Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips

Abstract

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.

Introduction

Since the pioneering work of Charles and Francis Darwin, monocot coleoptiles have long been used in research for indole-3-acetic acid (IAA) biosynthesis.¹ For maize, a number of studies have indicated that the coleoptile tip is the site of IAA biosynthesis, and the amount of IAA, and its direction of transport, are controlled by environmental stimuli.^2-7 In our previous work, we measured endogenous free and conjugated IAA in the tip, and transported free IAA from the tip to lower regions, and deduced that the main site of IAA biosynthesis was within the top 2 mm of the tip.⁶ Furthermore, ₁₃C₁₁¹⁵N₂-tryptophan (Trp) tracer experiments showed that incorporation of the stable isotope of Trp into IAA mainly occurred within the top 0–1 mm region.⁷ These results indicated that the most active site of IAA synthesis is within the 1 mm tip.

The mechanisms of IAA biosynthesis and the site of de novo IAA synthesis are not fully understood. However, recent advances in molecular genetic analysis, mainly using Arabidopsis, have led to the isolation of several convincing candidate genes involved in putative IAA synthetic pathways.^8-12 YUCCA was proposed to catalyze the conversion of tryptamine to N-hydroxy-tryptamine, a rate-limiting step in the YUCCA pathway.⁸ However, another recent report suggested the possibility that N-hydroxy-tryptamine is not the substrate for the YUCCA protein.¹³ In addition, TAA1, an aminotransferase, was shown to catalyze the formation of indole-3-pyruvic acid from Trp in the indole-3-pyruvic acid pathway.^10,11 In maize, SPI1 encodes a YUCCA-like protein that is essential for normal inflorescence development.¹⁴ In addition, vt2 was identified as a homologous gene of Arabidopsis TAA1.¹⁵

High resolution measurements of endogenous IAA, and monitoring of IAA distribution and movement in tissues, are necessary to identify the mechanisms and specific sites of IAA biosynthesis and its role in plants’ physiological events. In particular, the localization and concentration of IAA at the cellular level, which must control cell activity and/or its fates, are critical. The IAA distribution in plants, mainly studied in Arabidopsis, has been deduced by the expression patterns of artificial promoter:reporter constructs, such as DR5:GUS and DR5:GFP.^16-18 These constructs contain a synthetic auxin response element coupled to the reporter gene, and have been shown to be affected by cellular IAA concentration. However, for some cell types, direct IAA quantification indicated a lower or higher IAA level than what would have been expected from the DR5 promoter:reporter expression pattern.¹⁹ Furthermore, a report showed that the DR5 promoter element responds to brassinolides as well as IAA,^20,21 making it difficult to deduce whether the GUS or GFP signals accurately correspond to the amount of IAA in tissues. Immunolocalization of IAA has also been used to visualize IAA distribution in several plants.^22-26 However, there are many analogous compounds to IAA, such as conjugated IAA and IAA-derivatives, including intermediates and catabolites, in plant tissues. In addition, IAA deficient plants are not available. Therefore, it is difficult to assess the specificity of an antibody against IAA. It is particularly important to verify the specificity of an antibody to conjugated IAA, which exists ubiquitously in plant tissues at levels more than 10-fold higher than free IAA.

Here, we first established the immunohistochemistry of IAA within maize coleoptiles by comparing the amounts of endogenous free and conjugated IAA (total IAA minus free IAA) to the IAA signal obtained from IAA antibodies. Using an anti-IAA-C-antibody, we detected IAA immuno-signals whose intensities were strongly co-related with free-IAA levels, but not with conjugated-IAA, precursor Trp, and one of putative intermediates, indole-acetamide (IAM). Using this method, we also observed the precise IAA distribution through the coleoptile tissues, and the sites of IAA accumulation and reduction after 1-N-naphthylphthalamic acid (NPA) and 5-methyl-tryptophan (5-mT) treatments, respectively. The results indicated that IAA is mainly synthesized in the 0–2.0 mm region of coleoptiles and that IAA synthetic activity is high in the outer epidermis of the approximately 0.5 mm region. We also investigated intra-cellular IAA localization in the outer-epidermal cells by electron-microscopy using an anti-IAA-N-antibody. IAA was localized in the cytoplasm, plastids, and mitochondria, but not in vacuoles or cell walls.

