Soil and Biota of Serpentine: A World View 2009 Northeastern Naturalist 16(Special Issue 5):351–365 Root Ultrastructure of Senecio coronatus Genotypes Differing in Ni Uptake Jolanta Mesjasz-Przybyłowicz1,*, Alban Barnabas1, and Wojciech Przybyłowicz1,2 Abstract - Root ultrastructure and histochemistry of Ni-hyperaccumulating and nonhyperaccumulating genotypes of Senecio coronatus were compared using transmission electron and light microscopy. Distinct groups of inner cortical cells in the Nihyperaccumulator had an organelle-rich cytoplasm, while indistinct groups of these cells in the non-hyperaccumulator had few organelles. The inner cortical-cell groups and adjacent endodermis in both genotypes appeared to be sites for the synthesis of an alkaloid which was produced more abundantly in the Ni-hyperaccumulator. Casparian bands in exodermal cells were better defined in the non-hyperaccumulator, suggesting a more efficient barrier for exclusion of Ni. Results are discussed in relation to the differential uptake of Ni by the genotypes and ultrastructural aspects of alkaloid production. Introduction Ultramafic or serpentine soils have a world-wide distribution and are characterized by disproportionate amounts of Mg in relation to Ca and elevated concentrations of heavy metals such as Ni (Brooks 1987, Kruckeberg 1984). These soils harbor a distinct, often endemic plant community (Brooks 1987). Most of the plants adapted to these metal-rich soils exclude metals from their shoots, as excessive accumulation of heavy metals is toxic to most plants (Baker and Brooks 1989, Baker and Walker 1989). This exclusion strategy is based on reduced uptake into roots, storage of metals in root vacuoles, and restricted translocation into shoots (Lasat and Kochian 2000). However, about 1–2% of plants on ultramafic soils take up and accumulate large quantities of metals in their shoots: a phenomenon known as hyperaccumulation. Plants are defined as hyperaccumulators if they contain more than 0.1% of metal in the dry matter (for Pb, Ni, Cu, Co, Cr) or more than 1% ( for Zn and Mn ) in aboveground parts (Baker and Brooks 1989). Hyperaccumulating plants cope with elevated concentrations of toxic metals inside their tissues through cellular and sub-cellular compartmentation (Kupper et al. 1999), production of metal-binding compounds, and use of detoxification mechanisms involving selective ligands (Callahan et al. 2006). Nickel is the most common heavy metal accumulated by plants. Of the approximately 400 plant species reported to accumulate metals, 318 accumulate Ni (Baker et al. 2000, Reeves and Baker 2000). 1Materials Research Group, iThemba LABS, Somerset West, 7129, South Africa. 2On leave from the Faculty of Physics and Applied Computer Science, University of Mining and Metallurgy, Kraków, Poland. *Corresponding author - mesjasz@tlabs.ac.za. 352 Northeastern Naturalist Vol. 16, Special Issue 5 There are five nickel hyperaccumulating plants in South Africa (Morrey et al. 1992, Smith et al. 2001), and one of these species, Senecio coronatus (Thunb.) Harv. Asteraceae (Hilliard 1977), is an interesting example of a plant’s adaptation to different ecological conditions. The species is widespread in grasslands in South Africa, but is also found on ultramafic outcrops. Populations of S. coronatus occurring on ultramafic soils differ in terms of Ni uptake and represent Ni-hyperaccumulating and non-hyperaccumulating genotypes. Mesjasz-Przybyłowicz et al. (1997) reported the existence of three genotypes of S. coronatus growing on ultramafic outcrops in Mpumalanga, South Africa: two populations growing at a distance in separate localities (Agnes Mine and Kaapsehoop) (Mesjasz-Przybyłowicz et al. 1994) hyperaccumulated nickel, whilst the third population, also geographically isolated (Songimvelo Game Reserve), showed lower concentrations of this element. The amount of Ni in leaves of this latter group was below the hyperaccumulation threshold, but higher than normally found in plants and typical of flora growing on ultramafic soils. Our unpublished results showed that plants of the hyperaccumulating genotypes maintained their ability to take up high amounts of Ni when grown on ultramafic soil from Songimvelo Game Reserve under controlled laboratory conditions, whilst non-hyperaccumulating plants from Songimvelo, transplanted into soil from Agnes Mine or Kaapsehoop, did not hyperaccumulate