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Accumulation of Nickel in Trichomes of a Nickel Hyperaccumulator Plant, Alyssum inflatum
Rasoul Ghasemi, Seyed Majid Ghaderian, and Ute Krämer

Northeastern Naturalist, Volume 16, Special Issue 5 (2009): 81–92

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Soil and Biota of Serpentine: A World View 2009 Northeastern Naturalist 16(Special Issue 5):81–92 Accumulation of Nickel in Trichomes of a Nickel Hyperaccumulator Plant, Alyssum inflatum Rasoul Ghasemi1,2, Seyed Majid Ghaderian1,*, and Ute Krämer2 Abstract - Compartmentation of metals in specific tissues, cells, and subcellular compartments is considered a metal-tolerance mechanism in metal hyperaccumulator plants. In this study, we investigated the accumulation of Ni in the trichomes of a serpentine endemic Ni hyperaccumulator plant, Alyssum inflatum, native to western Iran. Elemental analysis of plants from their natural habitat showed that the Ni concentration of trichomes was not higher than in the shoot, suggesting that Ni does not preferentially accumulate in trichomes. Treatment of plants by adding different concentrations of Ni to the growth medium showed that staining of trichomes with dimethylglyoxime (a specific stain for Ni) increased as concentrations of external Ni increased. Accumulation occurred in the base of trichomes and, by increasing the concentration of Ni, accumulation extended to the rays and cell walls. The results showed that trichomes can accumulate high concentrations of Ni and that Ni accumulation can be under the control of Ni concentration in the shoot. Introduction Plants have two different strategies in response to elevated concentrations of metals in soil. Most plants respond by excluding excessive uptake of metals into their shoot (Baker 1981), but some plants respond by accumulating and detoxifying high concentrations of metals in their shoot (Baker and Brooks 1989). Hyperaccumulator plants, a term introduced by Brooks et al. (1977), can accumulate >1000 μg g-1 of metal in aboveground dry matter for Ni, Cr, Co, Pb, and Cu and >10,000 μg g-1 of metal for Mn and Zn (Baker and Brooks 1989). Serpentine (ultramafic) soils contain relatively high concentrations of Ni, Cr, and Co, and these metals are potentially toxic to plants. Among all known metal hyperaccumulator plants (more than 450 species), about 75% are Ni hyperaccumulators (Baker et al. 2000). In temperate regions, most Ni hyperaccumulators belong to the Brassicaceae, the largest number being in the genus Alyssum, section Odontarrhena, in which all species are perennials (Brooks 1998, Brooks et al. 1979, Reeves et al. 2001). Most Ni hyperaccumulators in the genus Alyssum are distributed on serpentine soils and are considered as strict metallophytes (Pollard et al. 2002). Metal hyperaccumulator plants are highly metal tolerant at the cellular level. After translocation of heavy metal to the shoot, some mechanism detoxifies the extraordinary concentrations of metal. Krämer et al. (1997a), by comparing two species of Thlaspi, determined that Ni translocation rates to 1Department of Biology, University of Isfahan, Isfahan, Iran. 2BioQuant, INF 267- BQ 23, University of Heidelberg, 69120 Heidelberg, Germany. *Corresponding author - ghaderian@sci.ui.ac.ir. 82 Northeastern Naturalist Vol. 16, Special Issue 5 shoots are very similar between Ni hyperaccumulator and non-accumulator species. They concluded that the extraordinary degree of Ni tolerance in hyperaccumulator species allows them to accumulate Ni. Several mechanisms for heavy metal detoxification and tolerance have been found (Clemens 2006, Hall 2002). Metal complex formation and compartmentation in cellular compartments or specialized cells are mechanisms that prevent interruption of normal activities of cells. Many localization studies have shown that heavy metals in hyperaccumulator plants accumulate in epidermal tissue (Asemaneh et al. 2006, Bidwell et al. 2004, Frey et al. 2000, Küpper et al. 1999, 2000; Psaras et al. 2000; Tappero et al. 2007; Zhao et al. 2000) and surface appendages such as trichomes (Broadhurst et al. 2004a, 2004b, 2009; de la Fuente et al. 2007; Krämer et al. 1997b; Küpper et al. 2001; McNear et al. 2005; Tappero et al. 2007). Some studies have shown that subcellular locations of accumulated metals include the apoplast and vacuoles (Asemaneh et al. 2006, Bidwell et al. 2004, Frey et al. 2000, Krämer et al. 2000, Küpper et al. 2000). Other reports have shown accumulation of heavy metals in other specialized compartments. In Berkheya coddii Roessler, the cuticle of the upper epidermis is the main compartment for accumulation of Ni in the leaves (Robinson et al. 2003). In some species of Euphorbiaceae, laticifer tubes of stems and epidermal cells of leaves are locations