INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Arabidopsis ecotype Columbia has been shown to contain 23 different glucosinolates (Hogge et al., 1988). Myrosinase (EC 3.2.3.1), also known as -thioglucoside glucohydrolase, catalyzes the hydrolysis of glucosinolates into Glc and an unstable intermediate that undergoes nonenzymatic rearrangement to form sulfate and isothiocyanates, thiocyanates, nitriles, epithioalkanes, or elementary sulfur dependent on the concentration of H⁺, metal ions, epithiospecifier protein, and/or other cofactors (Bones and Rossiter, 1996; Foo et al., 2000). The complexity of the myrosinase-glucosinolate system suggests a diverse and multifunctional role in the cruciferous plants. Glucosinolates are a diverse group of sulfur-containing glycosides that may serve as a sink for nitrogen and sulfur, and the hydrolysis products may have important roles in the defense of the plant against microorganisms and insects (Bones and Rossiter, 1996; Rask et al., 2000). Previous studies have shown that myrosinase is located in idioblasts named myrosin cells, which have been found in several species of Brassicacea, including oilseed rape (Brassica napus), white mustard (Sinapis alba), cauliflower (B. oleracea), and Chinese cabbage (B. campestris; Bones and Iversen, 1985; Thangstad et al., 1990, 1991; Bones et al., 1991; Höglund et al., 1991, 1992; Geshi et al., 1998). Myrosin cells occur as scattered cells in radicles, stems, leaves, petioles, seeds, and seedlings (Bones and Rossiter, 1996; Rask et al., 2000). The myrosinase enzyme has been localized to myrosin cells by immunocytochemical methods (Bones et al., 1991; Thangstad et al., 1990, 1991; Höglund et al., 1991; Geshi et al., 1998), and myrosinase transcripts have been detected by in situ hybridization (Lenmann et al., 1993; Falk et al., 1995).

The cellular localization of glucosinolates has been subjected to considerable debate, and the involvement of these compounds in growth, development, and defense could be more clearly defined following their cellular and subcellular localization (Kelly et al., 1998). Thangstad et al. (2001) used microautoradiography to study the in vivo localization of a titrated labeled desulfoglucosinolate precursor after pulse-chase feeding of oilseed rape plants. A cell-specific localization was found in radicles and cotyledons of the maturing embryo resembling the pattern of the myrosin cells known to contain myrosinase (Bones et al., 1991). Based on energy-dispersive x-rays and cell sap analysis, Koroleva et al. (2000) reported that specific cells next to the phloem bundles of the Arabidopsis flower stalk (S cells) were rich in glucosinolatessubstrates of myrosinase.

The existence of multiple forms of myrosinase genes have been shown in various Brassicacea species (Xue et al., 1992; Thangstad et al., 1993; Falk et al., 1995). In Arabidopsis, two myrosinase functional myrosinase-encoding genes, thioglucoside glucohydrolase 1 (TGG1) and TGG2, have been reported (Chadchawan et al., 1993; Xue et al., 1995).

In this study, we used -glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes, and immunocytochemical localization to examine the expression pattern of the TGG1 myrosinase in Arabidopsis. Our results show that the TGG1 promoter directs a high level of reporter gene expression in guard cells and phloem cells, and that both cell types are myrosin cells. It is interesting that myrosin phloem cells in flower stalk are located as the nearest neighbors to S cells reported to be rich in glucosinolates (Koroleva et al., 2000). This supports a cellular separation of the enzyme and the substrate in Arabidopsis flower stalk.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

To investigate the spatial expression pattern of the TGG1 myrosinase gene, transgenic plants carrying constructs containing 0.5 or 2.5 kb of the TGG1 gene promoter region fused to the GUS gene were made (constructs were named TGG1Pro:GUS and TGG1Pro:GUS, respectively). In addition, plants carrying a PBI construct containing the full TGG1 2.5-kb promoter fused to the GFP was made and named TGG1Pro:sGFPCt. The TGG1Pro:sGFPCt construct encodes a GFP:TGG1 fusion protein that contains amino acids from the N-terminal (38 amino acids) and C-terminal (58 amino acids) part of the TGG1 myrosinase. In addition, the construct contains 3'-untranslated region signals to allow correct cellular trafficking and three poly adenylation sites (Fig. 1). Promoter activity was detected as histochemical GUS staining in material pretreated in acetone for better GUS substrate penetration and as GFP fluorescence in living plants. Myrosinase-containing cells were immunocytochemical localized using the polyclonal K089 and monoclonal 3D7 myrosinase antibodies (Thangstad et al., 1990; Höglund et al., 1992).