Ni. Boyd et al. (2002, 2008) reported the presence of populations of S. coronatus representing both genotypes on other ultramafic localities in the vicinity of Badplaas, Mpumalanga. Ultrastructural studies on metal hyperaccumulating and non-hyperaccumulating plants from ultramafic and non-ultramafic habitats have been undertaken mainly in relation to damage of subcellular structures caused by increasing concentrations of heavy metals. Using this information as one of the indices of evaluation, mechanisms of heavy metal tolerance in plants have been proposed (Bernal et al. 2006, Ni et al. 2005, Sresty and Rao 1999); the upper limit of tolerance of heavy metals by plants has been determined (Liu and Kottke 2003a, Molas 2002, Molas 1997); the effects of the presence of excess heavy metals in soils supporting economically important crop plants have been assessed (Panou-Filotheou et al. 2001); and the suitability of plants as candidates for phytoremediation of heavy metal-contaminated soils has been determined (Islam et al. 2008; Liu and Kottke 2003b, 2004). Few ultrastructural studies have been undertaken comparing the ultrastructural morphology of heavy metal hyperaccumulating and nonhyperaccumulating ecotypes or genotypes of the same species. As far as we are aware, ecotypes of only two species, and genotypes of just one other species, have been investigated thus far regarding their responses to heavy metal exposure. Cadmium-hyperaccumulating and non-Cd-hyperaccumulating ecotypes of Sedum alfredii Hance, exposed to various Cd concentrations, resulted in ultrastructural changes in root meristem and leaf mesophyll cells in both ecotypes, but damage was more pronounced in the non-Cd-hyperaccumulating ecotype even when Cd concentrations were one tenth of those applied to 2009 J. Mesjasz-Przybyłowicz, A. Barnabas, and W. Przybyłowicz 353 the Cd hyperaccumulator (Jin et al. 2008). Islam et al. (2008) investigated the effects of various Pb concentrations on two ecotypes of Elsholtzia argyi Leveille from Pb/Cu-contaminated mining and non-contaminated agricultural areas, respectively. In both ecotypes, Pb caused membrane damage, but it was more obvious in the ecotype from the non-contaminated agricultural area, indicating that the ecotype from the Pb/Cu mining area was more tolerant to high lead concentrations. Two genotypes of Cajus cajan (L.) Millsp., grown under various concentrations of Zn and Ni, showed a differential response in the ultrastructure of root cortical cells (Sresty and Rao 1999). Ultrastructural alterations to metal toxicity at high concentrations occurred mainly in the membranes, but to a lesser extent in one genotype, showing that the latter was more tolerant to higher concentrations of Zn and Ni. In a study of two populations of the Ni-hyperaccumulating plant Dianthus repens Willd., one growing on soil with a high Ni concentration and the other growing on typical acidic soil, leaf ultrastructure of both populations was compared (Kravkina 2000). Large osmiophilic inclusions were found in the mesophyll and bundle sheath cells of plants growing on soil with high Ni concentration but not in plants on acidic soil. The author suggested that these inclusions may be protein-nickel complexes which could be involved in metal detoxification. None of the above-mentioned studies, however, have compared the ultrastructural morphology of hyperaccumulating and non-hyperaccumulating genotypes of the same species growing in their natural environment on ultramafic soil. The purpose of the present investigation was to examine this aspect, as well as ultrastructural aspects of the genesis, transport, and storage of an alkaloid known to be synthesized in roots of species of Senecio. This study also extends our previous work (Mesjasz-Przybyłowicz et al. 2007) in which root cytology at the light microscope level and elemental distribution using a nuclear microprobe were examined in both genotypes of S. coronatus growing on ultramafic outcrops. Methods Collection and sampling Plants were collected from two ultramafic sites in Mpumalanga, South Africa: Agnes Mine (Ni-hyperaccumulating genotype) and Songimvelo Game Reserve (non-hyperaccumulating genotype). Ten plants from each site were collected. They were transported to the laboratory, and the roots were rinsed thoroughly and quickly in distilled water to remove excess soil. Samples were taken about 10 cm away from the