Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

Tip growth in xylogenic suspension cultures ofZinnia elegans L.: Implications for the relationship between cell shape and secondary-cell-wall pattern in tracheary elements

Summary The relationship between cell expansion, cortical microtubule orientation, and patterned secondary-cell-wall deposition was investigated in xylogenic cell suspension cultures ofZinnia elegans L. The direction of cell expansion in these cultures is pH dependent; cells elongate at pH 5.5–6.0, but expand isodiametrically at pH 6.5–7.0. Contrary to our expectations, indirect immunofluorescence revealed that cortical microtubules are oriented parallel to the long axis in elongating cells. Pulse labeling of the walls of isolated cells with the fluorochrome Tinopal LPW demonstrated that xylogenic Zinnia mesophyll cells elongate by tip growth in culture. These results confirm that cortical microtubules in developing tracheary elements reorient before bundling to form transverse cortical microtubule bands. This rearrangement may allow the secondary cell wall pattern to conform to cell shape, independent of the direction in which the cell was expanding prior to reorientation.Abbreviations CMT cortical microtubules - Mes 2-[N-morpholino]ethanesulfonic acid - TE tracheary element     

Cytodifferentiation in isolated single cells

Isolated mesophyll cells ofZinnia elegans synchronously differentiate to tracheary elements at a high frequency when cultured in a liquid medium containing naphth-aleneacetic acid and benzyladenine. An experimental system with the isolatedZinnia mesophyll cells has been extensively used as an efficient model system for the study of cytodifferentiation in higher plants, providing many valuable results on cytodifferentiation. In this review, I introduce the results obtained with theZinnia system and discuss the induction and the sequence of differentiation. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.     

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

Summary. The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O₂⁻ and H₂O₂) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O₂⁻ production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl₃ assays, which monitor H₂O₂ production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl₃) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H₂O₂ is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H₂O₂ in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H₂O₂ was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H₂O₂ produced by this in vitro culture system is destined for use during lignin biosynthesis. Correspondence and reprints: Departamento de Biologia Vegetal, Facultad de Biologia, Universidad de Murcia, 30100 Murcia, Spain.     

Possible involvement of DNA-repair events in the transdifferentiation of mesophyll cells ofZinnia elegans into tracheary elements

In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [³H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [³H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.     

Ectomycorrhiza formation ofTricholoma matsutake onPinus densiflora

Mycorrhizal association ofTricholoma matsutake withPinus densiflora was studied. A naturally establishedP. densiflora stand (age: ca. 45 yr) where occurrences ofT. matsutake sporocarps had been confirmed was studied in lbaraki Prefecture, Japan. Pine root systems connected withT. matsutake sporocarps via the fungal white mycelia were sampled in October 1997. The sampled pine roots were covered overall with mycelia. Under a dissecting microscope, the mycelia were confirmed to form fungal sheaths on the lateral roots. Under a light microscope, transverse and longitudinal sections of these roots showed the presence of both fungal sheaths and Hartig nets, which are typical of ectomycorrhizas. The fungal sheath was ca. 1.5–20 μm. in thickness, and felt prosenchymatous in texture. Hartig nets developed continuously at the cortex and extended to the boundary between cortical cells and endodermal cells. The same ectomycorrhizal morphotype on the pine was also recovered from inside the same mycelial colony (i.e., “shiro”) ofT. matsutake from winter to summer. These results suggest thatT. matsutake has a perennial ectomycorrhizal association withP. densiflora.     

Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Summary Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy     

Contribution ofAzospirillum brasilense to the nitrogen needs of grain sorghum (Sorghum bicolor (L) Moench) in humid sub-tropics

Summary Field experiments were conducted to assess the contribution ofAzospirillum brasilense to the N needs of grain sorghumcv. CSH — 5 during monsoon (June–October) seasons of 1978 and 1979.A. brasilense contributed to the N uptake by crop in the range from 5.8 to 19.6 kg N/ha. However, the contribution ofA. brasilense to the N needs of sorghum was more when sorghum was manured with farmyard manure at the rate of 10 tons/ha. Publication of G.B.P.U.A.T. Expt. Station, Pantnagar — 263145, India.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Transport of rosettes from the golgi apparatus to the plasma membrane in isolated mesophyll cells ofZinnia elegans during differentiation to tracheary elements in suspension culture

Summary Rosettes of six particles have been visualized by freeze-fracture in the protoplasmic fracture (PF) faces of: a) the plasma membrane, b) Golgi cisternae, and c) Golgi-derived vesicles in mesophyll cells ofZinnia elegans that had been induced to differentiate synchronously into tracheary elements in suspension culture. These rosettes have been observed previously in the PF face of the plasma membranes of a variety of cellulose-synthesizing cells and are thought to be important in cellulose synthesis. InZinnia tracheary elements, the rosettes are localized in the membrane over regions of secondary wall thickening and are absent between thickenings. The observation of rosettes in the Golgi cisternae and vesicles suggests that the Golgi apparatus is responsible for the selective transport and exocytosis of rosettes in higher plants, as has been previously indicated in the algaMicrasterias (Giddings et al. 1980). The data presented indicate that the Golgi apparatus has a critical role in the control of cell wall deposition because it is involved not only in the synthesis and export of matrix components but also in the export of an important component of the cellulose synthesizing apparatus. The rosettes are present in the plasma membrane and Golgi vesicles throughout the enlargement of the secondary thickening, suggesting that new rosettes must be continually inserted into the membrane to achieve complete cell wall thickening.Abbreviations EF Golgi vesicles, exoplasmic fracture; the plasma membrane, extracellular fracture - PF protoplasmic fracture     

Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

Role of root hairs in phosphorus depletion from a macrostructured soil

One rape (Brassica napus cv. Wesroona) plant and four cotton (Gossypium hirsutum cv. Sicot 3) plants were grown in plastic cells containing soil labelled with 407 kBq of³³P g⁻¹ soil. After 5–8 days of growth, the³³P depletion zones of all plants were autoradiographed and³³P uptake by plants was measured. The autoradiographs were scanned with a microdensitometer and the optical densities at several places within the³³P depletion zones of roots were obtained. The volume of soil explored by root hairs was estimated from measurements of root diameters and lengths of roots and root hairs. About half of the total³³P depleted by cotion roots came from outside the root hair cylinder whereas most of³³P taken up by rape was from within the root hair cylinder. Plants grown in a macrostructured soil may have roots growing in voids, within aggregates or on the surfaces of aggregates. The results of this study demonstrate that root hairs have a strong influence on the accessibility of phosphorus to roots in such a soil, and thus on the phosphorus nutrition of plants.     

Materials for the fungus flora of Japan (56)Mariannaea camptospora andM. elegans var.punicea from Japan

One additional species and a variety ofMariannaea, M. camptospora andM. elegans var.punicea, were recorded for the first time in Japan.Mariannaea camptospora formed two types of conidiophores. One type was characterized by simple verticillate phialides sometimes with punctuate walls at the base, producing long oblique conidial chains, and symmetrical spindle-shaped conidia. The other type was characterized by more crowded and shorter phialides with small conidial droplets and hemispherical to concave smaller conidia.Mariannaea elegans var.punicea was characterized by distinct red purple pigmentation in agar media. (55): Udagawa, S. and Uchiyama, S., Mycoscience41: 263–267, 2000.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

A simplified medium for in vitro tracheary element differentiation in mesophyll suspension cultures from Zinnia elegans L.

A simplified medium has been developed for the differentiation of tracheary elements in suspension cultures of mesophyll cells of Zinnia elegans L. All inorganic salts contained in media used previously were retained in the simplified medium, but most were reduced in concentration. The only organic supplements required for optimum differentiation were thiamine and nicotinic acid, in addition to the plant growth regulators N6-benzylaminopurine and -naphthyleneacetic acid, and sucrose as a carbon source. Mannitol, an osmoticum, was necessary for rapid, synchronous differentiation. This simplified medium is particularly suitable for studies of the role of Ca²⁺ in tracheary element differentiation due to the elimination of myo-inositol, an intermediate in the phosphatidyl inositol signal transduction pathway and reduction in the concentrations of Mg²⁺ and Mn²⁺, which block calcium channels. It is also possible to eliminate EDTA from the medium, enabling studies using specific calcium chelators. Additional culture variables for the optimization of differentiation are discussed.Abbreviation TE tracheary element     

Immunofluorescent and calcofluor white staining of developing tracheary elements inZinnia elegans L. Suspension cultures

Summary Xylogenesis has been studied in primary suspension cultures ofZinnia elegans L.: The wall patterns produced in culture closely resemble those described for intact tissues (annular, spiral, reticulate, scalariform, pitted). Using fluorescence microscopy and immuno-cytochemical techniques we have followed both the changes in wall deposition and microtubule organization during xylogenesis. Calcofluor white has been used to detect secondary wall deposition before it can be observed using either phase contrast or polarization optics. The development of tracheary elements can be divided into three stages: 1. microtubules grouped into bands without secondary wall deposition evident; 2. groups of microtubules subtending wall material only visible using Calcofluor white; 3. a complex microtubule pattern reflected by well developed wall thickenings detected using Calcofluor, phase contrast and polarization optics.     

Colonization of the root zone ofAmmophila arenaria by harmful soil organisms

The role of harmful soil organisms in the degeneration ofAmmophila arenaria at coastal foredunes was examined by the growing of seedlings ofA. arenaria in soil samples collected from its root zone. Three sites, each representing a successive stage in foredune succession were examined: (1) a highly mobile dune (sand accretion of 80 cm year⁻¹) with vigorousA. arenaria, colonizing only the upper 30-cm of the annually deposited layer of sand, (2) a mobile dune with vigorousA. arenaria (sand accretion of 22 cm year⁻¹) and a 1-metre soil profile completely colonized by roots and (3) a stable dune (no sand accretion) with degeneratedA. arenaria and young roots mainly present in the upper 0–10 cm. In the upper part of the highly mobile site, the presence of harmful soil organisms was confined to the root layers and at the mobile site for all depth layers a significant growth reduction ofA. arenaria was observed due to the activity of harmful soil organisms. At the stable site, however, growth had only been reduced in some of the depth layers. At all sites newly formed roots ofA. arenaria had been colonized by harmful soil organisms within one year. If present in sand prior to root growth harmful soil organisms reduced root length and root hair formation severely and they enhanced branching of the roots. It is concluded that harmful soil organisms initiate degeneration ofA. arenaria in stable dunes by attack of the root system, which makes the plants suffer from abiotic stress.