The kinesin-immunoreactive homologue from Nicotiana tabacum pollen tubes: Biochemical properties and subcellular localization

In plant cells, microtubule-based motor proteins have not been characterized to the same degree as in animal cells; therefore, it is not yet clear whether the movement of organelles and vesicles is also dependent on the microtubular cytoskeleton. In this work the kinesinimmunoreactive homologue from pollen tubes of Nicotiana tabacum L. has been purified and biochemically characterized. The protein preparation mainly contained a polypeptide with a relative molecular weight of approx. 100 kDa. This polypeptide bound to animal microtubules in an ATP-dependent manner and it further copurified with an ATPase activity fourfold-stimulated by the presence of microtubules. In addition, the sedimentation coefficient (approx. 9S) was similar to those previously shown for other kinesins. Immunofluorescence analyses revealed a partial co-distribution of the protein with microtubules in the pollen tube. These data clearly indicate that several properties of the kinesin-immunoreactive homologue are similar to those of kinesin proteins, and suggest that molecular mechanisms analogous to those of animal cells may drive the microtubule-based motility of organelles and vesicles in plants.Abbreviations AE-LPLC anion-exchange low-pressure liquid chromatography - AMPPNP 5-adenylylimidodiphosphate - PKH pollen kinesin homologue - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

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.     

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.     

Effect of NaCl on Biomass and Contents of Sugars, Proline and Proteins in Seedlings and Leaf Explants of Nicotiana tabacum Grown in vitro

Effects of NaCl on growth in vitro and contents of sugars, free proline and proteins in the seedlings and leaf explants of Nicotiana tabacum cv. Virginia were investigated. The fresh and dry mass of the seedlings decreased under salinity. These growth parameters in leaf explants decreased at 50 mM NaCl and increased up to 150 mM NaCl and then decreased at higher level of salinity. Free proline content in both seedlings and leaf explants increased and polysaccharide content decreased continuously with increasing of NaCl concentration. Reducing sugars, oligosaccharides, soluble sugars and total sugars contents in both seedlings and leaf explants decreased up to 150 mM NaCl and then increased at higher concentrations of NaCl.     

γ-Glutamyl-transpeptidase in tobacc suspension cultures: Catalytic properties and subcellular localization

γ-Glutamyl-transpeptidase activity (EC 2.3.2.2) was found in ammonium sulfate precipitates of extracts from cultured cells of Nicotiana tabacum L. var. Samsun. Specific activity up to 3.2 nmol (mg protein)⁻¹ min⁻¹ was achieved, using the artificial substrate γ-glutamyl- p -nitroanilide (K_m 0.6 m M ) instead of glutathione. Optimal enzyme activity was obtained at pH 8.0–8.5 and 45°C. The enzyme reaction was inhibited competitively by γ-glutamyl analogs (6-diazo-5-oxo-L-norleucine: K_i 0.76 μ M ; L-azaserine: K_i 0.23 m M ) or the inorganic ion m -periodate (K_i 0.43 m M ). Cell fractionation and in vivo experiments revealed that 77% of the γ-glutamyl-transpeptidase activity is localized in the soluble cytoplasmic fraction, while 20–23% of the enzyme is found on the outer surface of the plasmalemma.     

γ-Glutamylcyclotransferase in tobacco suspension cultures: Catalytic properties and subcellular localization

Steinkamp, R., Schweihofen, B. and Rennenberg, H. 1987. γ-Glutamylcyclotransfer-ase in tobacco suspension cultures: Catalytic properties and subcellular localization.
γ-Glutamylcyclotransferase activity (EC 2.3.2.4) in ammonium sulfate precipitates (40–70% saturation) of extracts from cultured tobacco cells ( Nicoliana tabacum L. cv. Samsun) was analyzed as liberation of 5-oxo-proline from L-γ-glutamyl dipeptides. The enzyme was highly specific for the sulfur containing γ-glutamyl dipeptides γ-glutamyl-L-methionine and γ-glutamyl-i.-cysteine. Maximum activity was obtained at pH 8.7 and 35°C. As also observed with animal γ-glutamylcyclotransferase, the tobacco enzyme exhibited a relatively low substrate affinity (γ-glutamyl-i.-methionine: K_m 2.2 ± 0.4 mM ). In contrast to animal γ-glutamylcyclotransferase, the tobacco enzyme was not inhibited by the D-isomerof the substrate D-γ-glutamyl-i.-methionine; it also did not use the D isomer as a substrate. γ-Glutamylcyclotransferase of tobacco cells was shown to be a soluble enzyme entirely localized in the cytoplasm.     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

