BACKGROUND:The aim of this study was to assess similarities and differences between the trout eye and the human eye.MATERIALS AND METHODS:Gross and microscopic examinations of the formalin-fixed eyes of each five trout and human eyes (donor eyes not suitable for keratoplastic) were carried out.RESULTS:Compared to the human eye, the trout showed a flattening of the anterior-posterior axis, and cartilage-stabilized sclera. The peripheral cornea was much thicker than the central, had a multilayered thick epithelium, a distinct Bowman layer, and an implied Descement membrane. A ring-shaped ligament filled up the angle of the anterior chamber and linked the iris to the cornea. The lens showed a spherical aspect with a thick capsule and missing zonular fibres, however, a suspensory ligament of a superior part of the lens was present. Ventrally, at the end of the falciform process, a small, pigmented structure was in contact with the lens. The retina was similarly differentiated, but the choroid showed special structures like choroidal gland, falciform process and the argentea compared to the human eye.CONCLUSIONS:Great variations between the ocular anatomy of the trout and the human exist. However, the retina of the trout is fully differentiated and remarkably similar to that of human eyes.

Different arbuscular mycorrhizal fungi (AMF) alter growth and nutrition of a given plant differently. Plant gene expression patterns in response to fungal colonization show a certain overlap when colonized by fungi of the Glomeraceae. However, little is known of plant responses to fungi of different fungal taxa, e.g. the Gigasporaceae. We therefore compared the impact of colonization by three taxonomically different AMF species (Glomus intraradices, Glomus mosseae and Scutellospora castanea) on Medicago truncatula at the physiological and transcriptional level using quantitative-PCR. Each AMF developed a species-typical colonization pattern, with a colonization degree of 60% for G. intraradices and 30% for G. mosseae. Both species developed appressoria, intraradical hyphae, arbuscules and vesicles. S. castanea showed a colonization degree of 10% and developed appressoria, intraradical hyphae, arbuscules and arbusculate coils. All AMF enhanced the plant biomass accumulation and nutritional status although not in correlation with the colonization degree. The expression of 10 mycorrhiza-specific or mycorrhiza-associated plant genes could be separated into two clusters. The first cluster, containing arbuscule-induced genes, was highly induced in interactions with G. intraradices and G. mosseae but also slightly induced by S. castanea. The second cluster of genes contained genes that were induced primarily by S. castanea. In conclusion, genes that respond to colonization by fungi of the genus Glomus also respond to Scutellospora. However, there is also a group of genes that is significantly induced only by Scutellospora and not by Glomus species in this study. Our data indicate that genes may be differentially regulated in response to the different AM fungi.

Expression of Mtchit 3-3, a class III chitinase gene, is specifically induced by arbuscular mycorrhizal (AM) fungi in roots of the model legume Medicago truncatula and its transcripts accumulate in cells containing arbuscules. Agrobacterium rhizogenes-transformed roots and root-organ cultures of M. truncatula were used to study effects of Mtchit 3-3 on AM fungi. * This work provides evidence for enzymatic activity of the Mtchit 3-3 gene product and shows with promoter:gus fusions that a 2 kb fragment located 5' upstream from the translational start codon of Mtchit 3-3 is sufficient to confer arbuscule-dependent gene expression. By fusing the Mtchit 3-3 coding region to the CaMV 35S promoter the expression pattern was disrupted. Surprisingly, disruption stimulated spore germination of Glomus intraradices and Glomus constrictum, and in the case of G. intraradices resulted in a higher probability of root colonization and spore formation. However, no effect on the abundance of arbuscules within colonized roots became apparent. These observations demonstrate that disruption of the tight arbuscule-dependent expression pattern of Mtchit 3-3 has effects on the early interaction between roots and AM fungi.

The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti -induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4 , was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5 , was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4 , expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium -induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.

A class III chitinase gene. Mtchitinase III-3, has been found to be induced in roots of Medicago truncatula specifically upon colonization by the arbuscular mycorrhizal fungi Glomus mosseae and Glomus intraradices. Here we show by in situ hybridization techniques that Mtchitinase III-3 is only expressed in cortical root cells containing arbuscules formed by either of the fungi. Transcripts of Mtchitinase III-3 were detected in cells containing developing as well as mature arbuscules. They were absent from cells adjacent to arbuscule-containing cells, from cells in contact with intraradical hyphae and from cortical cells in non-colonized areas of the root. These results indicate that the product of the Mtchitinase III-3 gene has a role within individual cortical cells during formation and functioning of arbuscules.

Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

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.

The diaminobenzidine (DAB) staining technique was used to examine the accumulation of H2O2 in parts of roots of Medicago truncatula Gaertn. colonized by the arbuscular mycorrhiza (AM)-forming fungus Glomus intraradices Schenk and Smith. At the cellular level, the combination of bright-field and fluorescence microscopy revealed that a brownish stain, indicative of H2O2 accumulation was present within cortical root cells in the space occupied by arbuscules. Accumulation of H2O2 was especially pronounced in cells containing arbuscules that were clumped and less branched. Moreover, H2O2 accumulated around hyphal tips attempting to penetrate a host cell. In contrast, no H2O2 accumulation was observed in hyphal tips growing along the middle lamella, or in appressoria or vesicles. On the basis of these findings we suggest that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices.

