Ferulic acid: a substrate for two isoperoxidases from Nicotiana tabacum tissue cultures

An anodic isoperoxidase (A₂) from tobacco tissue culture W-38 and a cathodic isoperoxidase (C₄) from tobacco tissue suspension culture WR-132 have been separated and characterized. Both isoperoxidases catalysed oxidation of ferulic acid in the presence of H₂O₂. When the reaction mixture was subjected to TLC, ferulic acid was found to have been converted to an unknown compound which, after treatment with ammonia, fluoresces green in UV light. Both the isoperoxidases A₂ and C₄ appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The Kms for guaiacol are 4 and 4·5 mM for isoperoxidases C₄ and A₂, respectively. The pH optimum for both enzymes is about 6·0. The effect of various phenolic and related compounds on the activity of each isoperoxidase is reported and discussed.     

β-d-glucosidase activity in developing leaves of Nicotiana tabacum

Tobacco (Nicotiana tabacum L. cv. Burley 21) leaves were assayed for β-d-glucosidase activity, using esculin as substrate. The enzymatically produced esculetin was silylated and quantitatively measured by GLC, using a tritium foil electron-capture detector. In field-grown plants, the activity in mid-stalk leaves increased with plant maturation; conversely, the activity in the top leaves decreased.     

A damascone derivative from Nicotiana tabacum

A new member of the damascone series, 4-hydroxy-β-damascone, was identified in the steam distillable oil from Virginia tobacco.     

Volatile acids of sun-cured greek Nicotiana tabacum

The volatile acids of sun-cured Greek tobacco have been studied. Examination of this material by GC-MS supplemented by other spectroscopic methods and in some instances by synthesis, has permitted the identification of nearly a hundred compounds. About half of them have not been encountered previously in tobacco or tobacco smoke, and the majority of the new compounds are straight and branched-chain unsaturated acids and aromatic acids. Five of the oxygenated acids are evidently seco- or nor-terpenoids.     

Scopoletin: A substrate for an isoperoxidase from Nicotiana tabacum tissue culture W-38

Scopoletin was found to be a substrate for a single anodic isoperoxidase isolated from tobacco callus tissue W-38. Isolation of this peroxidase was accomplished using DEAE-cellulose chromatography. This isoperoxidase catalysed the destruction of scopoletin in the presence of H₂O₂ only. An enzyme assay for the scopoletin reaction was developed. The pH optimum of the enzyme was 5·5 and the apparent K_ms for scopoletin and H₂O₂ were 0·6 and 0·9 rnM respectively.     

Pectolytic enzyme activity from Nicotiana tabacum pollen

Ungerminated pollen of Nicotiana tabacum contains a pectolytic enzyme which has its optimal activity between pH 5.5 and 6.5. Pectic lyase was not detected.     

An acidic xylan from extracellular polysaccharides of suspension-cultured cells of Nicotiana tabacum

An acidic xylan was isolated from the extracellular polysaccharides of suspension-cultured tobacco cells. Its structure was investigated by methylation analysis and ¹³C NMR spectroscopy and was shown to consist of a main chain of β-(1→4)-linked D-xylopyranosyl residues to which were attached as side-chains, α-D-glucuronic acid residues at O-2.     

Sesquiterpenoid phytoalexins from suspended callus cultures of Nicotiana tabacum

Treatment of suspended callus cultures of Nicotiana tabacum with commercial cellulase elicited four principal stress metabolites including the phytoalexin capsidiol and a second eremophilane-type diol, shown on the basis of chemical and spectroscopic evidence to be 4-epieremophil-9-ene-11 /gx, 12-diol (without assignment of absolute configuration). This diol appears to be structurally identical with debneyol isolated from N. debneyi (see accompanying paper). Among minor metabolites were an isomer and a dehydro-analogue of the diol. GC/MS of cyclic derivatives (boronates and di-t-butylsilylene derivatives) of vicinal diols was useful for their detection and characterisation. The remaining two major metabolites appeared to be phytuberol and phytuberin.     

Floral morphogenesis in thin-layer tissue cultures of Nicotiana tabacum

The morphological changes in thin-layer tissues of Nicotiana labacum L. cv. Samsun, cultured on Murashige and Skoog medium with 1 μ M each of naphthalene acetic acid (NAA) and benzyladenine (BA), were studied during the first 8 days of culture with light and scanning electron microscopy. The first three days of culture arc characterized by enlargement of all cells and cell divisions starling in the cortical parenchyma cells adjacent to the medium. Between days 3 and 6, epidermal and/or subepidermal cells start to divide, resulting in division centers, which lead to flower bud formation. The hormones NAA and BA in different concentrations affect the formation and distribution of flower buds, bud morphology and callus formation. BA influences particularly bud formation and bud morphology, while NAA affects callus formation in particular. In addition, polarity may occur in the formation of both callus and flower buds, the degree of which depends upon the hormone concentrations.     

Inhibition of O-acetylserine sulfhydrylase by fluoroalanine derivatives

O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.     

Biosynthesis of N2-(1,3-dicarboxypropyl) ornithine [nopalinic acid] in crown gall tumour tissue

Protein extract from crown gall tumour tissue, induced on Nicotiana tabacum by Agrobacterium tumefaciens strain T37, synthesized nopalinic acid [N²-(1,3-dicarboxypropyl)ornithine] from l-ornithine and α-ketoglutarate in the presence of NADPH. Label was incorporated into nopalinic acid from both l-ornithine-[¹⁴C] and α-ketoglutarate-[¹⁴C] in vivo. Nopaline [N²-(1,3-dicarboxypropyl)arginine] did not appear to be metabolized to nopalinic acid in vivo.     

Timing of morphological and histological development in premeiotic anthers of Nicotiana tabacum cv. Xanthi (Solanaceae)

The tobacco stamen has been the object of many developmental studies, and the organ has more recently become a model for molecular genetic studies of anther differentiation. However, the spatial and temporal details of cellular differentiation of early anther development have never been thoroughly characterized. In the present study, the age of 15 tobacco flowers from plants grown under constant light and temperature was estimated using growth analysis. Prior to tissue fixation for light microscopy, moulds of stamen and anther primordia were made with a dental impression polymer so morphological and histological observations could be made on each tissue sample. Flower ages spanned an 8-d interval during which petal and stamen initiation occurred, and sporogenous cells reached the leptonema stage of meiosis. The initial development of the tetrasporangiate anther shape largely preceded periclinal division of archesporial initials. Anatomically, periclinal divisions in the hypodermal ∗∗∗(l₂) layer were observed before archesporial initials began to divide. These data indicate differences in the cellular basis of tobacco anther development compared to earlier clonal analyses of Datura. The pattern of mitotic cell division associated with microsporangial development suggested modal peaks in division over time. The ability to estimate developmental time in the tobacco anther has implications for future studies directed at understanding mechanisms of anther evolution via heterochrony.