root-hair zone for histochemical and ultrastructural studies. The dominant root of each plant collected was sampled. Embedding in resin Root samples for resin-embedding were fixed in 3% glutaraldehyde, post-fixed in 2% osmium tetraoxide, dehydrated in a graded ethanol series and embedded in Spurr’s resin (Spurr 1969). Ultrathin sections were mount354 Northeastern Naturalist Vol. 16, Special Issue 5 ed on copper grids, stained with 2% aqueous uranyl acetate followed by lead citrate (Reynolds 1963) and examined and photographed with a JOEL 1200 transmission electron microscope. Histochemical tests A 0.5% solution of aniline blue in distilled water was used for differential staining of root tissues. The stain was applied to hand-cut cross-sections of fresh roots. Sudan Black B was used to test for lipids (O’Brien and McCully 1981) in cross-sections of both fresh and resin-embedded root material. Wagner (Furr and Mahlberg 1981) and Dragendorff (Svendsen and Verpoorte 1983) reagents were used as indicators to test for alkaloids. The indicators were applied directly to hand-cut cross-sections of fresh roots on microscope slides and observed with a light microscope. Negative controls for both lipids and alkaloids were also used. Results At the light-microscope level, groups of cells in the inner cortex of the hyperaccumulator adjacent to the endodermis and phloem, stained intensely with aniline blue (Fig. 1A, large arrows). These specialized cells often contained accumulated material (Fig. 1A, small arrows). In the same region of the non-hyperaccumulator, such specialized cells were not obvious. The small groups of inner cortical cells here did not stain as intensely with aniline blue (Fig. 1 D, double arrows). At the transmission-electron-microscope level, the specialized inner cortical cells of the Ni-hyperaccumulator were characterized by relatively large vacuoles and an organelle-rich cytoplasm (Fig. 1B, C). An extensive network of endoplasmic reticulum (ER) (both smooth and rough) permeated the cytoplasm, and numerous ribosomes, microbodies with crystalline inclusions, mitochondria, Golgi bodies, membranous vesicles, and spherical bodies were present (Figs. 1C, 2A, 2B, 3E). In contrast, in the non-hyperaccumulator, the small groups of inner cortical cells possessed very large vacuoles and a thin parietal layer of cytoplasm with few of the organelles that were present in cells of the Ni-hyperaccumulator. (Figs. 1E, 1F, 3A). Fewer spherical bodies were also evident in these cells (Fig. 1E, arrowheads). Spherical bodies also occurred in endodermal cells that were located adjacent to the inner cortical-cell groups in both genotypes (Figs. 3G, 4A). A closer examination of the spherical bodies in cortical cells of both genotypes showed that they originated within the cytoplasm in close association with the ER (Figs. 1G, 1H, 2A, 3A). Young spherical bodies resembled lipid bodies and had homogeneous contents of moderate electron density. A membrane-like bounding layer enclosed them (Figs. 1G, 1H, 2A). As spherical bodies increased in size, cytoplasmic vesicles, generally of two sizes (very small and larger) were incorporated into their matrix giving the spherical bodies a multivesicular appearance (Figs. 2B, 3A, 3E). Vesicles appeared to originate from rough ER (Figs. 2A, 3A). Vesicles frequently had an electron2009 J. Mesjasz-Przybyłowicz, A. Barnabas, and W. Przybyłowicz 355 dense bounding membrane enclosing its contents. Smooth ER was also often intimately associated with the spherical bodies (Figs. 1G, 3E). As multivesicular spherical bodies continued to increase in size, they were gradually extruded from the cytoplasm into the vacuoles (Figs. 2B, 3A) and into the intercellular air spaces between cells of the inner cortex (Fig. 3A, C, F) where they coalesced to form larger deposits. Extrusion of the multivesicular spherical bodies into the vacuoles appeared to occur by a localized breakdown of the tonoplast and its reformation after their passage (Fig. 2B). Exit of the spherical bodies via the plasma membrane into the extracytoplasmic space probably occurred in a similar manner. Passage of spherical body contents through the cell walls into the intercellular airspaces seemed to be facilitated by a partial dissolution of the middle lamella and cell wall (Fig. 3B, arrows). Occasionally, the faint outlines of the spherical bodies which had coalesced within the