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.

Two chitinases (EC 3.2.1.14) and two beta-1,3-glucanases (EC 3.2.1.39) were purified from the culture medium of spruce (Picea abies [L.] Karst.) cells to study their role in modifying elicitors, cell walls, growth, and hyphal morphology of ectomycorrhizal fungi. The 36-kD class I chitinase (isoelectric point [pI] 8.0) and the 28-kD chitinase (pi 8.7) decreased the activity of elicitor preparations from Hebeloma crustuliniforme (Bull. ex Fries.) Quel., Amanita muscaria (L.) Pers., and Suillus variegatus (Sw.: Fr.) O.K., as demonstrated by using the elicitor-induced extracellular alkalinization in spruce cells as a test system. In addition, chitinases released monomeric products from the walls of these ectomycorrhizal fungi. The beta-1,3-glucanases (35 kD, pi 3.7 and 3.9), in contrast, had little influence on the activity of the fungal elicitors and released only from walls of A. muscaria some polymeric products. Furthermore, chitinases alone and in combination with beta-1,3-glucanases had no effect on the growth and morphology of the hyphae. Thus, it is suggested that apoplastic chitinases in the root cortex destroy elicitors from the ectomycorrhizal fungi without damaging the fungus. By this mechanism the host plant could attenuate the elicitor signal and adjust its own defense reactions to a level allowing symbiotic interaction.

. Rapid reactions comprising efflux of K + and Cl − , phosphorylation of a 63-kDa protein (pp63), extracellular alkalinization and synthesis of H 2 O 2 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.

Elicitors from the ectomycorrhizal fungi Hebeloma crustuliniforme (Bull.) Quel., Amanita muscaria (L.) Pers., Suillus variegatus (Sw.: Fr.) O. K. and the spruce pathogenic fungus Heterobasidion annosum (Fr.) Bref. induced an extracellular alkalinization response in suspension-cultured cells of Picea abies (L.) Karst. within 4 to 6 min. In addition, phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein were induced in spruce cells by 4 min after elicitor application. Both the alkalinization and the protein phosphorylation were inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. Furthermore, the elicitors induced the synthesis of chitinase and extracellular ß-1,3-glucanase as well as the accumulation of peroxidase in the walls of spruce cells. On the other hand, auxins prevented the induction of peroxidase accumulation in spruce cell walls. This inhibiting effect was found for elicitors from Hebeloma Hebeloma crustuliniforme, Suillus variegatus and Heterobasidion annosum. Moreover, enzymes which were secreted by the spruce cells decreased the effectiveness of the elicitors from both the ectomycorrhizal and the pathogenic fungus. These findings indicate that no fundamental difference exists in the reactions which are induced in host cells by elicitors from the ectomycorrhizal fungi Hebeloma crustuliniforme, Amanita muscaria, and Suillus variegatus and the pathogenic fungus Heterobasidion annosum. We assume that the production of auxin by the invading fungus and the inactivation of fungal elicitors by extracellular enzymes of the plant itself could be inductive to root colonization and ectomycorrhiza formation.

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.

Heterotrophic spruce (Picea abies [L.] Karst.) callus cells were grown in dual culture with ectomycorrhizal fungi (Amanita muscaria [L.] Pers., Lactarius deterrimus Groger, Hebeloma crustuliniforme [Bull.] Quel., Suillus variegatus [Sw.: Fr.] O.K.) or with the pathogenic fungus Heterobasidion annosum [Fr.] Bref. Spruce cells stimulated growth of the mycorrhizal fungi, whereas growth of the pathogenic fungus was not affected. Two of the mycorrhizal fungi, S, variegatus and L. deterrimus, caused hypersensitive reactions in spruce cells. In contact with the surface of the plant cells the mycorrhizal fungi developed densely interwoven lobed, hand- and fan-shaped structures. They resembled morphological features occurring in the hyphal mantle and Hartig net of ectomycorrhizas. The ectomycorrhizal fungi did not show altered branching patterns when growing on cellophane sheets. These results demonstrate that callus cells of the host P. abies are able to stimulate fungal growth and to induce ectomycorrhizal fungi to form mycorrhiza-like structures which normally are only generated in the presence of host roots.

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 beta-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.

The role of apoplastic invertase (beta-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.

An der rechten Seite des Ductus venosus, bei der Einmündungsstelle in die Vena cava inferior, ist eine Klappe vorhanden. Sie ist eigentlich eine Trennwand zwischen Ductus venosus und Vv. hepaticae media und sinistra.

Die Öffnung am Abgang des Ductus venosus wird während des Fetallebens durch einen Sporn eingeengt. Dieser Sporn enthält nur sehr wenig glatte Muskulatur. Einen eigentlichen Sphincter habe ich nicht gesehen.