Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.     

Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by l-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by l-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.     

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

 Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [³H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins. Received: 5 November 1999 / Accepted: 18 January 2000     

Accumulation of aluminium in the cell wall pectin in cultured tobacco (Nicotiana tabacum L.) cells treated with a combination of aluminium and iron

In nutrient medium, aluminium (Al) accumulation in tobacco cells occurs only in the presence of ferrous ion [Fe(II)]. The localization of Al was examined to elucidate a mechanism of Al accumulation. After the digestion of Al-treated cells with cellulase and pectolyase together, the resulting spheroplasts contained as much Al as the intact cells. However, the cell walls isolated from Al-treated cells also contained as much Al as the intact cells. Comparison of sugar and Al contents in polysaccharide components extracted chemically from cell walls isolated from intact cells and spheroplasts revealed that the enzymes digested most of the cellulose and hemicellulose, but only half of the pectin, and that Al mainly existed in the pectin remaining in the spheroplasts. Gel-permeation chromatography of the pectin fraction (NH₄-oxalate extract) from the cell walls of the intact cells indicated that Al was associated with small polysaccharides of approximately 3–7 kDa. These results suggest that a minor part of pectin is a major site of Al accumulation. The content of cell wall pectin increased during Al treatment in nutrient medium. Taken together, we hypothesize that Al may bind to the pectin newly produced during Al treatment.     

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry

Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with K _m values of 0.7 mg ml^-1 for pectin with a DE of 70% and 17 mg ml^-1 for pectin with a DE of 25%. The sequences of the NH₂-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands. Received: 15 November 1997 / Accepted: 7 April 1998     

Identification of peptidases in Nicotiana tabacum leaf intercellular fluid

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration-centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum gamma-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.     

Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum

 The aquaporin NtAQP1 from Nicotiana tabacum L. is insensitive to heavy-metal ions. In addition to water, the transport of urea or glycerol is facilitated by this plasma-membrane-located water channel. Northern hybridization and whole-mount in situ hybridization revealed a high steady-state level of NtAQP1-RNA in roots, a decreased content in shoots and a low content in leaves. By immunolocalization with an antibody targeted to the N-terminus of the aquaporin, the localization of NtAQP1-protein at sites of expected high water transport rates from and to the apoplast or symplast could be demonstrated. The specific pattern of NtAQP1 distribution in petioles strongly indicates a transcellular movement of water. Received: 12 August 1999 / Accepted: 27 December 1999     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca²⁺ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca²⁺ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate. Received: 14 August 1996 / Accepted: 9 October 1996     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Acid invertase in Nicotiana tabacum crown-gall cells: Molecular properties of the cell-wall isoform

Cell-wall invertase (CWI; EC 3.2.1.26) was salt-eluted from non-disrupted Agrobacterium tumefaciens-transformed Nicotiana tabacum L. cells and purified to homogeneity. More than 90% of total cellular invertase activity (measured at pH 4.8) was recovered in the NaCl-eluted fraction whereas for the cytoplasmic marker glucose-6-phosphate dehydrogenase 96% of total activity could be extracted from the tissue after salt-elution, indicating absence of appreciable stress-induced cell disruption. Likewise, appreciable contamination of CWI with vacuolar acid invertase could be excluded. Tobacco CWI cross-reacted with an antiserum directed against deglycosylated carrot CWI; however, during some purification steps CWI enzyme activity did not correlate with CWI immunosignal. In fractions of low CWI activity and strong immunosignal, a putative inhibitor peptide with an apparent M_r of 17 kDa was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining (Weil et al. 1994, Planta 193, 438–445). The CWI of transformed tobacco cells has an apparent M_r of 69 kDa (SDS-PAGE) and is a basic (pI 9.5) glycoprotein. Gel-permeation chromatography indicated that enzymatically active CWI is a monomer. Deglycosylation of the denatured CWI by treatment with endo--N-acetylglucosaminidase, peptide-N-glycosidase F and trifluoromethanesulfonic acid indicated the presence of two high-mannose and two complex glycans. In partially purified CWI fractions the carrot CWI antiserum cross-reacted with one other tobacco cell-wall peptide (M_r 28 kDa). To address the possibility of a second invertase isoenzyme cross-reacting with the carrot antiserum, intact CWI and the 28-kDa peptide were digested in vitro with Staphylococcus aureus V8 protease and cyanogen bromide. A comparison of the resulting peptide patterns identified the 28-kDa polypeptide as a cleavage product of CWI. Running electroeluted CWI (69 kDa) on a second SDS-polyacrylamide gel led to substantial formation of the 28-kDa peptide. This suggests that the intrinsic 28-kDa cleavage product is the result of an intrinsic lability of tobacco CWI, rather than being a proteolytic degradation product.Abbreviations Con A concanavalin A - CWI cell-wall invertase - Endo H endo--N-acetylglucosaminidase - Glc-6-P-DH glucose-6-phosphate dehydrogenase - 1-OMG methyl -d-glucopyranoside - p17 17 kDa peptide - pI isoelectric point - PNGase F peptide-N-glycosidase F - TFMS trifluoromethanesulfonic acid This work was supported by grants from the Deutsche Forschungsgemeinschaft. The gift of an antiserum directed against carrot cellwall invertase from Dr. Arnd Sturm (Friedrich-Miescher-Institut, Basel, Switzerland) is kindly acknowledged. Furthermore, the authors thank Sigrid Ranostaj for excellent technical assistence.     

