INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Arbuscular mycorrhizas (AMs) are the most common type of mycorrhizas (for review, see Smith and Read, 1997). AMs are formed between roots of more than 80% of all terrestrial plant species and Zygomycete fungi from the order Glomales. The fungus is able to grow into the root cortex by forming intraradical hyphae, which are subsequently differentiated into highly branched structures, the arbuscules, within cortex cells. Intraradical hyphae and arbuscules are responsible for exchange of nutrients between the plant and the fungus. The plant supplies the fungus with carbohydrates, whereas the fungus assists the plant with the acquisition of phosphate and other mineral nutrients from the soil (Harrison, 1998). The beneficial effects of the AM symbiosis result from a complex molecular dialogue between the two symbiotic partners (Harrison, 1999). Some processes occurring in this dialogue are known to be mediated by phytohormones on the plant side. However, most of these phytohormone effects were suggested from application experiments (Barker and Tagu, 2000). A possible role for abscisic acid in the establishment of mycorrhiza was suggested from the fact that the endogenous content of abscisic acid was increased in mycorrhizal roots, but not in nonmycorrhizal roots (Bothe et al., 1994). In a previous study, the establishment of AM in barley (Hordeum vulgare) roots was shown to be accompanied by the accumulation of putrescine and agmatine amides of 4-coumarate and ferulate, respectively, compounds that are also accumulated upon treatment of nonmycorrhizal barley roots with jasmonates (Peipp et al., 1997). This suggests a possible role of jasmonates in AM formation.

Jasmonic acid (JA) and its derivatives, commonly termed jasmonates, are hormonal regulators involved in plant responses to abiotic and biotic stresses, as well as in plant development (Creelman and Mullet, 1997; Wasternack and Parthier, 1997). The role of jasmonates is well established as part of a complex signal transduction pathway activated upon local wounding of leaves (Ryan, 2000). Levels of endogenous jasmonate increase upon wounding and are followed by activation of genes involved in plant defense responses such as those coding for proteinase inhibitors, enzymes of phytoalexin synthesis, vegetative storage proteins, thionins, and defensins (Creelman and Mullet, 1997; Farmer et al., 1998; Ryan, 2000). However, it is less well understood how the rise of jasmonates is regulated. The elevation of jasmonate levels is usually correlated with the activation of genes coding for JA biosynthetic enzymes (for review, see Wasternack and Hause, 2002).

The biosynthesis of JA (Fig. 1) starts with the insertion of oxygen at position 13 of -linolenic acid catalyzed by 13-LOX. The resulting hydroperoxide is converted by AOS into an unstable allene oxide that can rapidly be degraded in vitro by chemical hydrolysis. Under cellular conditions, the allene oxide is preferentially, if not exclusively, converted by AOC into (9S, 13S)-oxophytodienoic acid (OPDA). According to the present knowledge, this enantiomer is the unique precursor for the naturally occurring (+)-7-iso-JA, which is formed by reduction of OPDA catalyzed by an OPDA reductase and three subsequent steps of -oxidation. The more stable (-)-JA is then formed by spontaneous isomerization.

View larger version (10K): [in this window] [in a new window]   Figure 1.   Metabolic scheme of JA biosynthesis. The enzymes involved in JA biosynthesis are 13-lipoxygenase (13-LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and oxophytodienoic acid reductase (OPR).

In barley, as in other plants, the genes coding for 13-LOX (Vörös et al., 1998), AOS (Maucher et al., 2000), and AOC (H. Maucher, personal communication) are transcriptionally activated upon treatment with jasmonates. Osmotic stress, caused experimentally by sorbitol treatment or appearing tissue specifically in seedling development, also induces the expression of AOS and AOC, and is strictly correlated with elevation of JA levels (Maucher et al., 2000; H. Maucher, personal communication). Such an endogenous rise in jasmonates is functionally relevant because jasmonate-induced gene expression occurs. The most abundant gene product occurring in barley leaves upon JA treatment or upon endogenous rise of JA is a 23-kD protein (JIP23; Andresen et al., 1992; Lehmann et al., 1995). JIP23 is always detectable after the elevation of jasmonate levels (Kramell et al., 2000). Therefore, the occurrence of JIP23 is a valuable reporter of endogenous rise of jasmonates as used for the analysis of the pathogenic interaction of barley leaves with powdery mildew (Hause et al., 1997). Also, in other tissues of the barley plant, there is a strict correlation of the expression of JIP23 and enhanced endogenous JA levels. JIP23 is expressed constitutively in the root tip, the scutellar node, and the leaf base, which are tissues that show enhanced JA levels (Hause et al., 1996; Maucher et al., 2000). Furthermore, the elevated JA level in these barley tissues correlates with AOS expression, suggesting a causal link between expression of genes coding for JA biosynthetic enzymes, elevation of JA levels, and expression of JA-induced genes (Maucher et al., 2000). Therefore, simultaneous recording of the expression of AOS and JIP23 and JA levels represents a tool for asking whether an increase in JA biosynthesis is correlated with expression of JA-biosynthetic genes and JA-dependent processes.

