The role of peroxidase isoenzyme groups of Nicotiana tabacum in hydrogen peroxide formation

Three peroxidase isoenzyme-groups found in cell walls of tobacco were tested for their capacity to form H₂O₂. Isoenzyme-group G_I, located only in cell walls (G_II and G_III are also found in protoplasts) showed the highest K_app-value for H₂O₂-formation. The lowest K_app-value, i.e., maximal H₂O₂-formation was received for group G_III which is ionically bound to the cell wall. As shown before, G_I yields maximal polymerization rates for coniferyl- and p-coumarylalcohol. These facts indicate that each of the peroxidase isoenzyme groups of the cell wall is involved with different catalytic functions within the same pathways of H₂O₂-formation and succeeding lignification. H₂O₂-formation catalyzed by all 3 groups was increased by very low concentrations of Mn²⁺-ions. The required amount of Mn²⁺ leading to maximal stimulation was in each case dependent on the basic rate of H₂O₂-formation. Maximal stimulation of H₂O₂-formation by phenolic compounds was achieved by coniferylalcohol at a concentration of 10^-4M for all groups. Stimulation by p-coumaryl-and by sinapylalcohol was not as significant.     

Peroxidase-mediated integration of tyramine into xylem cell walls of tobacco leaves

When [2-¹⁴C]tyramine was fed in vivo by petiolar uptake to Nicotiana tabacum Xanthi n.c. leaves partially inoculated with tobacco mosaic virus, radioactivity accumulated in inoculated areas bearing necrotic lesions, mainly in the veins and around the lesions. Light-microscopic autoradiography showed that integration of radioactivity was especially evident in xylem cell walls. This was confirmed in sections of petiole by electron-microscopic autoradiography. Study of the mechanism of insolubilisation of tyramine showed that the amine was integrated in regions in which peroxidase activity could be located cytochemically using 3,3-diaminobenzidine and H₂O₂ as substrates. When sections of petiole were incubated with labelled tyramine and H₂O₂ after fixation in glutaraldehyde, a distribution of radioactivity similar to that obtained after feeding tyramine by petiolar uptake was observed. It is concluded that simple phenols such as tyramine can be integrated in vivo into cell walls because they are oxidised by peroxidases. This result illustrates the difficulty of studying the metabolism of exogenous phenols in plants, especially in lignifying tissues which contain active wall-bound peroxidases.Abbreviations DAB 3,3-diamino-benzidine - TMV tobacco mosaic virus     

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl₂. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent M_r approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent M_r approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed. Received: 9 January 1997 / Accepted: 14 March 1997     

Altered feedback sensitivity of acetohydroxyacid synthase from valine-resistant mutants of tobacco (Nicotiana tabacum L.)

Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from leaves of three valine-resistant (Val^r) tobacco (Nicotiana tabacum) mutants, and compared with the enzyme from the wild-type. The enzyme from all three mutants is appreciably less sensitive to inhibition by leucine and valine than the wild-type. Two of the mutants, Val^r-1 and Val^r-6, have very similar enzymes, which under all conditions are inhibited by less than half that found for the wild-type. The other mutant, Val^r-7, has an enzyme that only displays appreciably different characteristics from the wild-type at high pyruvate or inhibitor concentrations. Enzyme from Val^r-7 also has a higher apparent K_m for pyruvate, threefold greater than the value determined for the wild-type and the other mutants. The sulphonylurea herbicides strongly inhibit the enzyme from all the lines, though the concentrations required for half-maximal inhibition of enzyme from Val^r-1 and Val^r-6 are higher than for Val^r-7 or the wildtype. No evidence has been found for multiple isoforms of acetohydroxyacid synthase, and it is suggested that the valine-resistance of these mutant lines is the result of two different mutations affecting a single enzyme, possibly involving different subunits.     

Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum)

Here we show that fructose 2,6-bisphosphate cannot be reliably measured in mature leaves of tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.), or stinging nettle (Urtica dioica L.) using conventional extraction techniques, since the recoveries of fructose 2,6-bisphosphate added during extraction are poor. However, fructose 2,6-bisphosphate could be extracted by boiling leaves in ethanol and aqueous buffer. Evidence for the reliability of this technique is provided by high recovery measurements of fructose 2,6-bisphosphate added to the leaves before extraction. This extraction method was used to measure changes in the level of fructose 2,6-bisphosphate throughout the photoperiod in tobacco and potato leaves. These changes are compared with the rate of accumulation of sucrose and starch in the leaf samples. Variations in the levels of fructose 2,6-bisphosphate, and the relationship between this metabolite and sucrose and starch accumulation in these leaves during the photoperiod are similar to the pattern observed in leaves of other plant species.Abbreviations BSA bovine serum albumin - Fru-2,6-P₂ fructose 2,6-bisphosphate This research was supported by the Agricultural and Food Research Council (Grant no. PG43/531), and the Royal Society.     

Chemical inhibition of cell wall formation and cytokinesis,but not of nuclear division,in protoplasts of Nicotiana tabacum L. cultivated in vitro

The effect of cytochalasin B, colchicine, coumarin and 2,6-dichlorobenzonitrile on cell wall formation and cellular division was studied by light and electron microscopy with tobacco mesophyll protoplasts cultivated in vitro. 2,6-dichlorobenzonitrile was found to be the most effective and reversible inhibitor of cell wall formation. The other inhibitors caused irreversible damage and/or inhibited mitosis. In protoplasts cultivated in the presence of 2,6-dichlorobenzonitrile the total inhibition of cell wall formation had no effect on nuclear division, but cytokinesis was totally inhibited so that multinucleate protoplasts were obtained.Abbreviations DB 2,6-dichlorobenzonitrile=dichlobenil - CB cytochalasin B     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

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     

Isolation and characterization of Populus isoperoxidases involved in the last step of lignin formation

Peroxidases (EC 1.11.1.7) from Populus x euramericana were investigated during the dormant and growing seasons using histochemical and biochemical methods. The activities of syringaldazine oxidase and p-paraphenylenediamine-pyrocatechol oxidase in sections of branches were maximal during spring in both phloem and young xylem. Cytoplasmic and cell-wall peroxidase activities from different lignified tissues were estimated in vitro. Pronounced differences were noticed between fractions isolated during spring and winter. Gel electrophoresis showed the presence of an anionic fast-migrating isoperoxidase group with a high syringaldazine-oxidase activity. The isoenzymes of this group were different in winter and in spring. The properties of these isoperoxidases (kinetic constants, pH optimum, resistance to heat) were investigated after isolation by ion-exchange chromatography.Abbreviations PPD-PC p-phenylenediamine-pyrocatechol - syr-oxidase syringaldazine-oxidase     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Influence of p-dimethylaminoazobenzene and doxorubicin on tobacco cell growth (Nicotiana tabacum L.)

Callus growth, in tobacco pith tissue culture, is activated by p-dimethylaminoazobenzene (p-DAAB), whose carcinogenic properties are well known. Ultrastructural changes, appearing in a period prior to initiation of cell proliferation, occur earlier and more intensely in the presence of the carcinogen. This also influences changes in non-histone chromatin proteins (NHCP). Doxorubicin (DR), an anti-tumor drug, inhibits callus growth and modifies the pattern of NHCP.     

Long-distance transport of sulfur in Nicotiana tabacum

Sulfur reduction in tobacco plants is a light-enhanced process that predominantly takes place in the leaves rather than the roots. The amount of sulfate reduced in mature leaves can exceed their own requirement and enables an export of reduced sulfur, both basipetal toward the roots as well as acropetal toward the growing parts of the stem. Evidence is presented that translocation of reduced sulfur toward the roots occurs in the phloem. TLC and paper chromatography reveal that glutathione is the main transport form of reduced sulfur in tobacco plants; 67–70% of reduced ³⁵S was confined to glutathione, 27–30% to methionine, and 2–8% to cysteine.Abbreviation TLC thin-layer chromatography     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

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.     

