Elicitor‐induced changes of wall‐bound and secreted peroxidase activities in suspension‐cultured spruce (Picea abies) cells are attenuated by auxins

In ectomycorrhizae auxins are proposed to attenuate elicitor-induced defence reactions in the host plant. To examine this hypothesis we compared the elicitor-induced accumulation of peroxidase isoforms between suspension-cultured spruce (Picea abies[L.] Karst.) cells incubated in media with and without auxins. In spruce cells changes in ionically and covalently wall-bound as well as symplasmic peroxidase (EC 1.11.1.7) activities were observed when elicitors from the following fungal species were applied: (1) Hebeloma crustuliniforme, an ectomycorrhizal partner of spruce; (2) Suillus variegatus, an ectomycorrhizal fungus incompatible with spruce; (3) Heterobasidion annosum, a spruce pathogen. Activity staining after SDS-PAGE and western blotting showed an accumulation of an ionically wall-bound 38-kDa peroxidase isoform. In addition, two covalently wall-bound isoforms (34 and 53 kDa) that could be released from spruce cell walls by cellulase and pectinase treatment were also induced by elicitors from these fungi. Moreover, in cells cultured without auxins all the elicitors triggered a rapid and transient accumulation of ionically wall-bound peroxidases, which reached a maximum activity 48 h after elicitor application. This early and transient peroxidase accumulation was diminished and delayed in cells cultured in the presence of auxins. In contrast, activity of peroxidases released into the culture medium of spruce cells or into the medium of protoplasts was suppressed by the elicitors of Hebeloma crustuliniforme. However, this suppression was attenuated by the action of auxins. It is suggested that under natural conditions, in infected spruce roots, the elicitors of the compatible fungus cause both suppression of the peroxidase (which is secreted to the free space of the roots), and induction of wall-bound and symplasmic peroxidases. On the other hand, auxins synthesized by the fungus could weaken these different elicitor-mediated effects.     

Characterization of wall-bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that function as β-d -fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin-labeled protein. The K_m values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase K_m= 16 mM, tightly bound invertase K_m= 8.6 mM). Activity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.     

Sucrose Utilization of the Ectomycorrhizal Fungi Amanita muscaria and Hebeloma crustuliniforme Depends on the Cell Wall-bound Invertase Activity of their Host Picea abies

The role of apoplastic invertase (β-d -fructofuranoside — fructohydrolase, EC 3.2.1.26) of the host Picea abies for carbohydrate uptake and growth of two of its natural ectomycorrhiza partners was studied. For that purpose, hyphae of Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quell., as well as roots and suspension cultured cells of Picea abies (L.) Karst. were used. Apoplastic invertase activity was demonstrated on roots and suspension cultured cells of spruce (in the latter case with 21.7 nkat (g fresh weight)^?1). Inhibition of the root cell wall invertase activity (pH optimum 4.5) by increasing the apoplastic pH allowed determination of the permanent release of sucrose from the root. However, under in vivo conditions at a lower cell wall pH the hydrolysation products glucose and fructose were predominantly found. In contrast to spruce cells and certain fungi, such as Saccharomyces (Novick et al., 1981) or Phycomyces (Ruiz-Herrera et al., 1989) invertase activity of the mycorrhizal fungi Hebeloma and Amanita was negligibly low. Furthermore, sucrose could not be consumed by Amanita and Hebeloma. As a consequence, cultures of these mycorrhizal fungi starved when kept on media with sucrose as sole carbohydrate source. But addition of invertase initiated hyphal growth immediately. Studies on carbohydrate uptake of host and fungal cells confirmed that the monosaccharides glucose and fructose were readily incorporated by spruce and fungal cells, with a clear preference for glucose. From these results it is suggested that apoplastic invertase activity of the host Picea abies is a precondition for the utilization of sucrose by the studied mycorrhizal fungi during the nutritional interaction of the symbiotic partners.     

The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies

A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [¹⁴C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of ¹⁴C-labelled feruloyl groups. Cultured maize and spinach cells took up [¹⁴C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [¹⁴C]feruloyl-polysaccharides and, simultaneously, low-M_r [¹⁴C]feruloyl esters. When all free [¹⁴C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-M_r pool into the polysaccharide fraction. A proportion of the [¹⁴C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [¹⁴C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [¹⁴C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8–5′-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H₂O₂, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [¹⁴C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.     

Carbon and nitrogen flow in silver birch and Norway spruce connected by a common mycorrhizal mycelium

 Spruce and birch seedlings were grown together in boxes filled with unsterile peat. Both seedlings were colonized by the ectomycorrhizal fungus Scleroderma citrinum. The two plants thus shared a common external mycelium. ¹⁵N-labelled ammonium was supplied exclusively to the fungus, while the birch or the spruce plant was continuously fed with ¹³C-labelled CO₂ for 72 h. The carbon and nitrogen transfer rates were strikingly different for birch and spruce seedlings. The mycorrhizal mycelium received carbohydrates mainly from the birch plant and the nitrogen transfer by the fungus to the plants was largely directed towards the birch. Carbon assimilates were also transferred in both directions between birch and spruce; however, there was no conclusive evidence for a net transfer of carbon between the plants. Accepted: 20 September 1996     

Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+ and Cl− release, extracellular alkalinization and H2O2 synthesis of Picea abies cells

Rapid reactions comprising efflux of K⁺ and Cl⁻, phosphorylation of a 63-kDa protein (pp63), extracellular alkalinization and synthesis of H₂O₂ are equally induced in cells of Picea abies (L.) Karst. by chitotetraose, colloidal chitin and cell wall elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. an ectomycorrhizal partner of spruce. Cleavage of fungal cell wall elicitors and of artificial chitin elicitors to monomeric and dimeric fragments by apoplasmic spruce chitinases (36-kDa class I chitinase, pI 8.0, and 28-kDa chitinase, pI 8.7; EC 3.2.1.14) equally prevented induction of these rapid reactions. Also, N-acetylglucosamine oligomers and elicitors from the fungal cell walls showed a similar dependence of their activity on the degree of polymerisation. From these results it is suggested that, during ectomycorrhiza formation, only some of the chitin-derived elicitors reach their receptors at the plant plasma membrane, initiating reactions of the hypersensitive response in the host cells. The remaining fungal elicitors will be degraded to varying extents by wall-localized chitinases of the host root, reducing the defence reactions of the plant and allowing symbiotic interactions of both organisms. Received: 6 January 1997 / Accepted: 14 March 1997     

Limited transfer of nitrogen between wood decomposing and ectomycorrhizal mycelia when studied in the field

Transfer of ¹⁵N between interacting mycelia of a wood-decomposing fungus (Hypholoma fasciculare) and an ectomycorrhizal fungus (Tomentellopsis submollis) was studied in a mature beech (Fagus sylvatica) forest. The amount of ¹⁵N transferred from the wood decomposer to the ectomycorrhizal fungus was compared to the amount of ¹⁵N released from the wood-decomposing mycelia into the soil solution as ¹⁵N-NH₄. The study was performed in peat-filled plastic containers placed in forest soil in the field. The wood-decomposing mycelium was growing from an inoculated wood piece and the ectomycorrhizal mycelium from an introduced root from a mature tree. The containers were harvested after 41 weeks when physical contact between the two foraging mycelia was established. At harvest, ¹⁵N content was analyzed in the peat (total N and ¹⁵NH₄ ⁺) and in the mycorrhizal roots. A limited amount of ¹⁵N was transferred to the ectomycorrhizal fungus and this transfer could be explained by ¹⁵NH₄ ⁺ released from the wood-decomposing fungus without involving any antagonistic interactions between the two mycelia. Using our approach, it was possible to study nutritional interactions between basidiomycete mycelia under field conditions and this and earlier studies suggest that the outcomes of such interactions are highly species-specific and depend on environmental conditions such as resource availability.     

Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, -glucosidase and acid phosphatase, secreted by the cells during growth, and also -amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H₂O₂ from the cells (oxidative burst). H₂O₂ released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H₂O₂ is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H₂O₂ is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H₂O₂. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H₂O₂ and, when present together with elicitor, induction was synergistically increased.     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane     

Effects of aluminium on growth and nutrient uptake of small Picea abies and Pinus sylvestris plants

Summary The effects of aluminium concentrations between 0.2 and 30 mM at pH 3.8 ±0.2 on small plants of Norway spruce [(Picea abies (L.) Karst], Scots pine (Pinus sylvestris L.), and Scots pine infected with the ectomycorrhizal fungus Suillus bovinus (L. ex Fr.) O. Kuntze were investigated. The plants were grown at maximum relative growth rate (R_G % day^–1) with free access but very low external concentrations of nutrients. Steady-state conditions with respect to relative growth rate (R_G) and internal nutrient concentrations were achieved before addition of aluminium, which was added as AlCl₃ and/or Al(NO₃)₃. There were reductions in rg at aluminium concentrations of 0.3 mM in spruce, 6 mM in pine and 10 mM in ectomycorrhizal pine, i. e. at aluminium concentrations considerably higher than those normally occurring in the top layer of the mineral soil where most fine roots are found. Nutrient uptake rate per unit root growth rate was calculated for different nutrient elements. The uptake rate of calcium and magnesium was reduced at aluminium concentrations of 0.2 mM (spruce), 1 mM (pine) and 3 mM (ectomycorrhizal pine), without influencing R_g. The results question the validity of the hypothesis of aluminium toxicity to forest tree species at low external concentrations.     