In the present work, we show for the first time, to our knowledge, that the interaction of barley roots with a mycorrhizal fungus leads to marked increases of JA levels. To analyze the possible involvement of jasmonates in the establishment of AMs, we recorded JA levels, as well as the temporal and spatial expression patterns of genes coding for AOS and JIP23. The data revealed expression of both genes within arbuscule-containing cells after the onset of arbuscule formation. We discuss a possible link between the enhanced sink function of mycorrhizal roots compared with nonmycorrhizal roots and the induction of JA biosynthesis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Elevated Jasmonate Levels in Mycorrhizal Roots Are Accompanied by Expression of a Gene Encoding a Pivotal Enzyme of JA Biosynthesis

To analyze a possible link between mycorrhization and endogenous levels of jasmonates, mycorrhization rates, and contents of JA, JA-Ile and OPDA were determined during the time course of mycorrhization of barley roots with the AM fungus Glomus intraradices. Inoculation with fungal spores according to the standard protocol (Maier et al., 1995) led to an increase of the mycorrhization rate from 20% at week 3 to 60% at week 8 (Fig. 2A). The JA level increased in the mycorrhizal roots up to 4-fold between weeks 3 and 4 of cultivation, but remained constant at a low level in nonmycorrhizal roots (Fig. 2A). Also, the level of the major amino acid conjugate of JA, JA-Ile, exhibited a transient rise upon mycorrhization. The level of the JA precursor OPDA remained nearly constant at a basal level of about 0.2 nmol g¹ fresh weight during mycorrhization.

View larger version (50K): [in this window] [in a new window]   Figure 2.   Accumulation of endogenous jasmonates and of transcripts of AOS and JIP23 upon inoculation of barley roots with spores of G. intraradices. A, Accumulation of free JA and JA-Ile in nonmycorrhizal and mycorrhizal barley roots. Jasmonates and the conjugate were extracted and quantified from roots pooled from five different plants as indicated in "Materials and Methods." Three independent extractions and analyses were performed giving similar values that varied by about 15%. One series of data is given. The mycorrhization rate is given as the percentage of colonized root segments pooled from five different plants. B, Accumulation of transcripts of genes coding for AOS and JIP23 analyzed by reverse transcriptase (RT)-PCR. Ubiquitin transcripts were used as control to confirm constant levels of amplified fragments for all samples.

Figure 2B illustrates AM-induced AOS mRNA accumulation. Three-week-old nonmycorrhizal roots exhibited some AOS mRNA accumulation, presumably due to the high percentage of AOS-expressing root tips in young roots (Maucher et al., 2000). However, at later stages, only mycorrhizal roots exhibited AOS mRNA accumulation, suggesting an increase in capacity of JA biosynthesis. This is indicated by the rise in JA level and by the expression of the JA-responsive gene JIP23 (Fig. 2B), both occurring exclusively in mycorrhizal roots.

AOS and JIP23 Transcripts and Protein Accumulate within Arbuscule-Containing Cells

In extraction procedures for mRNA analyses and JA measurements, mycorrhizal root cells are mixed with nonmycorrhizal cells. Therefore, we analyzed whether or not only the mycorrhizal roots cells exhibit the respective altered gene expression by performing in situ hybridization for AOS and JIP23 expression with young mycorrhizal roots from plants of nurse-pot cultures at 12 d after transplantation (see below). As shown in Figure 3, A through D, hybridizations with the antisense probes revealed occurrence of AOS mRNA and JIP23 mRNA only in arbuscule-containing cortex cells. Hybridizations with the AOS and JIP23 sense probe did not exhibit any label as exemplified for AOS (Fig. 3, E and F). This result could be confirmed by immunocytochemical detection of AOS and JIP23. Using mycorrhizal roots 8 weeks after inoculation, immunolabeling was performed with monospecific polyclonal antibodies raised against AOS and JIP23. As expected, nonmycorrhizal roots did not exhibit staining with anti-AOS antibodies (Fig. 4A) or with anti-JIP23 antibodies (Fig. 4, D, E). AOS protein was clearly and exclusively detectable in arbuscule-containing cells of the inner root cortex (Fig. 4, B, C), whereas the central cylinder and the rhizodermis were free of label. Using parallel cross sections, JIP23 was localized within the same cells (Fig. 4, F, G). However, an additional immunodecoration was found within the central cylinder (Fig. 4G, arrows). Here, the companion cells of the sieve element complex of the phloem exhibited label. The controls performed with preimmune sera showed only faint background staining as shown in Figure 4H for JIP23. These data support the assumption that JA biosynthesis (shown by expression of AOS) and the expression of JA-induced genes occur within cells harboring arbuscules.