Results

Immunohistochemical Detection of IAA with an Anti-IAA-C-Antibody

The anti-IAA-C-antibody (IAA-Carboxyl linked-antibody; Agdia, mouse anti-IAA monoclonal antibody, IAA-17–2-A) was raised against carboxyl-linked IAA. Therefore, to conserve the antigenicity of IAA, 1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDAC) treatment is necessary as the fixative.²³ EDAC crosslinks the carboxyl group of IAA to proteins in tissues, and preserves the antigenicity of IAA to this IAA-C-antibody. The cross-reactivity of the antibody for IAA-related compounds had been determined using ELISA tests (see Product information). However, ELISA tests cannot fully verify the specificity of an antibody for immunohistochemical detection in plant tissue sections. Therefore, to corroborate the specificity of the monoclonal IAA-C-antibody for IAA within maize coleoptiles, IAA signals were observed under several coleoptile conditions containing different levels of free and conjugated IAA (total IAA minus free IAA). Furthermore, maize coleoptiles contain high levels of tryptophan (Trp) and indole-3-acetamide (IAM), but almost no indole-3-acetonitrile (IAN) and indole-3-acetaldoxime (IAOx).^4,12 It was reported that 11.1 ng/gFW of IAM was determined in the coleoptile tip, whereas IAN and IAOx were not detectable by LC-MS/MS analysis.¹² In addition, Trp exists in the tip at more than 50-fold higher levels than free IAA.⁴ It was therefore important to investigate the cross-reactivity of the IAA-C-antibody to Trp and IAM. Initially, we checked IAA signals of externally applied IAA, IAM, and Trp in coleoptiles and roots using the antibody. The results showed that the antibody successfully detected externally applied IAA in a concentration-dependent manner, but did not detect Trp and IAM (Figs. 1A, B).

Figure 1.

Immunolocalization of several concentrations of external IAA, Trp and IAM. (A) Immunolocalization of several concentrations of external IAA, 100 ?M Trp, and 100 ?M IAM in (A) coleoptiles and (B) root. After 30 min of IAA-depletion of the 2–4 mm region of the coleoptiles and root tip, they were treated with the indicated concentration of IAA, Trp and IAM for 60 min. ((A) Scale bar = 1 mm, (B) bar = 200 ?m)

When 100 μM NPA was applied to the inside of the top 2 mm region of coleoptiles, or when the top 2 mm of coleoptiles was excised, the amount of free IAA in the 2–4 mm region of the coleoptiles decreased to less than 20% of the level after 60 min of treatment, compared with the non-treated control. However, the amount of conjugated IAA did not change under these treatments (Fig. 2A). Immunolocalization of IAA in non-depleted control sections showed that the auxin signal was mainly localized in the coleoptile outer-epidermis and mesophyll cells, between vascular bundles and the inner-epidermis, and weakly in the inner-epidermis (Fig. 2B). When either the primary antibody or the secondary antibody was omitted from the complete procedure, no signals were observed (data not shown). Omitting EDAC pre-fixation resulted in staining only in vascular bundles (Fig. 2B), indicating cross-reaction of the antibody to some component (s) in vascular tissues (Fig. S1). In the samples that were depleted of IAA by NPA or by 2 mm tip excision, almost no IAA signals were detected in sections in which conjugated IAA remained at the same level as the control (Fig. 2A (control), Figure 2B). Furthermore, IAA redistribution after 60 min of gravity stimulus was detectable (Fig. S2), using the method reported previously by Nishimura et al., 2009.⁷ These results showed that the intensity of IAA signals basically correlated with the free IAA levels in the tissue. Thus, we concluded that the monoclonal IAA antibody specifically recognizes free IAA, but not conjugated IAA or other IAA-structurally-related compounds, at least in maize coleoptile tissues.