Initial signalling processes induced by elicitors of ectomycorrhiza-forming fungi in spruce cells can also be triggered by G-protein-activating mastoparan and protein phosphatase-inhibiting cantharidin

The first responses in spruce [Picea abies (L.) Karst.] cells induced by elicitors (N-acetylglucosamine oligomers) from ectomycorrhizal fungi have been described as follows: efflux of Cl⁻ and K⁺, influx of Ca²⁺, extracellular alkalinization, phosphorylation of a 63-kDa protein (pp63), dephosphorylation of a 65-kDa protein (pp65) and synthesis of H₂O₂ (Salzer et al. 1996, Planta 198: 118–126). In order to obtain new insights into the triggering mechanism and the sequence of these rapid responses we used compounds which are known to activate or block specific steps within an elicitor-induced signal transduction cascade in plant cells. Comparable to elicitors the two protein phosphatase inhibitors, cantharidin and calyculin A, as well as mastoparan, an activator of trimeric G-proteins, were able to induce the release of Cl⁻ and K⁺ from spruce cells and the alkalinization of the medium. Half-maximal activation of the alkalinization occurred at 133 nM calyculin A, 2.3 μM cantharidin and 1.6 μ mastoparan. The structural analogue of mastoparan, Mas 17, which has no G-protein-stimulating properties, was unable to trigger the above-mentioned reactions. In addition, cantharidin and calyculin A induced an increased synthesis of H₂O₂ in spruce cells which was prolonged in comparison to the elicitor-induced transient formation of H₂O₂. Also, the cantharidin-induced release of K⁺ was more pronounced and longer lasting than that caused by elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) and N-acetylglucosamine oligomers. Furthermore, cantharidin, calyculin A and mastoparan induced the phosphorylation of pp63. Remarkably, the protein kinase inhibitor, staurosporine, inhibited all the rapid responses described above, no matter whether they were triggered by fungal elicitors or by the protein phosphatase inhibitors. These results indicate that in the initial signalling events in spruce cells, essential protein phosphorylations occur either as an (auto) phosphorylation of a membrane-bound receptor kinase prior to the activation of a G-protein or (and) immediately downstream of the activated G-protein in a phosphorylation cascade and are the basic requirements for the ion fluxes following downstream. Received: 24 April 1998 / Accepted: 23 July 1998     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-M<Subscript>r</Subscript> apoplastic inhibitor

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H₂O₂ in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-α-L-arabinofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-D-xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl−¹⁴C]FAXX and [pentosyl−³H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [¹⁴C]feruloyl groups were converted to [¹⁴C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H₂O₂-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H₂O₂ and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M_r, was destroyed by heating, and remained in the aqueous phase (pH ≈3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.     

Estimating the relative nutrient uptake from different soil depths in Quercus robur, Fagus sylvatica and Picea abies

The distribution of fine roots and external ectomycorrhizal mycelium of three species of trees was determined down to a soil depth of 55 cm to estimate the relative nutrient uptake capacity of the trees from different soil layers. In addition, a root bioassay was performed to estimate the nutrient uptake capacity of Rb⁺ and NH₄⁺ by these fine roots under standardized conditions in the laboratory. The study was performed in monocultures of oak (Quercus robur L.), European beech (Fagus sylvatica L.) and Norway spruce [Picea abies (L.) Karst.] on sandy soil in a tree species trial in Denmark. The distribution of spruce roots was found to be more concentrated to the top layer (0–11 cm) than that of oak and beech roots, and the amount of external ectomycorrhizal mycelia was correlated to the distribution of the roots. The uptake rate of [⁸⁶Rb⁺] by oak roots declined with soil depth, while that of beech or spruce roots was not influenced by soil depth. In modelling the nutrient sustainability of forest soils, the utilization of nutrient resources in deep soil layers has been found to be a key factor. The present study shows that the more shallow-rooted spruce can have a similar capacity to take up nutrients from deeper soil layers than the more deeply rooted oak. The distribution of roots and mycelia may therefore not be a reliable parameter for describing nutrient uptake capacity by tree roots at different soil depths.     

The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination,is not readily released,and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots

The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.     

Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L.

Arachidonic acid, cellulase, CuSO₄, a sonicate of Phytophthora infestans mycelium and a spore suspension of Penicillium chrysogenum all elicited the formation of the sesquiterpenoid phytoalexins lubimin, 3-hydroxylubimin and rishitin in fruit cavities of Datura stramonium. 3-Hydroxylubimin was the predominant phytoalexin formed after treatment of the fruits with arachidonic acid, cellulase and the P. infestans preparation. Copper sulphate was a potent elicitor of lubimin but not 3-hydroxylubimin. The fungus P. chrysogenum metabolized lubimin and 3-hydroxylubimin to 15-dihydrolubimin and 3-hydroxy-15-dihydrolubimin respectively, both in fruit cavities inoculated with spores of this fungus and in pure culture. The 15-dihydrolubimin formed in the fruits by the fungus was further metabolized (by the fruits) to both isolubimin and 3-hydroxy-15-dihydrolubimin. The precursor-product relationships between all of the subject compounds was investigated by feeding experiments with ³H-labelled compounds. 2-Dehydro-[15-³H₁]lubimin was rapidly and efficiently incorporated into lubimin and may be the direct precursor of lubimin in planta. 3-Hydroxy[2-³H₁]lubimin was incorporated into the nor-eudesmane rishitin but 10-epi-3-hydroxy[2-³H₁]lubimin was not. An updated scheme for the biosynthesis and metabolism of lubimin and related compounds in infected tissues of solanaceous plants is presented.We thank Mr Vic Swetez for the provision of plant material, Mrs Margaret Huffee for technical assistance, Dr David Ewing for help with obtaining NMR spectra, and the Agricultural and Food Research Council for financial support.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase