Myrosin Cells and Myrosinase Expression Pattern in Nasturtium (Tropaeolum majus L.)

Abstract

Plants from the Brassicales order are known for the presence of a glucosinolate–myrosinase link, which is an important protection strategy against multiple stressors. The main goal of this study was to investigate the presence of the myrosinase enzyme and reveal the myrosin cell ultrastructure in the vegetative organs of nasturtium. The presence, localisation and expression of the enzyme myrosinase type 1 (TGG1) at different developmental stages of Tropaeolum majus L. (nasturtium) were investigated using immunohistochemical and immunofluorescent techniques. The expression of myrosinase was detected in the vegetative organs of T. majus. During plant development, within four consecutive weeks, a decrease in myrosinase expression was noticed in all studied plant organs. The location of greater myrosinase accumulation and activity is shown to be the root, contrary to the nasturtium stem and leaf, where we found the lowest myrosinase expression. Transmission electron microscopy was used to reveal the ultrastructural features of the myrosin cells of nasturtium. Myrosin cells are usually scattered between parenchyma cells and S-cells. Mostly, they are rectangular or slightly elongated in shape and can be recognised by an electron-dense large central vacuole and an expanded rough endoplasmic reticulum. The results of this study provide new data on myrosin cell morphology and the expression pattern of myrosinase in T. majus.

1. Introduction

The nasturtium plant (Tropaeolum majus L.) belongs to the Tropaeolaceae family and the Brassicales order and has many benefits for humans since it contains a variety of macro- and microelements and biologically active compounds, such as polyphenols [1], anthocyanins, natural pigments [2,3] and ascorbic acid [2,4,5]. Its beneficial effects are used in alternative medicine to cure various human diseases [2,4].

Although T. majus L. has been the subject of several biological studies, the data on the myrosin expression and ultrastructure of its myrosin cells are still scarce or missing. The enzyme myrosinase (thioglucoside glucohydrolase, TGG) catalyses the hydrolysis of a group of low-molecular-weight compounds known as glucosinolates (GSLs) [6,7]. GSLs are hydrophilic compounds that are biologically inactive if they are separated from other components in plant cells. Myrosinase, a member of the family of β-glucosidases, is an enzyme that catalyses the cleavage of the S-glycosidic bond found in all GSLs [8,9]. In plants, this myrosinase–GSL bond is a chemical protection system against herbivores and pathogens [8,10]. Myrosinase from myrosin cells catalyses the hydrolysis of GSLs to make toxic components, such as isothiocyanates or thiocyanates, against herbivores. This defense strategy is referred to as the ‘mustard oil bomb’ theory [11]. The subsistence of the GSL–myrosinase bond is found in the Brassicales order and officiates as a defense system against biotic and abiotic stress factors. Myrosinase is described in all plants containing GSLs, especially in the Brassicaceae family [8], but also in some mammal tissues [12], fungi [13,14,15], insects [16,17] and microorganisms [8,13]. Myrosinase is localised in idioblasts called myrosin cells [7], which are mostly found peripherally in phloem tissue [6,8,18]. Myrosin cells are abnormal in shape, size and content, thus differing from neighbouring cells [19]. In various tissues and organs, even at various stages of development, myrosin cells show different morphology [20]. These cells appear scattered among the cells of roots, stems, leaves, petioles, seeds and seedlings [21]. Myrosinases and their substrates, glucosinolates, are spatially stored in myrosin cells and sulphur-rich cells (S-cells), respectively [6]. S-cells are usually grouped and placed between endodermis and phloem cells [22]. They are in direct contact with myrosin cells or can be found very close to them [6,17]. GSLs are found in the vacuoles of S-cells, but after tissue disruption, they act together with myrosinase producing a variety of bioactive products [8]. The presence of myrosin cells has been shown in several plant tissues, including the cotyledons and axis of embryos [17], cortex cells of radicles and hypocotyls, parenchymatic cells of cotyledons and leaves [18,23,24,25,26,27], epidermis and vascular cambium [28] and guard cells [6,17,20,29], as well as leaves, stem, root and petals [6,23].

A large amount of myrosinase accumulates inside the vacuoles of myrosin cells [19]. Myrosin cells are replenished with spherical myrosin granules that contain a homogeneous electron-dense material [6]. Myrosin grains can differ inside the same cell and between different species [25]. Their grain size varies from 2.5 to 10 nm. While myrosin cells differentiate, myrosin granules fuse to form a trabecular network in the cell to communicate with each other [6,21]. Myrosinase has so far been mostly investigated in three plants from the Brassicaceae family: oilseed rape (Brassica napus L.), white mustard (Sinapis alba L.) and arabidopsis (Arabidopsis thaliana L.) [11]. In the plant Arabidopsis thaliana, there are two types of myrosinases stored in myrosin cells, namely, TGG1 and TGG2 [19]. Most of the recent research carried out on the localisation of the myrosinase enzyme has focused on seeds and embryos [25], and there are few results on the areas of roots, stems and leaves [30,31,32,33,34].

Therefore, our research goal was to study the expression pattern of myrosinase in vegetative plant organs and reveal the ultrastructural features of nasturtium myrosin cells in comparison to those of other plants containing myrosinase. These results could contribute not only to the basic biological knowledge about this promising and still insufficiently researched plant but also improve knowledge about localisation and myrosinase expression in nasturtium. The obtained data could be used for further research on this species and its biological effects.

2. Materials and Methods

2.1. Planting and Growing of Nasturtium

Nasturtium (T. majus L.) used in this research was grown from seeds in a thermoregulation room under conditions of 12 h day/12 h night at a temperature of 24 °C. The seeds of the T. majus plant were planted in three large separate pots under the specified controlled conditions because their planting time is in the spring (planted in the second half of April). T. majus seeds are large and resemble peas. They were planted 2 cm deep in loose, poor soil and required regular watering. In addition to being decorative, the T. majus plant is also edible. There were at least 30 young plants (n ≥ 30) in each pot, and at least three plants (n ≥ 3) were taken from each pot as an independent measurement of each plant’s developmental stage. Tissues were sampled for four consecutive weeks at regular intervals to obtain tissue pieces at different developmental stages after growth. All three vegetative parts of the plant—the root, the stem and the leaf—were sampled. Nasturtium tissues were cut into small pieces with the dimensions of 0.5 cm × 0.5 cm.

2.2. Immunofluorescence and Immunohistochemistry Procedures

The specimens were fixed in 4% paraformaldehyde in phosphate buffer with a pH of 6.8, dehydrated in an ascending series of ethanol, cleared in xylene, and then embedded in paraffin wax on HistoCore Arcadia H (Leica Biosystems, Wetzlar, Germany). The prepared paraffin blocks were left in the refrigerator overnight. Paraffin sections 4–6 μm thick and cut on a rotary microtome HistoCore BIOCUT (Leica Biosystems, Wetzlar, Germany) were mounted on glass slides. After deparaffinisation in xylene (2x) and rehydration in descending concentrations of ethanol and water, the sections were heated in a citrate buffer (pH 6.0) for 10 min. After the sections were cooled at room temperature, they were washed in PBS (pH = 7.2) 4x. To exclude unspecific staining, a blocking buffer was applied for 30 min [35,36,37].

2.2.1. Immunofluorescence Staining

According to Racetin et al. [37], the sections were incubated overnight at room temperature in the dark in a humidity chamber with TGG1 myrosinase rabbit primary antibody (BGL38; Agrisera Part of Olink Group, Vannas, Sweden) diluted 1:1000 in PBS. After washing in PBS (pH = 7.2), Alexa Fluor^® 488-conjugated AffiniPure Donkey Anti-Rabbit IgG (H + L) (711-545-152, Jackson Immuno Research Laboratories, Inc., Baltimore, PA, USA) diluted 1:400 in PBS (pH = 7.2) was applied for 1 h and washed in PBS (pH = 7.2) 2x. The nuclei were stained with DAPI (4,6-diamidino-2-fenilindol). After final rinsing in PBS (pH = 7.2) 2x, the sections were mounted (Aqua/Poly Mount) and coverslipped, as described in detail by Kević et al. [35,36,37]. All slides were studied using an Olympus BX51 (Tokyo, Japan) epifluorescence microscope with a Nikon DS-Ri1 digital camera (Nikon Corporation, Tokyo, Japan).

2.2.2. Immunohistochemical Staining

For chromogenic immunohistochemistry, the primary antibody TGG1 myrosinase rabbit primary antibody (BGL38; Agrisera Part of Olink Group, Vannas, Sweden; 1:1000) was applied on tissue sections overnight in the dark. After washing in PBS (pH = 7.2) 3 × 2 min, the sections were incubated with biotinylated secondary antibody (Dako LSAB^®2 System–HRP, Dako North America, Inc., California, USA) in the dark at room temperature. The biotinylated LINK solution (biotinylated anti-rabbit and anti-mouse) was applied for 10 min. After washing in PBS (pH = 7.2), streptavidin–HRP was applied for 15 min. Subsequently, the sections were washed again in PBS (pH = 7.2), then stained with a diaminobenzidine tetrahydrochloride solution using a DAB substrate system (ZY0643, Agilent Dako, Santa Clara, CA, USA) for 5–10 min and counter-stained with haematoxylin for 1 min. Dehydration was performed in an ascending series of ethanol and then placed in xylene. After the final washing in PBS, the sections were mounted with NeoMount [38]. An Olympus Leica DM3000 LED (Leica, Wetzlar, Germany) light microscope with a Leica DMC4500 camera (Leica, Wetzlar, Germany) was used to observe and capture the slides.

In both staining procedures, negative controls with the omission of the primary antibody were included to exclude nonspecific staining. No significant staining was observed if the secondary antibody was applied alone.

2.3. Statistical Analysis

Image J software 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used for the quantitative analysis of myrosinase 1 expression. To quantify the immunoexpression of the myrosinase enzyme, ten non-overlapping representative visual fields of identical exposure time captured at an objective magnification of 20× were examined [39]. Image J software was used to detect the percentage of myrosinase 1 immunoreactive cell areas. For immunoreactivity analysis, photomicrographs were subtracted by the median filter and then prepared using colour thresholding to measure the section percentage area enfolded by a positive signal [39].

A one-way ANOVA test was used for statistical data processing, after which Tukey’s multiple comparisons test was performed to examine the difference in the root height, stem height, leaf length and total plant height in the four developmental stages. The results are expressed as mean (n = 5) ± standard deviation (SD). Significance was set at p < 0.05.

A two-way ANOVA test was used for statistical data processing, after which Tukey’s multiple comparison test was performed to examine the difference in the immunoexpression of the myrosinase type 1 enzyme between the root, stem and leaf areas in the four developmental stages. The results are presented as mean ± standard deviation (SD). Statistical significance was considered at p < 0.05. Analysis of the results was undertaken in GraphPad Prism 8.0.1. (GraphPad Software, Inc., San Diego, CA, USA).

2.4. Transmission Electron Microscopy (TEM) Procedure

The tissue samples of all three vegetative parts of the plant—the root, the stem and the leaf—from the first investigated period were fixed for 24 h at 4 °C in 2.5% glutaraldehyde and 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.0). The samples were washed out in a phosphate buffer and post-fixed in 1% osmium tetroxide in 0.1 M phosphate-buffered solution for 1 h. After washing in double-distilled water, the samples were dehydrated in an ascending series of acetone (30–100%) [35,40,41]. Tissue embedding was undertaken in Spurr’s epoxy resin [42]. Resin polymerisation was carried out at 65 °C for 48 h. To find the area of interest, the semi-thin sections were cut at 0.5 μm on the ultra-microtome (PowerTome XL, RMC Boeckeler, Boeckeler Instruments, Inc., Tucson, AZ, USA) and stained with 1% toluidine blue dissolved in 1% borax. Ultrathin sections were cut at 0.06 μm and counterstained with 2% uranyl acetate (5–15 min) and lead citrate (5–10 min). The ultrathin sections were observed under a transmission electron microscope (TEM) (JEOL JEM 1400 Flash) (Tokyo, Japan).

3. Results

3.1. Statistical Analysis of the Plant Growth Measurement

Each week, five healthy plants with preserved roots, stems and leaves were sampled. In the first week, the plants (Figure 1a) reached an average length of 25.7 ± 3.12 cm, with the stem being the most dominant part of the plants (Figure 2). Simultaneously, the roots were weak and underdeveloped, and the diameter of the leaves was no more than 1.8 ± 0.38 cm (Figure 2).

In the second week of sampling, significant changes were observed in the development of the roots. During this period, the roots grew by an average of 5.56 ± 1.42 cm to 9.34 ± 2.43 cm. The leaves were still small (Figure 1b), with the largest leaf reaching a diameter of 2.42 ± 0.6 cm (Figure 2).

During the last two weeks, the total plant length was significantly longer than at the beginning of the measurement (Figure 1c–e and Figure 2). The first two weeks’ total plant lengths were 25.7 ± 3.12 cm and 24.74 ± 4.94 cm, respectively, and the last two weeks’ plant lengths were 33.24 ± 2.19 cm and 36.58 ± 5.54 cm. Oscillations in the leaf morphology were observed in the third week. The larger (older) leaves took on a jagged shape, while some of the smaller (younger) leaves were round (Figure 1d). In the fourth week, it was observed that part of the stems turned brown due to senescence, while the majority remained green (Figure 1e).

3.2. Myrosinase Type 1 Expression Pattern

The expression pattern of the antibody myrosinase type 1 appeared in the parenchyma tissue and the vascular tissue of all examined plant parts (Figure 3).

In the root, the strongest myrosinase expression was observed in Phases 1 and 2 of plant development after germination. The highest percentage of the myrosinase immunoreactive cell area in the root was recorded in the meristematic zone of a young root (Figure 3a and Figure 4). Localisation of myrosinase in the xylem of the root parenchyma cells (Figure 3c) and the phloem (Figure 3d) showed high fluorescent signals. In the stem, large xylem vessels, which could be recognised in Phases 2 and 3 by empty lumens and tick cell walls (Figure 3e–g), showed a distinct expression of myrosinase. A similar intensity of fluorescent signal was observed in the phloem cells (Figure 3h). Positive myrosin phloem cells are characterised by a large diameter and length (Figure 3h). In the stem, the strongest expression of myrosinase type 1 enzyme was observed in Phase 2 and Phase 3, with significantly higher expression in the second phase (Figure 4).

No significant myrosinase expression was observed during all four developmental stages of the leaf (Figure 3i–l and Figure 4). Moreover, an extremely low-intensity myrosinase signal was observed in young cells of the parenchyma tissue in the vascular part of the leaf (Figure 3i–l), which we confirmed by statistical analysis of the percentage of myrosinase immunoreactive cell areas (Figure 4). However, it is noticeable that the expression decreases as the plant develops. Thus, in the fourth phase, the expression of myrosinase decreased significantly.

Statistical Analysis of Myrosinase Type I Expression

The percentage area of myrosinase expression in the investigated organs of nasturtium showed significantly different results. In the root, the percentage area of myrosinase expression was significantly greater compared to the other organs examined (Figure 4, p < 0.05; p < 0.0001). The highest expression of myrosinase in the root was observed in the developmental stages 1 and 2. As the developmental period progressed, the percentage of myrosinase-expressed surface area and the amount of myrosinase decreased. However, myrosinase was detected in the root at all four examined stages.

In the stem, the greatest extent of myrosinase expression was observed in the second and third phases in the period from 25 to 35 days after sowing, with the second phase clearly dominating. In the seven-day period between the first and second test phases, a sudden increase in expression can be observed. However, only Phase 2 showed a remarkable and significant expression of myrosinase. When comparing the expression in the stem with the root, which showed the most intense expression in the first phase, the strongest signal in the stem was observed seven days later in the second phase. The expressed percentage area in the stem was significantly higher in Phase 2 compared to all other phases but also significantly lower in comparison to the root (Figure 4, p < 0.05). Comparing the expression level of the stem in Phase 2 (the highest expression in the stem) and Phase 4 of the root (the weakest intensity of expression in the root), almost the same percentage of myrosinase expression area can be observed. Stem Phase 1 and Phase 4 showed almost the same but with surprisingly low percentages of expressed areas.

A very weak fluorescent signal of the enzyme myrosinase type 1 was observed in the leaf at all four developmental stages (Figure 3i–l). There was no statistically significant difference in the recorded percentage of myrosinase immunoreactive cell areas in the leaf region of nasturtium. Phase 3 showed the lowest percentage of expression area. Compared to the highest percentage area of myrosinase expression in the root, the statistical analysis confirmed a significant statistical difference (Figure 4, p < 0.0001). The highest expression of myrosinase was found in the idioblasts of the ground tissue in Phase 1 and Phase 2 (Figure 3 and Figure 4). In addition, a significant decrease in myrosinase expression was observed in the stem area, in both the root and leaf as the plants grew and aged, with the lowest expression occurring in the final fourth phase.

