A comparison of in vitro flowers to in vivo flowers of haploid and diploidNicotiana tabacum L.

Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.     

Glutathione synthetase in tobacco suspension cultures: catalytic properties and localization

Glutathione synthetase activity (EC 6.3.2.3) was analysed in ammonium sulfate precipitates of extracts l'rom photohetevotrophically grown cells of Nicotiana tabactm L. cv. Samsun by determination of glutathione as its monobromobimane derivative. Maximal enzyme activity was obtained at pH 8.0–9.0 in Tris-HCl and CHES as buffer systems. The enzyme showed an absolute requirement for Mg2+ and was slightly stimulated by K+. When Mg2+ was replaced by Mn2+ less synthetase activity was observed, and above 30 m M Mn2+ no activity was found. The enzyme was specific for glycine (KM = 0.308 m M ). No product formation was observed with ß -alanine and γ y-aminobutyrate using substrate conccntrations of 10 m M . The apparent KM values for γ -glutamylcysteine and γ -glutamyl- l -α-aminobutyrate were, respectively, 0.022 and 0.033 m M . By chloroplast Isolation ca 24% of the total glutathione synthetase activity of the cells could be shown to be localized in the chloroplasts, the rest being attributed to the cytoplasm of the tobacco cells.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Uptake and metabolism of benzyladenine in the early stage of flower bud development in vitro in tobacco

Benzyladenine (BA) was found to regulate the number of flower buds regenerated in vitro from pedicel tissue of tobacco. Flower bud induction was particularly sensitive to BA levels in the range of 0.45 to 1.0 μ M , where a two-fold increase in concentration caused a threefold rise in the number of buds. When tissues were fed radioactive BA for 24h, only 9–12% of the counts were recovered in the original compound. The rest was present in metabolites, tentatively identified as the mono-, di- and triribotides, 7- and 9-glucosides and 9-riboside of BA. The amount of growth regulator taken up and the quantities of BA and its metabolites in the explants were all linearly related to the concentration of the medium. The internal BA concentration was ca 60% of the level in the medium after 24 h. When the concentration in the medium was raised, relatively more BA remained in the non-conjugated form. However, this change in the equilibrium between BA and the conjugates is too small to account for the steep rise in the curve representing concentration vs effect between 0.45 and 1.0 μ M .     

Salicyloylaspartate as an endogenous component in the leaves of Phaseolus vulgaris

An amide conjugate of o-methoxybenzoic acid and aspartic acid has been isolated from bean leaves. After extraction and methylation of plant material, this compound was isolated as two isomeric monoethyl monomethyl esters. The ethylation of the aspartyl carboxyl groups was shown to be a likely result of an extraction procedure utilising acidified ethanol, the methylation of the aromatic hydroxy of the methoxy group to be due to the derivatisation procedure. Studies with pentafluorobenzylation confirmed that the endogenous compound is o-hydroxybenzoylaspartate.     

Absolute configurations of pittosporatobiraside A and B from Pittosporum tobira

The absolute configuration at C-12 of pittosporatobiraside A and B isolated from the leaves of Pittosporum tobira was determined to be S on the basis of the exciton chirality of their dibenzoate derivative. The structures of the two glycosides were thus established to be (1S,9S,10S,11S,12S,14R,16R)-12-[(Z)-2-methyl-1-oxo-2-butenyl]-6,14-dimethyl-2-methylene-9-(1-methylethyl)-15,17-dioxatricyclo[8.7.0.0^11,16]heptadec-5-en-13-one and (1S,9S,10S,11S,12S,14R,16R)-12-(3-methyl-1-oxo-2-butenyl)-6,14-dimethyl-2-methylene-9-(1-methylethyl)-15,17-dioxatricyclo [8.7.0.0^11,16]heptadec-5-en-13-one, respectively.     

Responses of stomata of senescing and nonsenescing leaves of Nicotians glauca to changes in intercellular concentrations of leaf carbon dioxide

Abstract. Gas exchange measurements were performed to test the hypothesis that failure of stomata to open in senescing leaves of Nicotiana glauca is caused by elevated concentrations of carbon dioxide in the intercellular spaces of leaf mesophyll tissue (c_i). Senescing leaves selected for experiments were completely chlorotic and lacked positive rates of photosynthesis. When stomata in detached epidermis from senescing leaves were illuminated in CO2-free air, they opened to similar apertures as those in detached epidermis from nonsenescing leaves. To compare the effects of changes in c_i on stomatal responses of the two leaf types, leaf 'flags' of either nonsenescing or senescing leaves were illuminated at a photosynthetic photon flux density of 500 μmol m⁻² s⁻¹ in a gas exchange cuvette. Leaf temperatures were maintained at 23.5 ± 0.5°C, and vapour pressure differences between leaves and the air were maintained between 0.70 and 0.75kPa. C_i was adjusted by changing external concentrations of carbon dioxide in air circulating through the cuvette. Conductances and photosynthetic rates of nonsenescing leaves changed in response to changes in c_i, but neither the conductances nor the photosynthetic rates of senescing leaves were affected significantly by changes in q. We conclude that guard cells of senescing leaves of Nicotiana glauca do not lose the capacity to respond to changes in carbon dioxide concentration and that increases in c_i resulting from declining rates of mesophyll photosynthesis are not the sole cause of maintenance of stomatal closure during leaf senescence. The data suggest that factors external to guard cells may prevent them from responding to changes in carbon dioxide concentrations in intact senescing leaves.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.