Figure 2.

Specificity of the anti- IAA-C-antibody for IAA. (A) Free and conjugated IAA (total IAA minus free IAA) levels in the coleoptile 2–4 mm region of the non-treated control and IAA-depleted (NPA treated 2 mm tip region and 2 mm tip excised) samples. After NPA was applied to the inside of the coleoptile 2 mm tip or coleoptile from which the 2 mm tip was excised, the coleoptiles were incubated for 60 min. Then, 2–4 mm of coleoptiles were collected for free IAA and conjugated IAA determination. (B) Immunolocalization of IAA in cross-sections of around 4 mm from the top of several coleoptiles containing different levels of free and conjugated IAA shown in (A). Control; Non-treated control. –EDAC; Without EDAC prefixation. NPA; coleoptiles that were NPA treated in the 2 mm tip region. Excised; coleoptiles from which the 2 mm tip was excised. Alkaline phosphatase-conjugated secondary antibody was used for signal detection. (Scale bar = 1 mm)

Distribution of IAA in Maize Coleoptiles

More precise distribution of IAA in the coleoptile tip was investigated by IAA immunolocalization. Our previous work indicated that high concentrations of IAA exist in the apical 0–1.0 mm of the tip, and that IAA levels decline to about one-third in the 1.0–2.0 mm section.⁶ Here, immunohistochemical IAA signals were detected in epidermal cells, with low levels in the mesophyll cells of the very tip region of coleoptiles (Figs. 3A, E). Intense IAA signals were observed in both mesophyll cells and outer-epidermis in the apical 1 mm region (Figs. 3B, F); the signals became weaker in the lower part of coleoptiles (Figs. 3D, G), which is consistent with our previously reported IAA quantification results.^6,7 In the transverse-sections of the sub-apical 2 mm, IAA was mainly restricted to the outer-epidermis and mesophyll cells, between the vascular bundles and inner-epidermis (Fig. 3D). The cellular localization of IAA in coleoptile tips was observed using a fluorescent second antibody and confocal microscopy. Omitting EDAC pre-fixation resulted in almost no signal staining (Fig. S3). In vacuolated inner-epidermal cells, IAA was distributed in the cytoplasm (Figs. 3I, K). The IAA signal was especially localized in the periphery of the nucleus in mesophyll cells (Fig. 3J, arrows). In the outer epidermis of the apical 0.5–1.0 mm region, cells retained more cytoplasm compared with other cells, and intense IAA signals were observed in the cytoplasm and in the periphery of the nucleus (Figs. 3L, M).

Figure 3.

Immunolocalization of IAA with the IAA-C-antibody in the coleoptile tip. Distribution of IAA in the coleoptile tip by alkaline phosphatase detection. IAA was localized in transverse sections (A-D) and in longitudinal sections (E-G) of the apical 2.0 mm region of coleoptiles. (A-D; scale bar = 0.2 mm, E-G; scale bar = 100 ?m) Immunofluorescence of IAA in longitudinal sections. (H) Longitudinal section of a coleoptile top 1 mm stained with Toluidine blue O. (I-M) Immunofluorescence of IAA in longitudinal sections. (I) Inner-epidermis of the top. (J) Mesophyll cells. (K) Inner-epidermis 1 mm from the top. (L) Outer-epidermis 1 mm from the top. (M) Outer-epidermis of the top region. IAA signals were observed mainly in the cytoplasm. Strong signals were observed around the nucleus (arrows in K). (Scale bar = 20 ?m)