The early light-induced protein is also produced during leaf senescence of Nicotiana tabacum

To better understand the genetic controls of leaf senescence, a tobacco (Nicotiana tabacum L. cv. SR1) mRNA that is up-regulated during senescence was isolated by the cDNA-amplified restriction fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/b-binding protein family that has been implicated in assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress. A protein antigenically recognized by antibodies to ELIP appeared during senescence with kinetics similar to those of its mRNA. The mRNA, designated ELIP-TOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-cyclopropane-1-carboxylic acid, and was detected later when senescence was retarded by benzyladenine. However, no ELIP-TOB mRNA was seen in the dark even though senescence was accelerated under these conditions. Furthermore, water stress and anaerobiosis stimulated the appearance of ELIP-TOB mRNA before losses of chlorophyll could be detected. We discuss the conditions that may lead to the up-regulation of ELIP-TOB during senescence and speculate as to the role of the gene product in this terminal phase of leaf development. Received: 18 May 2000 / Accepted: 24 June 2000     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Mosaicism in the organization of Con A binding sites on the membrane surface of female cells of Nicotiana tabacum

The presence of mosaicism in the organization of concanavalin agglutinin (Con A) binding sites on murine egg cells was first reported 30 year ago. This discovery has triggered extensive studies into the roles of glycoproteins in gamete interactions in animals. This report comprises the first account of the existence of the mosaicism in higher plants. The distribution of Con A binding sites on both egg cells and central cells of tobacco (Nicotiana tabacum) was found to be polar and apparently determined by the location of the nucleus of the cell. On central cells, Con A binding sites were distributed on the section of the plasma membrane surface near the nucleus. By contrast, the binding sites on egg cells were concentrated away from the nucleus. Therefore, polarity of the plasma membrane component of female cells was confirmed for the first time. It is proposed that such polarized ConA binding sites could be involved in sperm recognition.     

Characterization of pectin methyltransferase from soybean hypocotyls

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml⁻¹, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [¹⁴C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo. Received: 10 September 1999 / Accepted: 11 October 1999     

Association of carbonic anhydrase with a Calvin cycle enzyme complex in Nicotiana tabacum

Chloroplast-localized carbonic anhydrase (CA; EC 4.2.1.1), an enzyme which catalyzes the reversible hydration of CO₂, appears to be associated with other enzymes of the Calvin cycle in a large multienzyme complex. Gel-filtration fast protein liquid chromatography (FPLC) of soluble proteins obtained by osmotic lysis of tobacco (Nicotiana tabacum L. cv. Carlson) chloroplasts results in the co-elution of a protein complex of greater than 600 kDa which includes CA, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoribulokinase (PRK), and ribose-5-phosphate isomerase. Anion-exchange FPLC of chloroplast extracts indicates that there is an association of CA with other proteins that modifies its elution profile in a NaCl gradient, and that Rubisco co-elutes with the fractions containing CA. Following a protocol described by Süss et al. (1993, Proc Natl Acad Sci USA 90: 5514–5518), limited protease treatment of chloroplast extracts was used to show that the association of PRK with other chloroplast proteins appears to protect a number of lysine and arginine residues which may be involved in specific protein-protein interactions. A similar treatment of CA indicates some protection of these residues when CA is associated with other chloroplast polypeptides but the level of protection is not as profound as that exhibited by PRK. In concert with previously published immunolocalization studies, these data indicate that CA may be associated with Rubisco at the stromal periphery of a Calvin cycle enzyme complex in which PRK is more centrally located and associated with thylakoid membranes. Received: 2 June 1997 / Accepted: 28 June 1997     

Change in Cytokinin Levels in Reproductive Organs of Nicotiana tabacum Before and After Pollination

Immunoaffinity systems and indirect competitive ELISA were used to determine the changes of cytokinin (CTK) content in reproductive organs of Nicotiana tabacum before and after pollination. It was found that t-ZR and iPA existed in all reproductive organs tested, and their contents were higher in female than in male organs. On the 5th day before anthesis. CTK reached its peak value in anthers and filaments, then it declined. In styles, CTK content increased after pollination and reached its peak one day later. In ovaries, levels of CTK began to increase two days after pollination and reached its peak two days later, just at the time when fertilization took place. If unpollinated, CTK levels decreased both in styles and ovaries. Pollination and fertilization resulted in an increment of CTK in pistils, accompanied with the process of pollen germinfition and pollen tube growth. It could thus be inferred that t-ZR and iPA played some active role in pollination and fertilization.     

烟草ovate同源基因的全长cDNA克隆与序列分析

根据番茄中控制果实形状的主效数量性状基因ovate的序列,用生物信息学方法从茄科植物烟草中获得直系同源ovate基因(NTovate)的特异片段,经鉴定,此基因在烟草中至少有2个拷贝。在此基础上用cDNA末端快速扩增(RACE)方法,获得其中1个拷贝的1059bpNTovate全长cDNA序列。序列分析表明,NTovate cDNA序列编码352个氨基酸,其蛋白序列与番茄ovate蛋白序列和拟南芥ovate蛋白家族AtOFP7蛋白分别为70%和36%的序列一致率,而与此家族中其他蛋白以及水稻ovate蛋白仅在保守的ovate结构域有较低的同源性。此基因已在GenBank中登录(EU043369)。     

DREB类转录因子介导的烟草抗非生物胁迫特性研究

检测并分析了转BnDREB1-5基因烟草叶片的持水性能,上、下表皮气孔大小和密度及叶绿素含量,并在自然失水7 h后检测了细胞的离子泄漏状况。结果表明:转基因烟草叶片单位时间内每平方厘米的失水量是野生型烟草叶片的62%;上表皮的气孔大于野生型,而气孔开度小于野生型;野生型烟草叶片上的气孔密度接近转基因烟草的1.5倍;野生型烟草叶片的叶绿素含量比转基因烟草叶片高29%;野生型烟草叶片的质膜相对透性是转基因烟草叶片的1.4倍。