View larger version (110K): [in this window] [in a new window]   Figure 3.   Localization of AOS and JIP23 mRNA accumulating in mycorrhizal barley roots. Root segments from barley plants inoculated in nurse pot cultures for 12 d were processed for in situ hybridization using digoxigenin-labeled antisense RNA for AOS (A and B) and JIP23 (C and D). Note the occurrence of positive staining in arbuscule-containing cells (arrowhead in B and D). Negative controls performed by using DIG-labeled sense probes do not exhibit label as shown for AOS (E and F). Here, arbuscules are hardly detectable due to the absence of staining (arrows in E, and arrowhead in F). Bars = 50 µm.

View larger version (170K): [in this window] [in a new window]   Figure 4.   Localization of AOS and JIP23 in nonmycorrhizal and mycorrhizal barley roots. Immunochemical detection of AOS (A-C) and of JIP23 (D-G) in cross sections of roots of 8-week-old nonmycorrhizal (A, D, and E) or mycorrhizal (B, C, F, and G) plants, respectively. AOS protein was visualized by immunodecoration with a purified rabbit anti-AOS antibody; to visualize JIP23, polyclonal, monospecific rabbit anti-JIP23 antibodies were used. Both incubations were followed by a goat anti-rabbit immunoglobulin G antibody conjugated with alkaline phosphatase. Note the occurrence of both proteins within arbuscule-containing cortex cells. The immunodecoration with anti-JIP23 exhibits an additional label within companion cells of the central cylinder in mycorrhizal roots (arrows in G). A control (H), in which cross-sectioned mycorrhizal roots were incubated with JIP23 preimmune serum followed by the same secondary antibody, does not show labeling. Bars =100 µm.

Higher JA Levels Occur after the Initial Steps of the Establishment of Mycorrhiza

Because of the slow increase of mycorrhization rate upon inoculation with fungal spores, the stage of mycorrhization in which the initial rise in the JA level occurs cannot be analyzed precisely. Therefore, a nurse-pot culture system was used to achieve a rapid and near-synchronous mycorrhization (Roswarne et al., 1997). The first arbuscules could be detected after 6 d of cultivation (Fig. 5A), if 3-d-old barley seedlings were transplanted into the center of pots containing mycorrhizal leek (Allium porrum) plants. The frequency of arbuscules increased from 20% at d 6 to 45% at d 12 after transplantation. The mycorrhizal roots exhibited a drastic decrease of the arbuscule frequency at d 14. As a consequence, the time period used in the experimental setup covered the first generation of arbuscules, which usually remain active up to 14 d within the root cortex cells (Smith and Read, 1997).

View larger version (59K): [in this window] [in a new window]   Figure 5.   Accumulation of endogenous JA and of transcripts of AOS and JIP23 upon near-synchronous colonization of barley roots with G. intraradices. A, Accumulation of free JA in nonmycorrhizal and mycorrhizal barley roots. JA was extracted and quantified from roots pooled from five different plants as indicated in "Materials and Methods." Three independent extractions and analyses were performed giving similar values that varied by about 15%. One series of data is shown. The mycorrhization rate is given as the percentage of arbuscule-containing root segments pooled from five different plants. First arbuscules occurred at the 6 d after transplantation. B, Accumulation of transcripts of genes coding for AOS and JIP23. For northern-blot analysis, 20 µg of total RNA was loaded per lane. The positive control was performed by loading 2 µg of total RNA from barley leaf segments treated with 45 µM JA methyl ester for 24 h (LS/JM). Loading was checked by recording the ethidium bromide staining of rRNA.