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

Summary The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H₂O₂ and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3-diaminobenzidine both in the presence and in the absence of H₂O₂. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3-diaminobenzidine and coniferyl alcohol in the presence of H₂O₂. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.     

Biosynthesis of nicotianamine in the suspension-cultured cells of tobacco (Nicotiana megalosiphon)

Summary Seven kinds of suspension cell cultures from five species ofNicotiana were screened for the occurrence of nicotianamine. Nicotianamine was detected in the cultured cells ofN. megalosiphon andN. plumbaginifolia.l-[l-¹⁴C]Methionine, which is the precursor of the mugineic-acid-family phytosiderophores and nicotianamine in barley plants, was incorporated into nicotianamine by the cultured cells ofN. megalosiphon both in vivo and in vitro. The advantage of the cultured tobacco cells for the study of the biosynthesis of nicotianamine and the mugineic-acid-family phytosiderophores is discussed.     

De-novo synthesis and release of peroxidases in cell suspension cultures of Nicotiana tabacum L.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of ³H- and ¹⁴C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.Abbreviations AA^* radioactive amino-acid mixture - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

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     

A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase

When cell-wall invertase (CWI) from Nicotiana tabacum L. cell-suspension cultures, either non-transformed or transformed with Agrobacterium tumefaciens, was salt-eluted from intact cells and purified on Sulfopropyl-Sephadex (SPS) by pH-gradient elution, the enzyme lost about 50% of its activity during a 1-h incubation at pH 4.8. However, Western-blot analysis indicated no appreciable enzyme degradation. Re-chromatography of CWI peak fractions on SPS using NaCl-gradient elution showed the presence of a 17-kDa peptide (p17) in fractions with low CWI activity but strong CWI immunosignal (Weil and Rausch 1994, Planta 193, 430–437). When separating CWI from p17 by Concanavalin A (Con A)-Sepharose chromatography, inhibition could be restored by incubating the inhibitor-containing fraction with inhibitor-free CWI. More than 90% of CWI could be inhibited, suggesting that all CWI was susceptible to p17 binding. The presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺) during pre-incubation of CWI with p17 reduced CWI inhibition substantially. Also, sucrose protected CWI against inhibition by p17 (half-maximum protection at 1.3 mM). Binding of p17 to CWI during a 1-h pre-incubation was pH-dependent, pH 4.5 causing maximum inhibition, whereas at pH 6.5 no inhibition was observed. Gel-permeation chromatography revealed that the native inhibitor acts as a monomer. Immunoprecipitation of CWI co-precipitated p17, confirming direct binding of p17 to CWI. When fractions containing CWI and p17 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting a diffuse immunosignal of 86–90 kDa was observed (in addition to the prominent CWI signal at 69 kDa). Equilibration of this zone with urea-containing sample buffer prior to a second SDS-PAGE run resulted in a strong immunosignal at 87 (± 2) kDa, suggesting that during one step in the formation of the p17-CWI complex the two polypeptides became firmly aggregated. The distribution of CWI and glucose-6-phosphate dehydrogenase activities between the cell-wall protein fraction and salt-eluted cells shows that cells retained their structural integrity, thus indicating co-localization of p17 and CWI in situ (Weil and Rausch 1994). We have purified p17 to homogeneity and its N-terminus has been sequenced, revealing no similarity to other known protein sequences. Possible physiological roles of p17 are discussed.Abbreviations Con A concanavalin A - CWI cell-wall invertase - 1-OMG methyl -d-glucopyranoside - p17 17-kDa peptide - PMSF phenylmethylsulfonyl fluoride - PR pathogenesis related This work was supported by a grant from the Deutsche Forschungsgemeinschaft. The antiserum against the deglycosylated carrot cell-wall invertase was a gift from Dr. Sturm (Friedrich-Miescher-Institut, Basel, Switzerland). The antiserum against acidic tobacco PR1 proteins was obtained from Dr. Lotan (Weizmann Institute of Science, Rehovot, Israel). The antiserum against tomato hsp17 was a gift from Prof. Nover (J.-W.-Goethe-Universität, Frankfurt, Germany).