View larger version (7K): [in this window] [in a new window]   Figure 1.   Schematic presentation of the TGG1 promoter GUS and GFP fusion gene constructs. TGG1 promoter sequence (0.5 or 2.5 kb). Black boxes, Partial TGG1 protein encoding sequences. Broken box, TGG1 poly(A) signal. Nt, N-terminal; Ct, C-terminal; PolyA, poly(A) signal. Scale bar = 0.5 kb.

Spatial Promoter Activity

Six independent transgenic lines carrying TGG1Pro:GUS construct and 10 independent transgenic lines plants carrying the TGG1Pro:GUS construct with the partial 0.5-kb TGG1 promoter were investigated. The plants containing the full promoter were studied for three subsequent generations. No GUS expression was observed in plants containing the partial promoter. The distribution of cells showing TGG1 promoter-directed GUS expression at various stages and tissues is presented in Figure 2. GUS-positive guard cells are present in the hypocotyl, cotyledon, and rosette, and positive phloem cells are present in the cotyledon and rosette leaves in seedlings (Fig. 2, A, B, D, and E). No GUS-expressing cells are detected in root tissue (Fig. 2C). An identical cellular expression pattern is observed in adult tissues examined. GUS-expressing guard cells and phloem cells are present in the stem, cauline leaf, pedicle, gynoecium, pollen sack, sepal, and silique, respectively (Fig. 2, F-K). The developing seeds are GUS negative, and GUS expression appears only in the phloem cells of the silique (Fig. 2, K and L). The lack of GUS expression in plants carrying the partial TGG1 promoter verifies that it is the TGG1 promoter that directs GUS expression in guard and phloem cells.

View larger version (77K): [in this window] [in a new window]   Figure 2.   Spatial distribution of cells showing TGG1 promoter-directed GUS expression in various stages and tissues of Arabidopsis. A through E, Seedling 5 to 12 d after germination. F through K, Plants 1 to 2 weeks after bolting. A, Five day seedling; B, hypocotyl; C, root tip; D, cotyledon; E, rosette leaf; F, inflorescence; G, cauline leaf; H, gynoecium; I, pollen sack; J, sepal; K, transverse section of silique with seeds; L, silique.

M Cells in Phloem

The recent report from Koroleva et al. (2000) that glucosinolate-rich S cells are located close to the phloem bundle in the upper flower stalk led us to focus on GUS-positive phloem cells in this region. As a first step, hand-cut transverse sections were made (approximately 0.5 mm thick) and the location of GUS- and GFP-expressing cells relative in phloem was examined. In the sampled region, the phloem bundles appear clearly visible and vary between five and eight in number (Fig. 3). At the site of the phloem bundle, GUS expression appears in phloem cells facing the endodermis, and the pattern in which they appear is discontinuous. No GUS expression is observed in the phloem sieve element/companion cell complexes. Figure 3A shows distinct GUS-expressing phloem cells in association with individual phloem bundles. An identical location of GFP-expressing cells in phloem is observed in plants containing the TGG1Pro:sGFPCt construct (Fig. 3B). The apparent difference in intensity of staining or level of fluorescence observed between the individual phloem cells is due to the use of intact plant tissue where some cells are out of the focal plane.

View larger version (63K): [in this window] [in a new window]   Figure 3.   Transverse section of the upper main flower stalk showing TGG1 promoter-directed GUS and GFP expression. A, Hand-cut section of a GUS plant. B, Hand-cut section of a GFP plant combined with the respective lightfield image. White arrows, Myrosin guard cells; black arrows, myrosin phloem cells. Ph, Phloem; X, xylem.