3.3. Immunohistochemical Staining of Myrosinase Type 1

Chromogenic immunohistochemistry (IHC) applied to nasturtium tissue confirmed the results obtained by immunofluorescence (Figure 3). Myrosinase type 1 was found in all nasturtium tissue samples of the root, the stem and the leaf.

Myrosinase type 1 was expressed in the nasturtium root during all developmental phases. A weak signal indicated by less brown colour was found in the xylem parenchyma cells in the evolving Phase 1 (Figure 5a). In Phase 2, positive labelling was observed in the parenchyma tissue, the epidermis and the root cortex (Figure 5b). The myrosin cells were scattered among the cells of the root parenchyma (Figure 5b,c). The myrosin cells were mostly rectangular in shape and smaller or equal to the size of the neighbouring cells. In Phase 4, a strong expression of myrosinase type 1 was observed in the xylem and phloem parenchyma cells (Figure 5c). Immunohistochemical staining in the longitudinal section through the stem also confirmed expression along the entire xylem and phloem vessels (Figure 5d). The idioblasts of the phloem parenchyma showed positive staining for myrosinase. The myrosinase expression in the branching veins is composed of xylem and phloem cells embedded in parenchyma tissue in the leaf area, as can be seen in Figure 5e,f. Adjacent to the phloem and xylem, elongated myrosin is placed (Figure 5e,f). In addition, scattered myrosin cells potentially filled with myrosin grains can be found amongst the parenchyma cells of the xylem (Figure 5e).

3.4. Ultrastructure of Myrosin Cells in Nasturtium

Myrosin cells observed in the root, stem and leaf of nasturtium during Phase 1 have a similar morphology (Figure 6). These cells in all three vegetative parts of the plant are very similar in shape, being rectangular or slightly elongated when sectioned. The myrosin cells are scattered between the parenchyma cells and the S-cells and are smaller in comparison to these cells. Two large cells, close to the myrosin cells, are vessel elements (Figure 6a). The measured size of the myrosin cells is between 6 and 10 μm. Myrosinase cells are characterised by a large central vacuole and several small vacuoles at the periphery, as well as cytoplasm with a rich, rough endoplasmic reticulum. The vacuoles contain medium-dense granular content. Figure 6b shows myrosin grains in the small vacuole of the myrosin cell in the root. Homogeneous, electron-dense myrosin grains can also be seen in the cytoplasm of the myrosinase cells of the stem (Figure 6c,d). Near the cell wall of the myrosin cells, an extensive, rough ER with polysomes, Golgi apparatus as well as mitochondria, free ribosomes and numerous organelle-like, expanded cisternae can be seen (Figure 6b). S-cells are in close contact with the myrosin cells (Figure 6a,c,e) and are grouped around the myrosin cells. These cells are much larger than the myrosin cells, with a central vacuole and a thin cytoplasmic layer. In some S-cells, the cytoplasm is not visible at all due to the size of the vacuole. The transport between the myrosin and the S-cells is mediated through plasmodesmata, which can be seen along the cell wall between these two cells in the nasturtium leaf (Figure 6e). In the root, communication between two neighbouring S-cells is realised by an open pit chamber in which an opening pit membrane is clearly visible in the middle of the cell wall (Figure 6a). The sieve tube elements are rectangular and smaller in contrast to the much larger neighbouring phloem parenchyma cells, myrosin cells and S-cells. A companion cell with a dense cytoplasm, a vacuole and large nuclei can be seen next to the sieve tube element. The sieve tube element and companion cell communicate through plasmodesmata (Figure 6f). A characteristically branched plasmodesmata complex can be seen in the companion cell on the side of the shared wall (Figure 6f). In the phloem parenchyma cell, a chloroplast is located near the cell wall (Figure 6f).

4. Discussion

T. majus L. (nasturtium) has been the subject of several studies due to its bioactive compounds with antibiotic, antifungal, antiscorbutic, laxative and diuretic effects [2,3,4,5]. Like other species of the Brassicales order, nasturtium contains a GSL–myrosinase system whose bioactive products are important in the defence against biotic and abiotic stress factors. Myrosinase has been studied mainly in species of the Brassicaceae family, although it is also found in many other plant species. According to the available data, myrosinase expression and its localisation in plants of the Tropeolaceae family have not been studied at all, and the present study is the first report on the presence and expression of myrosinase in T. majus.

Tissue damage of any kind activates the enzyme myrosinase, causing the hydrolysis of GSLs. In contrast to many other plants that contain GSLs, only two types of GSL are found in nasturtium, namely, benzyl glucosinolate or glucotropeoline and sinalbin, both aromatic GSLs [43,44]. The hydrolysis that occurs through the substrate–enzyme defence mechanism and the chemical substances that are released are responsible for many benefits of this insufficiently researched plant. The localisation of myrosinase within the cell, particularly the localisation of GSLs, has been discussed for years. Husebye et al. presented a neighbouring position of myrosinase-containing cells and S-cells in the Arabidopsis flower stalk [17]. Our results are consistent with this report, as we found that myrosinase cells in nasturtium are in close contact with S-cells. The presence of myrosinase in the vascular tissue of A. thaliana L. and the different content of myrosinase in the seeds of A. thaliana L. and B. napus L. led us to speculate that myrosinase–GSL binding has a different function in different plant species [45,46]. In some vegetative organs and developing embryos of Arabidopsis, myrosin cells are found among the phloem parenchyma cells, but they were not observed in the parenchyma tissue [6]. Our results show myrosin cells in the nasturtium root, stem and leaves as independent, scattered cells among the surrounding cells. Ultrastructural analysis describes the localisation of myrosin cells between parenchyma tissue cells and S-cells. The close contact between the myrosin cells and the S-cells enables the plant’s fast defence response. Similar results have also been reported for Arabidopsis [6]. Since S-cells are found in all vascular parts of the plant, it is assumed that vascular tissue has an important role as the site of the GSL defence system [47]. The importance of the bond is shown by the fact that many authors consider it to be taxonomically significant. Therefore, one of the criteria for the classification of Brassicales plants is the presence and arrangement of myrosin cells [48].

It was shown that myrosin cells are developed independently of vasculature cells and differ in shape [49], which morphologically distinguishes them from the neighbouring ground meristem cells [50,51]. The shape of myrosin cells found in the mesophyll cotyledon tissue varies from isodiametric to elongated, and they are larger than neighbouring cells [25]. In addition, in hypocotyledons, myrosin cells are rectangular or cube-like, while in roots, they are elongated [21,25]. The myrosin cells reported in S. alba and R. sativus are morphologically very similar and round and smaller than cells from the surrounding tissue [25,52]. In the nasturtium leaf, the size of myrosin cells is mostly the same as the size of the surrounding cells in contrast to those in the root and the stem, which are much smaller than the neighbouring S-cells. Myrosin cells in nasturtium are single cells situated between other tissue cells, very similar to the localisation of the myrosin cells in the B. napus [23,25,26,29]. Morphologically, myrosin cells are easy to recognise by two characteristic features: the presence of the vacuoles and cytoplasm with distended rER. Our ultrastructural findings on myrosin cell morphology are like those previously reported on Arabidopsis [6,53], B. napus [6] and those reported on a few families in the Brassicales order [54]. In addition, our results correspond to previously reported data on vacuoles in myrosin cells [19,54].

Previous studies on myrosinase activity in plants from the order Brassicales have shown considerable differences between different species of the same family, as well as between different cultivars [55]. Except in the roots, the content of myrosinase decreases in adult plants, and myrosinase activity becomes lower [10]. Nevertheless, a drastic reduction in the content of GSL during the early development stage was reported, which is in accordance with reduced myrosinase expression [45]. This regulatory mechanism is caused by myrosinase hydrolysing GSLs for plant self-protection [45]. Our results confirm reduced expression of myrosinase during plant development. In previous studies, myrosinase activity was reported mostly in the root area. In B. napus, the myrosin cells were mostly found in the root cortex [23]. Also, as shown by Bones (1990), the root in B. napus has much more intense myrosinase activity in comparison to the stem and leaf. The root of nasturtium shows the strongest myrosinase expression according to our statistics, but in the leaf, we gained the weakest expression. Moreover, in the leaf, the presence of myrosinase was very low during all developmental stages.