普通烟草CESA基因家族成员的鉴定、亚细胞定位及表达分析

纤维素合成酶蛋白(cellulose-synthase proteins, CESA)是一类质膜定位蛋白,以蛋白复合体的形式存在于质膜上合成纤维素,在细胞壁建成和植物生长发育过程中起着非常重要的作用。本研究利用CESA蛋白保守域序列PF03552检索普通烟草(Nicotiana tabacum L.)蛋白序列,并通过拟南芥(Arabidopsis thaliana)10个CESA蛋白序列在普通烟草基因组数据库中利用TBLASTN程序进行比对,共获得21条NtCESA基因候选序列,对这些序列进行蛋白序列理化性质分析、系统进化树构建、基因结构分析、保守结构域及跨膜区分析和组织表达模式分析,并对NtCESA9和NtCESA14两个蛋白进行了亚细胞定位实验。结果表明：获得的21条NtCESA蛋白序列的理化性质相似;系统进化分析将21个NtCESA基因和10个AtCESA基因分成5个分支,每一个分支各成员之间的进化相对保守,基因结构类似,不同分支之间的基因结构差异也较小;NtCESA蛋白结构域相对保守,都含有CESA蛋白典型的N端锌指结构、C端跨膜区和DDD-QXXRW保守功能域;组织表达分析结果表明,大部分NtCESA基因在幼苗和成熟期烟草的根、叶、胚芽和愈伤组织中都有表达,同一个分支中的基因表达模式基本一致,并且NtCESA基因参与初/次生细胞壁纤维素的合成与该基因编码蛋白的跨膜区数目存在关联,表明NtCESA基因家族成员功能上的复杂性;亚细胞定位结果证实NtCESA9和NtCESA14为质膜定位蛋白。本研究为烟草CESA基因家族功能的深入研究奠定了基础。     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

Evidence for O-acetylserine in Nicotiana tabacum

O-Acetylserine, a precursor of cysteine in plants, was isolated from cells of Nicotiana tabacum cultured in liquid medium.     

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment. Electronic supplementary material  Supplementary material is available in the online version of this article at doi: and is accessible for authorized users.     

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.     

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.     

The Effects of Methylglyoxal on Glutathione S-Transferase from Nicotiana tabacum

Methylglyoxal (MG) is one of the aldehydes that accumulate in plants under environmental stress. Glutathione S-transferases (GSTs) play important roles, including detoxification, in the stress tolerance systems of plants. To determine the effects of MG, we characterized recombinant GST. MG decreased GST activity and thiol contents with increasing K _m. GST can serve as a target of MG modification, which is suppressed by application of reduced glutathione.     

The biotransformation of carvoxime and dihydrocarvoxime with cell suspension cultures of Nicotiana tabacum

In newly initiated cell suspension cultures of Nicotiana tabacum, (4R)-(?) and (4S)(+)-carvoximes and (1S,4R)(+)-dihydrocarvoxime were hydrolysed to the corresponding ketones and then the resultant ketones were reduced to the corresponding alcohols.     

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.     

烟草未授粉子房胚状体诱导的研究

对烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）未授粉子房胚状体的诱导进行了研究,结果发现,胚状体是单细胞起源,起源于大孢子或卵细胞,接种发育早期的子房,胚状体起源于大孢子;接种发育晚期的子房,胚状体起源于卵细胞;接种发育中期的子种,胚状多起源于大孢子,少数起源于卵细胞,不同的基因型,蔗糖浓度及光照强度等对胚状体诱导率的影响亦有不同。     

Identification of aneuploids in Nicotiana tabacum by isozyme banding patterns

A biochemical system was devised to identify aneuploids of Nicotiana tabacum. Leaf tissue from 6 nullihaploids, 4 nullisomics, and 10 monosomics was analyzed electrophoretically on slab acrylamide gels. The staining systems used were for peroxidase, esterase, superoxide dismutase, malate dehydrogenase, and glutamate oxaloacetate transaminase. Nullihaploids and nullisomics could be distinguished from each other and from haploid or disomic types by their unique isozyme banding patterns. The banding patterns of the monosomics closely resembled those of the disomic. Morphologically similar aneuploids from different populations had similar isozyme banding patterns.This paper represents research completed in partial fulfillment of the requirements for the Ph.D. degree by the senior author. The investigations reported in this paper (No. 81-3-119) are in connection with a project of the Kentucky Agricultural Experiment Station, and the paper is published with the approval of the Director.