intercellular airspaces were discernable (Fig. 3B, double arrows). Significantly larger deposits of material arising from coalescence of the spherical bodies in the intercellular airspaces formed in the Ni-hyperaccumulator (Fig. 3C) compared to the non-hyperaccumulator (Fig. 3F). Spherical Figure 1. Light micrographs (A, D), electron micrographs (B, C, E–H) of Ni-hyperaccumulating (H) and non-hyperaccumulating (NH) genotypes of Senecio coronatus. Distinct groups of specialized inner cortical cells in H (large arrows, A) with accumulating material (small arrows, A, B), relatively large vacuoles (B), organelle-rich cytoplasm (B, C), numerous spherical bodies (arrowheads, B). Indistinct groups of inner cortical cells in NH (double arrows, D) with very large vacuoles (E), thin parietal cytoplasm (arrows, E), few cytoplasmic organelles (F), few spherical bodies (arrowheads, E). Young spherical bodies (SB in G, H), with membrane-like bounding layer (arrowheads) closely associated with endoplasmic reticulum (ER). Microbodies (MB in G,H) with crystalline inclusions. En = endodermis, GB = Golgi bodies, IC = inner cortex, M = mitochondria, N = nucleus, P = plastid, Ph = phloem, R = ribosomes, and V = vacuoles. 356 Northeastern Naturalist Vol. 16, Special Issue 5 deposits resembling spherical bodies were also present in pericycle cells and sieve-tube elements of the phloem located adjacent to the endodermis and inner cortical cell groups in both genotypes (Figs. 3D, 3G, 4A). Casparian bands in the radial walls of exodermal cells of the nonhyperaccumulator (Fig. 4E) were better defined at the ultrastructural level compared to the hyperaccumulator (Fig. 4F). Treatment with Wagner and Dragendorff reagents resulted in reddishbrown staining of spherical bodies within the inner cortical and endodermal cells, spherical deposits in pericycle and phloem cells, and intercellular-accumulated material in both Ni-hyperaccumulating and non-hyperaccumulating genotypes, indicating the presence of alkaloids (Fig. 4B, C). In both genotypes, treatment with Sudan black stained the spherical bodies, spherical deposits, and intercellular-accumulated material black, demonstrating their lipid nature (Fig. 4D). Discussion The present study has revealed several ultrastructural differences between roots of the Ni-hyperaccumulating and non-hyperaccumulating genotypes, some of which may be related to their differential uptake of Ni. In addition, ultrastructural information obtained from both genotypes gave a good indication of the genesis, transport, and storage of an alkaloid known to be synthesized in the roots of species of Senecio. Figure 2. Electron micrographs of older spherical bodies (SB), with membrane-like bounding layer (arrowheads) in Ni-hyperaccumulator. Very small and larger vesicles (arrows, A) some with electron-dense bounding membrane and enclosed material, appearing to arise from rough ER (RER in A). Vesicles (double arrows, A) closely associated with SB. Multivesicular spherical body in B being extruded into vacuole (V). Small and larger vesicles some with electron-dense membranes within multivesicular spherical body (large arrows, B) resemble similar vesicles in cytoplasm (small arrows) contiguous with spherical body. M = mitochondrion, and T = tonoplast. 2009 J. Mesjasz-Przybyłowicz, A. Barnabas, and W. Przybyłowicz 357 Inner root cortical cells of both genotypes were characterized by the presence of large vacuoles, but the relative cytoplasmic volume of cells of the Ni-hyperaccumulator was greater than that of the non-hyperaccumulator. In the latter, the cytoplasm formed a thin parietal layer against the cell walls, and most of the cell volume was occupied by a large central vacuole. Increase in vacuolar size and reduction of cytoplasmic volume has also been reported in other plants in response to the presence of heavy metals. In an ultrastructural study of root cells of Allium sativum L. (Cultivated Garlic) exposed to various concentrations of Cd, Liu and Kottke (2003b) reported that the presence of Cd caused high vacuolation in root cortical parenchyma cells. Sanita di Toppi and Gabbriella (1999) indicated that a significant role in Cd tolerance is played by vacuolar compartmentation, preventing the free circulation of Cd ions in the cytosol and forcing these ions into a limited area. The non-hyperaccumulating Figure 3. Electron micrographs of non-hyperaccumulating-NH (A, F) and Ni-hyperaccumulating- H (B–D, E, G) genotypes. A spherical body (SB in A) within the cytoplasm in close proximity to the endoplasmic reticulum and a multivesicular spherical body (arrowhead in A), with faint outline of vesicles (arrows), being extruded into vacuole. Dissolution of portions of middle lamella and cell wall (arrows, B) for exit of spherical body material into intercellular air space. Note faint outline of coalescing spherical bodies (double arrows, B) in intercellular air space. Large deposits of alkaloidal material (arrow, C) in intercellular air space of H. Spherical deposits (arrows, D) in sieve-tube elements (SE). Portion of multivesicular spherical body (SB in E) with large and smaller vesicles (large arrows) similar to vesicles (small arrows) in cytoplasm contiguous with spherical body. Small deposits of alkaloidal material (arrows, F) in intercellular air spaces of NH. Intracytoplasmic spherical bodies (arrows, G) in inner cortical (IC) and endodermal (En) cells. Spherical deposits in pericycle (P) cells not intracytoplasmic (arrowheads, G). CB = Casparian bands, ER = endoplasmic reticulum, GB = Golgi body, M = mitochondrion, RER = rough ER, SB = spherical bodies, SER = smooth ER, and V = vacuole. 358 Northeastern Naturalist Vol. 16, Special Issue 5 genotype in the present study may be adopting a similar strategy probably because of a lack of tolerance for excess Ni. Another ultrastructural difference between the genotypes was the presence of a greater number of spherical bodies and more intercellular-accumulated material in the Ni-hyperaccumulator compared to the non-hyperaccumulator. Although histochemical tests are not definitive, the positive staining of spherical bodies and intercellular-accumulated material with Wagner and Dragendorff reagents indicates that they are alkaloids, in concurrence with the results of our previous study (Mesjasz-Przybyłowicz et al. 2007). Alkaloids are a diverse group of low molecular weight nitrogen-containing compounds that are derived mostly from amino acids (Facchini and De Luca 2008). As secondary metabolites, they are thought to play a defensive role in protecting a plant against herbivores and pathogens (Facchini and St-Pierre 2005). In species of the tribe Senecioneae (to which S. coronatus belongs) of the Asteraceae, a type of alkaloid known as pyrrolizidine alkaloid (PA), occurs (Hartmann and Dierich 1998, Hartmann and Toppel 1987, Toppel et al. 1987). Pyrrolizidine alkaloids are produced in the roots as seneciocine N-oxide, the primary product of PA biosynthesis (Hartmann et al. 1989). Figure 4. Electron micrographs (A, E, F) and light micrographs (B–D) of Ni-hyperaccumulating (H) and non-hyperaccumulating (NH) genotypes. Intracytoplasmic spherical bodies (arrows, A) in endodermal cells (En). Spherical deposits (arrowheads, A) in pericycle cells (P) and in sieve elements (SE), not intracytoplasmic. Spherical bodies (arrows, B, C), accumulated material in intercellular air spaces (double arrows, B, C) and spherical deposits (arrows) in endodermis (En), pericycle (P), and sieve elements (SE), stain positively for alkaloids. Spherical deposits/bodies (arrows, D) and accumulated material in intercellular air space (double arrow, D) in inner cortex (IC), endodermis (En), pericycle (P), and sieve elements (SE), stain positively for lipids with Sudan Black. Casparian bands (CB in E, F) better defined in NH compared to H. 2009 J. Mesjasz-Przybyłowicz, A. Barnabas, and W. Przybyłowicz 359 Senecionine N-oxide is synthesized from homospermidine, the first intermediate of the PA-specific pathway (Khan and Robins 1985). Homospermidine synthase, the entry enzyme of the PA pathway, catalyzes the formation of homospermidine from the primary metabolites putrescine and spermidine (Böttcher et al. 1993). Using polyclonal antibodies, Moll et al. (2002) demonstrated that homospermidine synthase was localized to specialized endodermal and neighbouring cortical parenchyma cells opposite the phloem in roots of Senecio vernalis. They therefore proposed that these specialized cells were also the intrinsic sites of the biosynthesis of senecionine N-oxide. Moll et al. (2002) also showed that the homospermidine synthase gene is expressed at high levels in the roots of S. vernalis, but not in the aerial parts of the plant. This result supported the biochemical evidence that roots of Senecio species are the exclusive site of PA biosynthesis, as was shown earlier with in vitro root cultures and detached plant organs (Hartmann et al. 1989, Hartmann and Toppel 1987, Toppel et al. 1987). On the basis of ultrastructural and histochemical findings in the present study, the inner cortical and endodermal cells adjacent to the phloem in both genotypes seem also to be sites for synthesis of senecionine N-oxide since the spherical bodies in both these cell types are generated within the cytoplasm. At the subcellular level, Moll et al. (2002) showed that immunogoldlabelled homospermidine synthase in roots of S. vernalis is localized in the cytoplasm and is not associated with any organelle. Although immunogold- labelling was not done in the present study, a cytoplasmic origin of homospermidine synthase is likely. Many other alkaloid biosynthetic enzymes have been found to occur in subcellular compartments other than the cytosol such as the vacuole (McKnight et al. 1991), tonoplast (Stevens et al. 1993), chloroplast thylakoid membranes (Dethier and De Luca 1993), endoplasmic reticulum (St-Pierre and De Luca 1995), and vesicles (Amman et al. 1986). In cultured opium poppy cells, two enzymes involved in the biosynthesis of sanguinarine were co-localized to the endoplasmic reticulum. Vesicles derived from the endoplasmic reticulum, carrying biosynthetic enzymes and/or alkaloids, were either engulfed by small vacuoles or aggregated within them, forming multivesicular bodies which subsequently fused to the central vacuole (Alcantra et al. 2005). A similar phenomenon was observed in the present study. Vesicles, with enclosed material, appeared to originate from the rough endoplasmic reticulum and were incorporated into the spherical bodies, thereby forming multivesicular bodies. It is possible that the vesicles may be carrying one or more components of the PA pathway to the spherical bodies, where PA production is completed. Alternatively, the vesicles may be transporting the completed primary product (senecionine N-oxide) of the PA pathway to the spherical body. Either way, this would afford the potential to sequester potentially toxic PA biosynthetic intermediates or a primary product away from sensitive areas of the cytoplasm. The multivesicular spherical bodies 360 Northeastern Naturalist Vol. 16, Special Issue 5 are eventually extruded into the vacuoles and intercellular spaces where they coalesce and form large storage deposits. Storage of alkaloids in discrete vesicles or in vacuoles has also been reported in other alkaloid-producing plants (Kutchan et al. 1986). In leaves of the opium poppy, alkaloids were sequestrated into vesicles of laticifers (Bird et al. 2003). Using root cultures, Toppel et al. (1987) and Hartmann (1994) found that species of Senecio with different PA patterns synthesize senecionine N-oxide as a common alkaloidal intermediate (Hartmann 1999). However, according to Hartmann (1999), PAs in the tribe Senecioneae can exist in two interchangeable forms: the non-toxic senecionine N-oxide and the pretoxic tertiary form, senecionine, which can become toxic after bioactivation. Senecionine N-oxide is easily reduced and converted into senecionine by a specific senecionine N-oxygenase present in the guts of certain insects. The enzyme has also been detected in plants, for example in seeds of Crotalaria scassellatii Chiov. (Chang and Hartmann 1998). Senecionine, according to Hartmann (1999), is lipophilic. Therefore, in the present study, the lipid-like nature of the spherical bodies and intercellular-accumulated material (evidenced by their positive staining with Sudan black) suggests that the PA may be in the pretoxic senecionine form, and this form could be characteristic for the species S. coronatus. According to Sander and Hartmann (1989) and Hartmann (1994), PAs to a limited extent may be transformed in roots into PA derivatives characteristic of the respective species (Hartmann 1994, Sander and Hartmann 1989). However, the major sites of the species-specific alkaloid transformations occur in the shoots (Hartmann and Dierich 1998). The other possibility in the present study is that lipids may be occurring in association with the PA, since spherical bodies during early stages of their development resemble lipid bodies. In addition, smooth endoplasmic reticulum, known to synthesize lipids, was also closely associated with the spherical bodies. Root-to-shoot translocation of senecionine N-oxide takes place in the phloem (Hartmann et al. 1989). Specific carriers are involved in phloem loading and unloading of the PA N-oxides because species that do not produce PAs are unable to translocate them via the phloem (Hartmann et al. 1989). The presence of spherical deposits that stain for both alkaloids and lipids in pericycle and phloem cells in both genotypes of S. coronatus suggests movement of PAs from their site of synthesis (specialized inner cortical and endodermal cells) via pericycle cells to the phloem for translocation. The role of the microbodies, which are closely associated with spherical bodies during their genesis and early development in both genotypes, is not known. Microbodies occurring in achlorophyllous tissues such as tubers and root cells are designated unspecialized microbodies with unidentified metabolic roles (Bosabalidis 1995, Huang et al. 1983, Newcombe 1982, Nishimura et al. 1996). The microbodies with their crystalline inclusions could be sequestering enzymes needed during periods of active metabolism (Olsen and Harada 1995), for example, when alkaloids are being produced. 2009 J. Mesjasz-Przybyłowicz, A. Barnabas, and W. Przybyłowicz 361 Mithofer et al. (2004) suggested that in higher plants, biotic as well as abiotic stress caused by the presence of heavy metals often induce the synthesis and accumulation of the same defense-related secondary metabolites. Both types of stresses result in the production of reactive oxygen species (ROS) involved in the oxidation of unsaturated fatty acids which initiate the formation of oxylipins, a highly variable class of lipid-derived signaling molecules. Oxylipins in turn induce expression of genes involved in the biosynthesis and accumulation of secondary metabolites such as alkaloids. It is possible therefore that in the present studies the greater production of PAs in the Nihyperaccumulator could be a response to heavy metal stress. In the non-hyperaccumulating genotype, the formation of some PAs is probably due in part to the fact that plants growing on ultramafic soils have a slightly higher than normal concentration of heavy metals such as Ni in their tissues. Its presence might cause some oxidative stress leading to the formation of PAs, although in far smaller quantities compared to the hyperaccumulating genotype. In our previous study (Mesjasz-Przybyłowicz et al. 2007), Casparian bands were identified in exodermal cell walls of both genotypes, but the bands fluoresced more intensely in the non-hyperaccumulator, suggesting differences in chemical composition and probably also in function, such as serving as a more efficient apoplastic barrier. Support for the latter was seen in nuclear microprobe-generated elemental maps which showed the distribution pattern of Ni in root tissues of both genotypes. The highest Ni concentration in the Ni-hyperaccumulating genotype was in the outer cortex: 20 times more than was present in the adjacent epidermis/exodermis, suggesting that the exodermis was probably not an efficient apoplastic barrier in this genotype. In contrast, in the non-hyperaccumulating genotype, the epidermis/exodermis had a higher Ni concentration compared to the adjacent outer cortex, indicating that the exodermis functioned as an efficient apoplastic barrier. The finding in the present study that Casparian bands in the non-hyperaccumulator were better defined at the ultrastructural level, compared to those in the Ni-hyperaccumulator, gives further support to our earlier finding that an exclusion mechanism for Ni may reside in the exodermis of the non-hyperacccumulating genotype. In summary, the main ultrastructural differences between roots of the Ni-hyperaccumulating and non-hyperaccumulating genotypes were: the presence of distinct groups of inner cortical cells with an organelle-rich cytoplasm and numerous spherical bodies in the Ni-hyperaccumulator compared to indistinct groups of these cells with fewer organelles and spherical bodies in the non-hyperaccumulator; a very narrow cytoplasmic layer and therefore larger vacuoles in inner cortical cells of the non-hyperaccumulator; greater deposits of alkaloidal material, arising from coalescence of spherical bodies, in the Ni-hyperaccumulator; and distinct Casparian bands in radial walls of exodermal cells of the non-hyperaccumulator. 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