For a higher resolution view, nonstained and GUS-stained material from the upper flower stalk was embedded in LR white and semi-thin sections were made (2-3 µM), some being stained with toluidine blue for better identification of the different cell types (Fig. 4). The toluidine blue-stained section in Figure 4A shows the cell types at the site of the phloem bundle. On the inner side of the clearly visible plastid-rich endodermis layer, cells with a phenotype of the glucosinolate-rich S cells described by Koroleva et al. (2000) are located. These cells are large and irregularly shaped, and are only weakly stained with toluidine blue. No plastids are observed. Next to the potential S cells opposite to the endodermis layer are the phloem parenchyma cells and the phloem sieve element/companion cell complexes, respectively. The GUS-positive phloem cells are located at specific positions in the phloem bundle overlapping the phloem parenchyma cells (Fig. 4, B and C). Therefore, the GUS-positive phloem cells are believed to be a subpopulation of phloem parenchyma cells. Based on convention, positive guard cells were named myrosin guard cells (MGCs) and positive phloem cells were named myrosin phloem cells (MPCs). Most of the observed GUS-positive MPCs are larger in diameter and length than the phloem sieve element/phloem companion cell complexes (up to 90 µm in length and 15 µm wide), but are smaller than the observed potential S cells (up to 500 µm long and 20 µm wide).

View larger version (121K): [in this window] [in a new window]   Figure 4.   Semi-thin section of the Arabidopsis flower stalk. A, Transverse section of wild-type flower stalk stained with toluidine blue. B and C, Transverse sections from GUS-expressing plants. D and E, Longitual sections from GUS-expressing plants. C, Cortex; CC, phloem companion cell; En, endodermis; Ep, epidermis; Ph, phloem; S, sulfur-rich cell (Korolova et al., 2000); SE, phloem sieve element; X, xylem. Bars = 20 µm.

Immunocytochemical Localization of Myrosinase

To verify that MPCs showing TGG1 promoter activity are true myrosin cells (myrosinase-containing cells), material from the upper flower stalk was sectioned and immunocytochemical labeled using the anti-myrosinase antibodies K089 and 3D7. MPCs show immunolabeling with K089 and 3D7 antibodies (Fig. 5, A-C). MGCs only show immunolabeling with the K089 antibody (Fig. 5D). No stained cells were observed in sections incubated under identical conditions without the K089 antibody (result not shown). In addition to MPCs and MGCs, distinct cells of the cortex, endodermis, and xylem were also labeled with the K089 antibody.

View larger version (158K): [in this window] [in a new window]   Figure 5.   Immunocytochemical localization of myrosinase in transverse and longitual sections of Arabidopsis flower stalks using the anti-myrosinase antibodies K089 and 3D7. A, B, and D, Transverse sections. C, Longitudinal section. A and B, Sections immunolabeled with the 3D7 antibody. C and D, Sections immunolabeled with the K089 antibody. Arrows indicate myrosinase-containing MGCs and MPCs. Bars = 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Our results show that the TGG1 myrosinase promoter is specifically expressed in guard cells and phloem cells at all the investigated developmental stages of the Arabidopsis plant. Immunocytochemical localization of myrosinase in cells of the flower stalk shows that the TGG1 promoter activity reflects the localization of the myrosinase enzyme in MGCs and MPCs. From Figure 4D, it appears that the sieve element and companion cells next to the MPCs also show GUS expression. From the transverse section (Fig. 4C), it is clear that these cells lack GUS staining in cytoplasm. Taken together with the lack of immunoreactivity of the sieve element and companion cells toward the myrosinase antibodies used, these cells are not myrosin cells, but they appear stained because of leakage of the stain from the neighboring MPCs.

The finding that the K089 antibody also show immunoreactivity toward other cell types than the GUS-expressing MGCs and MPCs suggests that additional myrosinases are expressed in the Arabidopsis flower stalk. The presence of myrosinase in other cell types than those expressing TGG1 is likely considering that a total of six myrosinase or myrosinase-like genes (of which two likely are pseudogenes) are present in the Arabidopsis genome.