Determination of Putative IAA Synthetic Cells in Maize Coleoptiles

IAA accumulates at the coleoptile tip after NPA treatments because IAA is constitutively produced at the coleoptile tip and its transport from the tip is inhibited by NPA.⁷ To determine the precise IAA synthetic region, and the IAA synthetic activity within the 2 mm tip of the coleoptile, we observed the site of IAA accumulation after NPA treatment using gas chromatography-mass spectrometry (GC-MS) (Fig. 4A). After NPA treatment, the amount of IAA (IAA pg/section) mainly increased in the 0.5–2.0 mm region of coleoptiles. There was almost no accumulation of IAA in the sub-apical 2.0–4.0 mm regions. Meanwhile, the concentration of IAA per tissue (IAA ng/gFW) was higher in the 0–1.0 mm region than in the 1.0–2.0 mm region, indicating that active IAA biosynthesis mainly occurs in top 0–1 mm region. To observe the site of IAA accumulation in this region, IAA was visualized by immunolocalization after NPA treatment. After 60 min of NPA treatment, strong IAA signals were observed in tissues corresponding to the results obtained from IAA determination by GC-MS (Fig. 4A). Intense signals were detected in the 1.0 mm region on transverse sections (Fig. 4B). Furthermore, the IAA signal was very intense in the outer epidermis in the 0.5 mm region from the top, under NPA treatment (Fig. 4B-a, b, arrows). The increased IAA signal in the outer epidermis of the 0.5 mm region might reflect the dense accumulation of IAA in the epidermis.

Figure 4.

Accumulation of IAA after NPA treatment. (A) Detailed determination of IAA levels in coleoptile tip sections after NPA treatment. The amount of IAA ((IAA pg/section) and (IAA ng/gFW)) was determined by GC-MS after 30 and 60 min of 100 ?M NPA treatment. (B) Immunolocalization of IAA with the IAA-C-antibody after NPA treatment. IAA was visualized by immunolocalization of IAA in cross-sections of the top 0.5, 1.0, and 2.0 mm of the coleoptile, with or without 60 min NPA treatment. (Scale bar = 0.5 mm) (a) and (b) Magnified images of the outer epidermis of 0.5 mm cross-sections in mock control (a) and 60 min NPA treatment (b). (Scale bar = 100 ?m) Intense IAA signals were observed in the inner-epidermis after NPA treatment (arrows in NPA).

When 5-metyl tryptophan (5-mT), a known Trp synthesis inhibitor, was applied to the inside of the coleoptile tip, the amount of IAA decreased in a dose-dependent manner (Fig. 5A). After 120 min of 500 ?M 5-mT treatment, the IAA level had decreased to approximately 50 pg/tip from 150 pg/tip at time zero. Subsequently, after 60 min of 500 ?M 5-mT treatment, 5-mT was washed out, and Trp was replaced in the inside of the tip. The IAA level was immediately recovered by the Trp treatment, while in the mock control no restoration of IAA level occurred (Fig. 5B). This 5-mT-mediated inhibition of IAA biosynthesis suggested that IAA is synthesized via a Trp-dependent pathway in the coleoptile tip. Immunolocalization of IAA showed that the IAA signal in tissues became weak after 5-mT treatment (Fig. 5C). In particular, the signal in the outer epidermis almost disappeared.

Figure 5.

Effect of 5-mT on the amount and distribution of IAA in coleoptile tips. (A) Effect of 5-mT on IAA levels in the coleoptile tip. 5-mT at the indicated concentrations was locally applied to the inside of the top 2 mm region of coleoptiles. (B) Recovery of IAA levels by Trp treatment. 5-mT (500 ?M) was applied to the inside, and the coleoptile sections were incubated for 60 min. 5-mT was then washed out and Trp (500 ?M) (1) or phosphate buffer (KPB) (2) was applied. (C) Effect of 5-mT on the distribution of IAA by immunolocalization. IAA was visualized by immunolocalization of IAA in cross-sections of about 0.5 mm from the top after 5-mT (500 ?M) treatment and in the control. The IAA signal in the epidermis became weaker after 5-mT treatment (arrows). (Scale bar = 100 ?m)

These results indicate that IAA is synthesized in the 2.0 mm apical region of coleoptiles. IAA synthetic activity is higher in the more apical 1.0 mm region of coleoptiles, and the outer epidermis around the apical 0.5 mm region has very high IAA synthetic activity.

Immunogold localization of IAA in apical epidermis

To verify the intracellular localization of IAA in IAA synthetic cells, IAA was visualized with immunogold-electron microscopy in the upper-epidermis of the 0.5 mm region of the coleoptiles proceeded by high-pressure freezing/freeze-substitution (HPF/FS). We considered that an outflow of low molecular mass compounds, such as IAA, would be prevented by the HPF/FS method. In this experiment, a rabbit IAA-N-polyclonal antibody was used to visualize IAA to avoid disruption of the cell structure caused by EDAC, in which the antibody is designed to recognize IAA fixed with aldehyde type fixatives.²⁵ The specificity of the antibody was confirmed with an ELISA test, which showed that the antibody specifically recognized IAA in maize roots.²⁵ Furthermore, immunohistochemical analysis with the antibody showed the same IAA distribution pattern as the IAA-C-antibody used in Figure 1–5 (Fig. 6A). The results of immunogold labeling of IAA with the IAA-N-antibody in the outer epidermis of the coleoptile tip are shown in Figure 6B-I. In the epidermal cells of the coleoptile tip, colloidal signals of IAA were clearly observed in the cytoplasm, but not in the vacuole (Fig. 6C) or cell wall (Fig. 6F). The signals were densely congregated in plastids without starch grains (Figs. 6D, E). Similarly, IAA signals were recognized in mitochondria (Figs. 6G, H). Some reports have indicated that IAA is transported inside cells via intracellular vesicle transport.^22,25 In some cells, the gold particles were localized in endomembrane structures, such as the Golgi apparatus and vesicle clusters (Figs. 6H, I). These results are the first report of the subcellular localization of IAA in cells that accumulate high levels of IAA.

Figure 6.

Immunogold electron microscopy analysis of IAA. (A) Immunolocalization with the IAA-N-antibody in cross-sections of the coleoptile tip. (B-I) Immunogold analysis of IAA with the IAA-N-antibody in the outer epidermis of the coleoptile tip. (B) Cell structure of a typical epidermal cell. (Scale bar = 3 ?m) (C) Signals in the cytoplasm, but not in vacuoles. (D-E) Signals in plastids. (F) A signal could not be detected in cell wall regions. (G-H) Mitochondrial localization. (Scale bar = 3 ?m) (I) Golgi-apparatus localization was also detected. (Scale bar = 2 ?m) Abbreviations: vacuoles (V), plastids (Pt), cell wall (CW), mitochondrion (Mt), and Golgi-apparatus (G). Asterisk in (I) shows trans-Golgi network (TGN), endosome, or mitochondrion.

Discussion

Evaluation of the Visualization of IAA in Maize Coleoptiles by Immunohistochemistry