In nonmycorrhizal barley roots (transplanted in between nonmycorrhizal leek plants), the JA level remains constant during the cultivation period, whereas mycorrhization led to an increase in the JA level (Fig. 5A). Here, a 3-fold increase occurs from d 8 to 14 after transplantation. The endogenous rise of JA was accompanied by AOS mRNA accumulation preferentially at d 12 after transplantation (Fig. 5B). In nonmycorrhizal roots exhibiting constant JA levels, constant AOS mRNA levels were also found. JIP23 mRNA, indicative of elevated JA levels, accumulated at d 12 and 14 after transplantation (Fig. 5B). The data reveal that upon near-synchronous mycorrhization, the elevated JA level is accompanied by transcript accumulation of genes coding for a JA-biosynthetic enzyme and a JA-inducible protein. However, the first arbuscules appeared 4 to 6 d prior to the accumulation of AOS and JIP23 mRNAs and of jasmonates.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The role of jasmonates in plant responses to wounding and pathogen attack is well established (for review, see Wasternack and Hause, 2002). However, in symbiotic interactions, putative signals, including jasmonates, are poorly understood. To date, only a few hints for a possible involvement of JA in mycorrhization have been shown: Treatment with JA stimulated the mycorrhizal development of an ectomycorrhiza (Regvar et al., 1996, 1997), and accumulation of secondary compounds occurring in AM barley roots is inducible in nonmycorrhizal roots by jasmonates (Peipp et al., 1997). Here, we analyzed the involvement of jasmonates in the formation of an AM symbiosis established by colonization of barley roots with G. intraradices.

Mycorrhization of barley roots is accompanied by a 5-fold elevation of the JA level and a 2.5-fold increase in the level of JA-Ile, both suggesting a causal link to mycorrhization. These levels most likely reflect the total amount of all JA-related compounds because a loss of JA-related compounds into the gaseous phase could not be observed in barley (W. Boland, personal communication) and catabolites have been not detected so far (O. Miersch, unpublished data). In barley leaves, OPDA was among JA-related compounds accumulating upon osmotic stress (Kramell et al., 2000). In other plants, OPDA accumulates in response to touch (Stelmach et al., 1998), elicitation of cell cultures (Parchmann et al., 1997), or wounding of leaves (Parchmann et al., 1997), usually together with JA. Both compounds were shown to function independently as signals in plant stress as shown by an Arabidopsis mutant unable to convert OPDA into JA (Stintzi and Browse, 2000). However, mycorrhization does not seem to be related to OPDA because it did not accumulate.

To date, there is no approach to directly localize the site of JA accumulation within plant tissues. Therefore, we used an indirect method by analyzing the temporal and spatial expression of a JA-biosynthetic gene, AOS, shown to be indicative of elevated JA levels (Maucher et al., 2000). These data were compared with the spatial expression pattern of a JA-responsive gene. As a consequence, the occurrence of the encoded protein is indicative of elevated JA levels. In situ hybridization and immunolocalization, performed with mycorrhizal barley roots, revealed an expression of both genes within cells that contain the main symbiotic interface by harboring arbuscules. This spatially coordinated expression suggests that elevated JA levels may occur specifically within arbuscule-containing cells. These results also suggest that the cell-specific and local rise of jasmonates by far should exceed the 4-fold increase measured from whole tissue extraction. The occurrence of JIP23 protein within the companion cells of mycorrhizal roots without detection of its mRNA may indicate persistence of JIP23 from a preceding JIP23 gene expression. Once synthesized, JIP23 protein might occur in these cells due to its negligible turnover known for barley leaf segments and seedlings (Hause et al., 1996).

In addition to the spatial link between mycorrhization and JA levels, a temporal correlation was found. However, higher JA levels occurred after the onset of mycorrhization, implying that a fully established mycorrhiza rather than the recognition of the interacting partners or the establishment of the symbiotic interface might cause AOS expression and elevation of JA levels. In addition to stress-induced changes, jasmonate levels rise at distinct developmental stages (Wasternack and Hause, 2002). Tissues accumulating jasmonates during development are the hypocotyl hook of soybean (Glycine max) seedlings (Creelman and Mullet, 1995), the scutellar node of barley seedlings (Hause et al., 1996), and ovules of tomato (Lycopersicon esculentum) flower buds (Hause et al., 2000). These elevated JA levels were accompanied by simultaneous increase in expression of AOS (Maucher et al., 2000) or AOC (Hause et al., 2000). It is interesting that all of these tissues exhibiting simultaneous rise in JA level and AOS/AOC expression are sink tissues. Arbuscule-containing cells also represent sinks for carbohydrates because a main feature of the mycorrhizal symbiosis is the supply of the obligate heterotrophic fungus with carbohydrates (Harrison, 1999).