Koroleva et al. (2000) reported that S cells of the Arabidopsis flower stalk are rich in glucosinolates. These cells are situated between endodermis and the cells belonging to the bundles of vascular phloem. Each of these bundles contains a phloem zone with phloem parenchyma and sieve element/companion cell complexes. The S cells were reported to be up to 1,000 µm long and 30 µm wide (Koroleva et al., 2000). Next to the MPCs in the Arabidopsis flower stalk, we found cells with an S cell appearance, which appears slightly smaller than described by Koroleva et al. (2000). We found these cells to be up to 20 µm wide and 500 µm long. Taken together, our observations indicate a location of myrosinase enzymes and glucosinolates in different but neighboring cells in the flower stalk of Arabidopsis.

Further support for glucosinolate-containing cells closely associated with phloem comes from the spatial expression pattern observed for the CYP79F1 and CYP79F2 genes involved in glucosinolate synthesis (Reintanz et al., 2001). These genes are exclusively expressed in or close to vascular phloem of the Arabidopsis, including the flower stalk. It is unfortunate that the identity of these cells was not described.

The role of glucosinolates and their hydrolysis products in plant defense is widely accepted (Bones and Rossiter, 1996; Rask et al., 2000). Fungistatic and bacteriostatic effects of glucosinolates have been reported, and the deterrence of nonspecialist pests has been well documented (Chew, 1998; Wallsgrove and Bennett, 1995; Tierens et al., 2001). Many specialized pests have overcome the toxicity of the myrosinase-glucosinolate system and even use glucosinolates and isothiocyanates to locate and identify their glucosinolate-containing host plants. The aphid specialist Brevicoryne brassicae can be induced to feed on nonhost plants when leaves are treated with 2-propenyl glucosinolate (Nault and Styler, 1972), and its intrinsic growth rate depends on the concentration of glucosinolates (Cole, 1997). The B. brassicae even expresses large amounts of its own endogenous myrosinase, which is believed to play a role in the insects defense and signaling against predators (Jones et al., 2001, 2002; Bridges et al., 2002).

The positioning of myrosinase-containing cells together with glucosinolate-containing S cells outside the phloem sieve elements is ideally suited for the protection of the phloem and its content against microbes and insects. During insect attachment, myrosinase and glucosinolate mix, resulting in the release of toxic hydrolysis products. The phloem parenchyma cells appear to be specialized in delivery of photosynthetic assimilate products such as Suc from the bundle sheath into the sieve tract (Haritatos et al., 2000). Similar mechanisms may enable efficient loading of products such as glucosinolates or hydrolyzed products into the phloem. The reactive, volatile, and/or cytotoxic effect of some of these products may serve different functions such as signaling and defense. Thangstad et al. (2001) showed that the glucosinolate precursor (desulphoglucosinolate) accumulates in specific cells in the embryo radicle and cotyledon, in a pattern that matches the distribution of myrosin cells in the embryo. This study did not address whether myrosinase enzyme and glucosinolate substrate were colocalized in the same cells or if they were located in different but adjacent cells, but it did provide evidence for a specific transport system for glucosinolates or desulfoglucosinolates.

There is now emerging evidence that the myro-sinase-glucosinolate system also is involved in processes other than plant defense. Knockout of the CYP79F1 gene results in a mutant where synthesis of short chain aliphatic glucosinolates is abolished, resulting in a bushy phenotype (Reintanz et al., 2001), indicating a role for glucosinolates in control of plant growth and development.

Our results show that the TGG1 myrosinase promoter is active in most organs of Arabidopsis except in the root and seeds. Promotor activity is especially high in young leaves, upper flower stalk, pedicle, and the transition zone between the pedicle and silique. In older tissue, TGG1 promoter activity is considerable lower. High myrosinase activity in young developing tissue may have dual functions. Myrosinase activity generates toxic components that may be beneficial to express in cells at the point of entry of microorganisms. The location of myrosinase in phloem and guard cells may indicate a role in communication, internally by hydrolysis of glucosinolates and transport in phloem, and externally by distribution of volatile degradation products from guard cells. The fact that myrosinase activity is especially high in developing tissue may also suggest that myrosinase is involved in the control of plant growth and development.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Constructs