Immunohistochemistry of IAA is indispensable for the visualization of IAA in plant tissues or cells. However, there is a serious drawback with this method: there is no reliable evidence to show the specificity of an anti-IAA antibody against free active IAA molecules in plant tissues. In particular, it is possible that the antibodies cross-react with conjugated IAA, which exists in plants at much higher levels than free IAA. Based on the accumulating data of free and conjugated IAA levels, the first challenge was to evaluate the immunological visualization method for IAA using the corresponding tissues. In the present study, we used anti-IAA-C-antibodies to detect tissue IAA,²⁴ and compared the results with localized IAA levels as determined by GC-MS. To verify the specificity of the IAA-C-antibody against free IAA within maize coleoptiles, several controls were prepared to check the correlation between the intensity of the immunoreactive signals and the levels of free and conjugated IAA (total IAA minus free IAA) (Figs. One and 2). More than 10-fold more conjugated IAA is present compared with free IAA in the coleoptile 2–4 mm region. When free IAA was depleted by NPA treatment or by excision, free IAA levels were reduced to less than 25% of those in control tissue, without any significant change in conjugated IAA levels. A clear correlation was observed between the intensity of the immunological signals and free IAA levels. Reduced and accumulated free IAA levels, as determined by GC-MS, after NPA treatment also reflected the intensity of immunoreactive signals (Fig. 4). Furthermore, the considerable decrease in IAA level induced by 5-mT treatments also correlated with weak immunological signals (Fig. 5). In addition, plant tissues, particularly maize coleoptiles, contain very high amounts of Trp. However, immune-staining using the antibody did not show any staining under 100 ?M Trp treatment, although the antibody recognizes IAA at a concentration of only 5 ?M (Fig. 1). The IAA-N-antibody used in immunogold analysis was shown to specifically recognize free IAA.25 Furthermore, immunolocalization with the IAA-N-antibody showed the same IAA distribution pattern as the IAA-C-antibody. This indicates that both the IAA-N-antibody and IAA-C-antibody specifically recognize free IAA, but not conjugated IAA and Trp. Thus, the immunolocalization method used in present study could successfully visualize the distribution of free IAA in maize coleoptiles.

Outer Epidermal Cells in the Apical 0.5 mm Region are Possible Active Sites of IAA Biosynthesis

In our previous work, we measured IAA using GC-MS and showed that the apical 2 mm tip region is the main IAA synthetic site.⁶ The present study showed that although the majority of the total mass of IAA is synthesized in the 0–2.0 mm tip region, the active IAA synthetic cells (where immunological intensity of the IAA signal was strengthened by NPA treatment because of the high accumulation of IAA in the cells) might exist in the 0–1.0 mm region of the tip (Fig. 4). Furthermore, in the approximately 0.5 mm tip region, intense immunoreactive signals were detected in the outer epidermis after NPA treatment. This was further confirmed by treatment with the IAA biosynthesis inhibitor 5-mT. IAA signals observed in outer-epidermal cells of control tissues almost disappeared in 5-mT treated cells (Fig. 5). Recently, it was reported that IAA may be synthesized in epidermis of the inflorescence meristem and the shoot apical meristem in maize.¹⁵ In our observation, the outer epidermis appeared to be composed of relatively cytoplasm-rich cells (see Figures 3H, and especially Figures 3L, M). We hypothesize that, in the coleoptile outer-epidermis, the outermost layer of coleoptiles, many environmental stimuli could be received, leading to regulation of auxin synthetic activity and directional auxin transport to inner cells. In fact, some reports have shown the presence of a blue light receptor, PHOT1, in the epidermal cells of Arabidopsis stems.^26,27

Subcellular distribution of IAA

To date, there has been no evidence concerning the intracellular site(s) of IAA synthesis. Here, we demonstrated immunogold labeling of IAA in the outer epidermis of the coleoptile tip (Fig. 6). IAA signals were clearly localized in the cytoplasm, but not in the vacuole, and clusters of gold particles were observed in the Golgi apparatus in some cells. This indicates that IAA is synthesized in the cytoplasmic region and is probably moved by vesicular transport, as has been previously reported.²⁵ Immunogold labeling was also observed in plastids and mitochondria. Plants and bacteria are known to have Trp synthetic ability. If Trp is synthesized in plastids or mitochondria in the coleoptile tip, it is possible that IAA is synthesized from Trp inside these plastids or mitochondria. Recently, ZmTSA, the maize tryptophan synthase α subunit, was reported to localize in the chloroplast of mesophyll protoplasts.²⁸ Therefore, IAA localized in plastids might be synthesized from Trp in organelles in the outer epidermis of the coleoptile tip. Determination of the intracellular IAA synthetic site(s) and its movement inside the cells will be important issues for the future of auxin biology.