It has been clearly shown that the uptake of hexoses takes place within the host root only (Shachar-Hill et al., 1995). Root cortex cells release Suc, which is converted into hexoses by acid invertases (Blee and Anderson, 1998). Hexoses can be subsequently taken up by the fungus within the apoplastic compartment (Bago et al., 2000). Thus, by supplying the fungus with carbohydrates, mycorrhizal roots represent a much stronger sink organ than nonmycorrhizal roots (Douds et al., 2000). This is supported by the observation that in mycorrhizal barley roots, JIP23 also occurred in companion cells, which are known to be osmotically stressed by active solute transport. Until now, the occurrence of JIP23 in companion cells has been found only in the scutellar node and the leaf base of germinating seedlings, where carbohydrates were transported from the endosperm to the developing tissues (Hause et al., 1996). Thus, the enhanced transport of carbohydrates into the root, and the inducibility of AOS and AOC expression by Glc (Hause et al., 2000; Maucher et al., 2000) suggest the following scenario: Sugars supplied by source tissues are translocated into sink tissues, here, mycorrhizal roots. The resulting putative osmotic stress or induction by the sugar itself may lead to expression of genes coding for enzymes of JA biosynthesis and finally to rise of JA levels.

Sugars are the substrate for heterotrophically growing tissues, including invading organisms such as symbiotic fungi, and they are also important regulatory signals for the metabolism of source as well as sink tissues (Roitsch, 1999). Sink tissues are usually potential targets for pathogens due to their high amounts of carbohydrates. However, a characteristic feature of sink tissues is the expression of sink-specific as well as defense-related genes (Roitsch, 1999). The latter may contribute to an increased defense status. Jasmonates may modulate such a defense status because they were shown to induce expression of pathogenesis- and stress-related genes (Wasternack and Hause, 2002). It is interesting that the induced systemic resistance of Arabidopsis triggered by nonpathogenic, root-colonizing Pseudomonas fluorescence bacteria is associated with jasmonate-responsive gene activation (Pieterse et al., 1998).

It is tempting to speculate that elevated JA levels occurring upon mycorrhization may enhance the defense status of mycorrhizal tissues, which were shown to be less sensitive to secondary infections by pathogens (Cordier et al., 1998) or to drought and osmotic stress (Augé, 2001). As a consequence, mycorrhizal plants should have maximal benefit from the symbiotic interaction. The data described in this report now provide the basis to analyze such a role of jasmonates in mycorrhizal roots by modulating endogenous JA levels via overexpression or suppression of JA biosynthetic genes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material and AM Inoculation

Cultivation and inoculation of barely (Hordeum vulgare cv Salome) and the propagation of the AM fungus, Glomus intraradices Schenck & Smith, were described previously (Maier et al., 1995). Mycorrhiza formation was induced by growing the plants in expanded clay (Lecaton, 2-5 mm particle size; Fibo Exclay, Pinneberg, Germany) mixed with 10% (v/v) of the fungal inoculum. To obtain nearly synchronous mycorrhization, 3-d-old barley seedlings were transplanted into the middle of pots containing mycorrhizal leek (Allium porrum) plants according to Roswarne et al. (1997). Mycorrhiza formation was determined microscopically with the gridline-intersection method at a magnification of ×20 after staining with trypan blue (Phillips and Hayman, 1970).

Extraction and Measurement of OPDA, JA, and JA-Ile

Fresh roots from at least five different plants were pooled to minimize biological differences and were immediately frozen in liquid nitrogen. One gram of root material was homogenized in a mortar and was extracted with 10 mL of 80% (v/v) methanol. For quantification of JA, appropriate amounts of (²H₆) JA were added to the extract, whereas in the case of JA-Ile, JA-[²H₃]-Leu was used as internal standard. The methanolic extracts were purified by chromatographic steps as described for the isolation of JA and JA methyl ester (Kramell et al., 1997). The final separation was performed by reverse phase-HPLC (column: LiChrospher 100, RP-18, 250 × 4 mm, 5 m; flow rate: 1 mL min¹; UV detection at 210 nm) using a 70:30 (v/v) mixture of methanol and water (containing 0.2% [v/v] acetic acid) as the mobile phase. The fractions corresponding to authentic JA (4-5 min) and JA-Ile/JA-Leu (6-8 min) were concentrated in vacuo. The content of JA-Ile was calculated on the basis of a calibration curve recorded with methylated JA-[²H₃]-Leu. The intensities of the molecular ions at m/z 340 for the deuterated compound and m/z 337 for the nonlabeled compounds were compared. For quantitation of OPDA, 0.5 g of plant material was extracted and prepared for gas chromatography-mass spectrometry analysis according to Mueller and Brodschelm (1994). Gas chromatography-mass spectrometry was performed as previously described (Hause et al., 2000).