Similar results were obtained in the radish (Raphanus sativus), where the root peel was described as the major myrosinase-containing organ and the leaf as the area of the lowest myrosinase activity [28]. Decreased myrosinase expression is also shown during germination in the cotyledons and radicles of seedlings of four species of the Brassicaceae family, B. napus L., S. alba L., R. sativus L. and B. oleracea L. [25]. Furthermore, we showed the solid myrosinase expression pattern in the early developmental phase in parenchyma cells, xylem parenchyma and phloem vasculature. In contrast to A. thaliana, the plant B. napus L showed the same result that myrosinase is found exclusively in the myrosin cells of the phloem ground tissue and protective cells of the vegetative tissue, while no cells were found in the parenchyma [56]. Nevertheless, parenchyma cells and vascular elements of all investigated parts of T. majus L. have shown a certain level of myrosinase activity. In addition, it seems that high myrosinase activity is supported by strong gene expression [28]. Our results correspond to the ones previously described, with the nasturtium root as the main tissue of myrosinase accumulation.

It has been shown in the present study that the expression of myrosinase in the stem becomes significantly lower as the plant grows. In contrast, in the plant A. thaliana, the myrosinase activity is stronger in the older parts of the stem [55]. Husebye et al. reported myrosinase expression in the stem cortex, the endodermis and the xylem in A. thaliana L., similar to the stem of B. napus L. [17]. These findings agree with those obtained in our study; however, no results related to the nasturtium stem cortex were found. In T. majus L., the most intense myrosin activity in the stem was recorded in vascular tissue, large xylem vessels and phloem cell walls. It has been found that positive myrosin cells found in the phloem were phloem parenchyma cells [6]. These cells were found facing the endodermis. Located outside of the phloem sieve elements, but in a neighbouring position, myrosinase-containing cells and glucosinolate S-cells provide excellent protection against harmful microorganisms and insects [6,17]. Husebye et al. showed that the sieve tract elements receive the photosynthetic assimilate products from the bundle sheath through phloem parenchyma cells; therefore, it is assumed that the myrosinase–GSL defence system in the phloem can function in the same manner [17]. The myrosinase expression pattern detected in the nasturtium stem and leaf correlates with these findings.

A special feature of myrosin cells is their location in the leaf vessels. Myrosin cells develop specifically next to phloem cells on the abaxial lower side of the leaf vasculature and are called phloem myrosin cells. Therefore, myrosin cells were originally thought to be a type of vascular cell [49,57]. Furthermore, in the leaves of a mature oilseed rape plant, myrosinase was found in cells symmetrically placed in the phloem parenchyma, while young leaves showed the myrosin cells always wider and longer than phloem ground tissue cells [6]. In the present study, the myrosin cells were located between phloem parenchyma cells, sieve tube elements and companion cells, as described in the phloem leaf area in Arabidopsis [6]. Furthermore, a stronger myrosinase activity was reported in young leaves than in older ones in A. thaliana L. [55] and B. napus L. [23], while in Arabidopsis, only scattered cells of seedling leaf tissue showed myrosinase expression [27]. However, no significant difference in myrosinase expression between young and old leaves in T. majus L. has been recorded, only low myrosinase expression in all developmental stages.

5. Conclusions

This study reveals the expression pattern of myrosinase enzyme type 1 and myosin cell morphology in T. majus for the first time according to the knowledge of the authors. Our results are based on immunohistochemical and electron microscopy methods and correspond to those previously described in other plant species containing the myrosinase–GSL system. We have shown that in T. majus, myrosinase type 1 expression decreases with the growth and ageing of the plant. The root is shown to be the place of greater myrosinase accumulation and activity. The most abundant areas of myrosinase expression in the stem were in the conducting tissue, large xylem vessels and phloem wall cells, with a clear dominance of expression in the second phase of development. The nasturtium leaf, compared to other investigated parts of the plant, showed the weakest expression of the myrosinase enzyme during all stages of growth. We also show, for the first time, the ultrastructure of nasturtium myosin cells, which are generally smaller than neighbouring cells and are located between larger S-cells. They have a rectangular shape, with a large central vacuole and some smaller vacuoles in the periphery. The results are new data for this species and represent a contribution to the biology of this understudied plant, whose beneficial biological effects are related to the enzymatic activity of myrosinase. The potential practical applications of the findings in this research are the use of T. majus in various industries (food industry, agro-industry, pharmaceutical industry and medicine) thanks to the glucosinolate–myrosinase system and the formation of glucosinolate degradation products because they divert numerous pests from fruits and vegetables and show different biological activities (antioxidant, antimicrobial, antifungal and cytotoxic activities). Nevertheless, further studies on myrosinase location and activities in T. majus are needed to confirm and extend the obtained results.