rate of the two sides of 10-mm apical segments prepared from primary roots and of intact primary roots of maize has been analyzed in both vertical and horizontal positions, using a filming method allowing continuous growth recording. The data showed that the georeaction began by a decrease in the overall elongation rate of the roots. This inhibition is effective on the lower side of the bending zone, where the growth is practically stopped during the period of maximum rate of geocurvature. In contrast, the growth is slightly enhanced on the upper part of the elongating zone.     

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K_m (photoheterotrophic cells: 16.0±2.0 M; heterotrophic cells: 11.8±1.8 M) and V_max (photoheterotrophic cells: 323±50 nmol·min^-1·g^-1 dry weight; heterotrophic cells: 233±3 nmol·min^-1·g^-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DNP dinitrophenol - DW dry weight - FW fresh weight - GSH reduced glutathione     

The cellular parameters of leaf development in tobacco: a clonal analysis

The cellular parameters of leaf development in tobacco (Nicotiana tabacum L.) have been characterized using clonal analysis, an approach that provides unequivocal evidence of cell lineage. Our results indicate that the tobacco leaf arises from a group of around 100 cells in the shoot apical meristem. Each of these cells contributes to a unique longitudinal section of the axis and transverse section of the lamina. This pattern of cell lincage indicates that primordial cells contribute more or less equally to the growth of the axis, in contrast to the more traditional view of leaf development in which the leaf is pictured as arising from a group of apical initials. Clones induced prior to the initiation of the lamina demonstrate that the subepidermal layer of the lamina arises from at least six files of cells. Submarginal cells usually divide with their spindles parallel to the margin, and therefore contribute relatively little to the transverse expansion of the lamina. During the expansion of the lamina the orientation and frequency of cell division are highly regulated, as is the duration of meristematic growth. Initially, cell division is polarized so as to produce lineages that are at an oblique angle to the midrib; later cell division is in alternating perpendicular planes. The distribution of clones generated by irradiation at various stages of development indicates that cell division ceases at the tip of the leaf when the leaf is about one tenth its final size, and then ceases in progressively more basal regions of the lamina. Variation in the mutation frequency within the lamina reflects variation in the frequency of mitosis. Prior to the mergence of the leaf the frequency of mutation is maximal near the tip of the leaf and extremely low at its base; after emergence, the frequency of mutation increases at the base of the leaf. In any given region of the lamina the frequency of mutation is highest in interveinal regions, and is relatively low near the margin. Thus, both the orientation and frequency of cell division at the leaf margin indicate that this region plays a minor role in the growth of the lamina.Abbreviation MF mutation frequency     

Metabolism of myo-Inositol and growth in various sugars of suspension-cultured tobacco cells

Tobacco (Nicotiana tabacum L.) cells were cultured in a liquid medium which contained sucrose as a source of carbon and energy. Various cell-wall constituents and wall precursors (L-arabinose, D-xylose, D-galactose, D-mannose, D-glucuronate, myo-inositol) were added to cells growing in this medium to by-pass possible rate-limiting steps in the relevant metabolic pathways. None of these compounds stimulated growth as measured by increase in fresh weight; myo-inositol did cause a slight increase and L-arabinose a decrease in dry weight accumulation compared to controls grown on sucrose only. Although myo-inositol was not needed for rapid growth, tracer level amounts of [2-³H]myo-inositol were rapidly absorbed and metabolized. Label was incorporated into the uronide and pentose residues of cell walls and exocellular polysaccharide.     

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N⁶-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Importance of the cap in maize root growth

A large population of primary roots of Zea mays (cv. LG 11) was selected for uniform length at zero time. Their individual growth rates were measured over an 8-h period in the vertical position (in humid air, darkness). Three groups of these roots with significantly different growth rates were then chosen and their cap length was measured. It was found that slowly growing roots had long caps whereas rapidly growing roots had short caps. The production by the cap cells of basipetally transported growth inhibitors was tested (biologically by the curvature of half-decapped roots) and found to be significantly higher for longer root caps than that for shorter ones.     

Flower development inAntirrhinum majus L. (Scrophulariaceae) with a comment upon corolla tube formation

The inflorescence apex is stratified and has a single layered tunica, bracts as well as all the floral organs develop acropetally. Except for stamens which are initiated with the activity of deeper layers of apex, all other floral appendages are initiated with the activity of second layer. On the basis of ontogenetic studies the floral organs have been homologised with the leaves. The adjacent calyx lobes develop and remain closely appressed and do not show post-genital fusion. Corolla tube is formed due to zonal growth preceeded by joint growth of hump on the abaxial face of stamen primordia and interprimordial region between petal primordia. Thus no post-genital fusion was observed. The placentae develop concomitantly with carpellary wall.