Extractions of RNA, Northern-Blot Analysis, and RT-PCR

Total RNA of frozen tissues was extracted by phenol:chloroform:isoamyl alcohol 25:24:1 (v/v) using modifications of Andresen et al. (1992). Because of the low amount of RNA isolated from several-week-old barley roots, RT-PCR was performed with 0.1 µg of total RNA by using the Titan One Tube RT-PCR System (Roche Diagnostics, Mannheim, Germany). Primers were designed according to the AOS sequences (Maucher et al., 2000), the JIP23 sequence (Andresen et al., 1992), and ubiquitin sequences (accession no. M60175/M60176) with the following combinations used: AOS: 5'-CCAGCGACCGCCTC-3' and 5'-GGAGCGGCTCCTCGAGG-3', resulting in a fragment of 600 bp from both AOS genes; JIP23: 5'-AATGGCCTCAGGAGTGTTTG-3' and 5'-TTCATGGTAGTGCCTTCACC-3', resulting in a fragment of 602 bp; and ubiquitin: 5'-CTCGCCGACTACAACATCC3' and 5'-GGTAAAAGAGCAGAGCAAAC-3', resulting in a fragment of 294 bp. The annealing temperature was 55°C for all reactions, and fragments were amplified by 35 PCR cycles.

In the experiments done with young barley roots from nurse-pot cultures, electrophoresis of 20 µg of total RNA per lane and northern-blot analysis were performed according to Sambrook et al. (1989). Blots were hybridized at 65°C for 16 h with ³²P-labeled fragments of the barley AOS1 cDNA or the JIP23 cDNA, both encompassing the full-length cDNA sequence. Gel loading was checked by comparing ethidium bromide-stained rRNA.

In Situ Hybridization and Immunocytochemistry

Small pieces of mycorrhizal and nonmycorrhizal roots were fixed with 3% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; 135 mM NaCl, 3 mM KCl, 1.5 mM KH₂PO₄, and 8 mM Na₂HPO₄). After dehydration in a graded series of ethanol, material was embedded in polyethylene glycol and cut as described (Hause et al., 1996). For in situ hybridization, cross sections of 10 µm thickness were collected in sieves, rinsed in Tris-HCl (pH 8.0), and incubated with 1% (w/v) bovine serum albumin (BSA) in the same buffer for 1 h. After acetylation, sections were dehydrated in graded series of ethanol and were air-dried. For hybridization, a solution of 0.3 M NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1× Denhardt's solution, 50% (v/v) formamide, 2 mg mL¹ tRNA, and 200 U mL¹ RNase inhibitor containing denaturated DIG-labeled sense or antisense RNA was applied and sections were incubated in a humid box at 45°C overnight. After two washing steps with 0.2× SSC at 55°C for 30 min each, sections were incubated with 20 µg mL¹ RNase A at 37°C for 30 min, followed by washing with 0.2× SSC at 55°C for 1 h. Immunological detection of DIG-labeled RNA hybrids was performed with an anti-DIG-fab fragment conjugated with alkaline phosphatase (Roche Diagnostics) according to the supplier's protocol. For localization of AOS protein and JIP23, sections of 5 µm thickness were immunolabeled with the purified rabbit-anti-AOS antibody (diluted 1:50 in PBS containing 5% [w/v] BSA; Maucher et al., 2000) or with the rabbit-anti-JIP23 antibody (diluted 1:5,000 in PBS containing 5% [w/v] BSA; Hause et al., 1996) followed by anti-rabbit immunoglobulin G antibody conjugated with alkaline phosphatase as described (Hause et al., 2000). The staining procedure was performed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Sections were mounted on poly-L-Lys-coated slides and were analyzed by bright field microscopy with an Axioskop microscope (Zeiss, Jena, Germany). Pictures were taken by a CCD camera (Sony, Tokyo) and were processed through Photoshop 4.0 (Adobe Systems, Seattle).