Materials and Methods

Plant Materials and Growth Conditions

Seeds of maize (Zea mays L. cv Golden Cross Bantam 70) were germinated at 25°C under red light for two days and then in darkness for one day, as described previously.6 For inhibitor treatment in Figures 2, 4 and 5 inhibitors were locally applied to the inside of the top 2 mm of coleoptiles, as described by Nishimura et al., 2009.⁷ The coleoptiles were abraded manually with a paste of fine aluminum oxide. The abraded coleoptiles were placed in 10 mM KPB (pH 6.7) with NPA or 5-mT, as previously described.^6,29 Endogenous free IAA and conjugated IAA (total IAA minus free IAA) were determined with GC-SIM-MS as described in previous reports.29 RT-PCR analysis was conducted as described by Nishimura et al., 2009.⁷

Immunolocalization of IAA

Excised coleoptiles were immediately fixed in freshly prepared 4% 1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide hydrochloride (EDAC) (Sigma, USA) in 0.1? PBS (pH 7.4) for 30 min at room temperature (RT). The coleoptiles were then washed in PBS, for 5 min, twice. Subsequently, the coleoptiles were post-fixed for 1–2 h in 4% paraformaldehyde in PBS at RT. After washing as above, the fixed tissues were sectioned as described previously (Nishimura et al., 2009). The sample sections were treated with detergent solution (10% DMSO and 3% Nonidet P-40 in PBS) for 30 min and washed three times in PBS for 10 min. The sections were then incubated overnight at 4°C with an anti-IAA-C-monoclonal antibody (Agdia) at a concentration of 0.1 mg/ml, or anti- IAA-N-polyclonal antibody²⁵ at a concentration of 0.01 mg/ml in PBS containing 0.05% Triton X-100. The sections were then rinsed three times for 10 min with PBS containing 0.1% Triton X-100. Samples were incubated with Alexa488-conjugated donkey anti-mouse IgG (Molecular Probes) at a 200-fold dilution, or alkaline phosphatase conjugated goat anti-mouse IgG (Invitrogen) at a 300-fold dilution, or alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma) at a 300-fold dilution for 3 h at RT. The prepared samples were observed with a light-microscope (model Olympus) or a laser scanning confocal microscope (model LSM5; Zeiss).

Immunogold Labeling of IAA Proceeded by HPF/FS

Coleoptiles were incubated in 0.1 M sucrose in 10 mM KPB (pH 6.7) for 1 h. The specimens were frozen in a high-pressure freezer (Leica EM-PACT) and the frozen specimens were fixed with anhydrous acetone containing 1% glutaraldehyde, as described previously.30 The fixed samples were embedded in LR-White resin and sectioned at 80 nm with an ultramicrotome. The ultrathin sections on nickel 200 mesh grids were blocked with 10% BSA in TBS for 10 min at RT. The sections were labeled with anti-IAA-N-polyclonal antibody²⁵ at a concentration of 0.06 mg/ml in TBS for 3 h. As a control, the sections were stained with non-immune rabbit serum. After washing with TBS containing 0.01% Triton X-100, the sections were indirectly labeled with 12 nm colloidal gold particles coupled to goat anti-rabbit IgG. Gold-labeled sections were then washed with TBS, rinsed in water, and stained with 4% aqueous uranyl acetate for 5 min. The grids were examined and photographed with a Gatan DualView camera and Gatan Digital Micrograph software.

Supplementary Material

Additional material

Supplementary PDF file supplied by authors.

Glossary

Abbreviations:

EDAC

1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide

GC-MS

Gas chromatography-mass spectrometry

GFP

Green fluorescence protein

GUS

?-glucuronidase

HPF/FS

High-pressure freezing/freeze-substitution

IAA

Indole-3-acetic acid

IAM

Indole-acetamide

IAN

Indole-3-acetonitrile

IAOx

Indole-3-acetaldoxime

IgG

Immunoglobulin G

KPB

Potassium phosphate buffer

LC-MS

Liquid chromatography-mass spectrometry

5-mT

5-methyl-tryptophan

NPA

1-N-naphthylphthalamic acid

PBS

Phosphate-buffered saline

RT

Room temperature

Trp

Tryptophan