Isolation of the TGG1 Promoter DNA Sequence

TGG1 promoter:GUS Fusion Construct, TGG1Pro:GUS

TGG1 Promoter:GFP Fusion Gene Construct, TGG1Pro:sGFPCt

Cloning of GFP:TGG1 fusion gene

PCR amplification and sequencing

Plants

Plant Transformation

Plant Growth and Sampling

Histochemical GUS Detection

The histochemical detection of GUS expression was performed essentially as described by Jefferson et al. (1987) with one important adjustment. Prior to incubation in substrate solution, plant material was incubated for 5 min in acetone at 20°C, air dried, and incubated for an additional 30 min in 0.1 M phosphate buffer, pH 7.0. GUS staining was performed using 0.1 to 0.2 mM 5-bromo-4-chloro-3-indolyl--glucuronic-acid and sodium salt (DUCHEFA, Haarlem, The Netherlands) at 37°C for 3 to 5 h (with the 0.5- to 1.5-mm sections) or 24 h (with the 1.0- to 1.5-cm sections), with an initial 3-min vacuum infiltration. After staining, the samples were examined and photographed using a stereo microscope (SMZ1500; Nikon, Tokyo) equipped with a digital camera (Coolpix 990; Nikon) or a research microscope (Eclipse 800; Nikon) equipped with a cooled digital camera (SPOT RT; Diagnostic Instruments, Burroughs, MI).

GUS-stained flower stalk material was embedded in LR white as described by Peleman et al. (1989). Sections (2-3 µm) were made on a ultramicrotome (type 4801A; LKB, Uppsala) and were placed on a film of water. The sections were transferred to a glass slide on a drop of water, dried, and mounted using DPX mountant (British Drug House; Laboratory Supplies, Poole, UK). Differential interference contrast images were obtained using a research microscope (Eclipse 800; Nikon).

GFP Fluorescence Detection

GFP fluorescence in the Arabidopsis flower stalk was monitored in intact tissues or in hand-cut transverse sections (approximately 0.5 mm). Care was taken not to allow the transverse sections to dry due to development of autofluorescence in dried samples. GFP fluorescence was visualized in live plant tissue using a GFP(R)-BP (EX 460-500 nm/DM 505 nm/BA 510-560 nm) filter.

Immunocytochemistry

Flower stalks were sampled 1 week after start of bolting, were cut into 0.5- to 2.0-mm pieces with a razor blade, and were fixed overnight in 4% (w/v) paraformaldehyde in 50 mM phosphate buffer (pH 7.2) at 4°C. After washing in 50 mM phosphate buffer, the pieces were dehydrated through graded series of ethanol, embedded in paraffin wax, and sectioned (6 µM) as described by Thangstad et al. (1990). The sections were transferred to poly-L-Lys-coated (0.1%, w/v) slides and were incubated overnight at 37°C and at 55°C for 15 min before wax removal. The wax was removed by a 10-min incubation in xylene followed by two washes in absolute ethanol. The sections were rehydrated through graded series of ethanol followed by a 10-min incubation in phosphate-buffered saline (PBS) prior to immunocytochemical labeling. The marker used for localization was a rabbit polyclonal antibody K089 (Thangstad et al., 1991) or a mouse monoclonal antibody 3D7 (Lenmann, 1990). To prevent nonspecific binding, sections were incubated in 10% (w/v) normal serum and 5% (w/v) bovine serum albumin (BSA) in PBS-0.5% (v/v) Tween 20 (PBS-T) for 15 min. Immunohistochemistry was performed at 37°C including 5% (w/v) BSA in the PBS-T/antibody solutions. The sections were incubated for 45 min with primary myrosinase antibody K089 diluted 1/10,000 or 3D7 diluted 1/500, 30 min with biotinylated secondary antibody anti-rabbit Ig diluted 1/500 or anti-mouse Ig diluted 1/300, and 30 min in an Avidin:Biotynilated enzyme complex solution (VECTASTAIN ABC kit, Vector Laboratories, Inc., Burlingame, CA). Finally, immunoreactive cells were visualized by a 10-min incubation in DAB-solution (3,3'-diaminobenzidine, Vector Laboratories, SK-4100) at room temperature. Cover slips were mounted using VectaMount (Vector Laboratories). Differential interference contrast photography was performed as described above.