for Ni accumulation (Berazain et al. 2007). The aim of this study was to determine the role of trichomes in accumulation of Ni in the shoot of an Iranian serpentine endemic plant, Alyssum inflatum Nyar. This plant, native to serpentine soils of western Iran, was described as a Ni hyperaccumulator by Ghaderian et al. (2007). Accumulation of Ni in trichomes under both natural and controlled conditions was determined using elemental analysis of isolated trichomes from leaves and stems of field collected plants and staining with dimethylglyoxime (DMG) of the whole leaves of plants grown in controlled conditions. Effects of different concentrations of Ni in the growth medium on the pattern of accumulation of Ni in trichomes were also investigated. Methods Alyssum inflatum is endemic to serpentine soils of western Iran (35°14'N, 46°28'E). The elevation of this area is about 1600 m above sea level. Average annual precipitation is more than 700 mm. The daily maximum temperature in summer reaches 42 °C, and the minimum temperature in winter reaches -20 °C. At the time of sampling, inflorescences were almost dried and seeds were mature. Whole plants were collected and air dried. Collecting of trichomes was performed by scraping the surface of leaves and stems; trichomes from inflorescences were not collected. Trichomes were 100–150 μm in diameter and star-shaped, with 4–5 dichotomous rays. The trichomes surfaces were rough and covered by nodules. Separated materials were then passed through a 200-μm sieve and then, under a binocular microscope, particles other than trichomes were removed. During this step, about half of the trichomes were undamaged, while the others were broken into rays and central parts. 2009 R. Ghasemi, S.M. Ghaderian, and U. Krämer 83 Seeds were collected from plants growing in their natural habitat. Seeds were cleaned by removing all other plant materials and then kept at 4 °C for 4 months. Before sowing the seeds, they were surface sterilized using 70% ethanol for 1 minute and a solution containing bleach with 3.5% NaOCl and 0.05% (W/V) Tween 20 for 15 minutes. After rinsing the seeds with sterile water, they were sown directly on the treatment medium in petri dishes and kept at 4°C for two days. The treatment medium was 25% strength Hoagland solution, which contained 1.5 mM Ca(NO3)2, 0.28 mM KH2PO4, 0.75 mM MgSO4, 1.25 mM KNO3, 0.5 μM CuSO4, 1 μM ZnSO4, 5 μM MnSO4, 25 μM H3BO3, 0.1 μM Na2MoO4, 50 μM KCl, 5 μM Fe-HBED (Iron [N,N’- di-(2-hydroxybenzoyl)-ethylenediamine-N,N’-diacetic acid]), and 3 mM MES-KOH pH 5.7 and 0.8 % (W/V) agarose. Final pH of the medium was adjusted to 5.7. Concentrations of Ni in the medium containing 0, 50, 100, 150, 200, 250, 300, and 350 μM were achieved by adding NiSO4. Culture medium and NiSO4 stock solutions were sterilized separately using autoclave and, before solidification, Ni was added to the culture medium and well mixed. Controlled growth conditions were 16/8 h light/dark and 21/18 °C for the light and dark periods, respectively. Light intensity was 60 μmol photon m-2 s-1 emitted by fluorescent tubes. Petri dishes were kept vertically, and plants were harvested after 23 days. Ten to twelve plants were grown in each petri dish. Plants were divided into two groups, one group was used for staining trichomes with DMG and another group was dried at 60 °C for 3 days and then kept at room temperature for 1 day, after which the dry weight was measured and then used for elemental analysis. Elemental analysis of plants from the natural habitat was performed on pooled samples of leaves and stems. The stem samples were similar to the stems from which trichomes were removed for elemental analysis. Elemental analysis of shoots and trichomes was performed using inductively coupled plasma-atomic emission spectrometry (ICP-AES). To prepare samples for elemental analysis, all shoot materials from 5–6 plants were mixed and then digested with 2 ml 60% nitric acid overnight in room temperature and then for 2 hours at 90 °C. After cooling, 1 ml H2O2 was added and again heated at 90 °C for 20 minutes or until clear. Final volume was made up using ultra pure water. Staining of trichomes for visualizing Ni accumulation was performed using dimethylglyoxime (DMG), which is a specific indicator for Ni (Reeves et al. 1999). In the presence of Ni, DMG forms a purple-colored complex. The solution used for staining contained 0.6 % (W/V) DMG (Merck) in 60% ethanol. Whole leaves or stems were immersed in DMG solution in 2 ml tubes and, after 8 h, the degree of staining of trichomes was compared in treated plants. The first fully expanded pair of leaves from the stem tip was used for staining. Separating the whole leaves from plants for staining with DMG was done very carefully to prevent any damage to leaves or trichomes. To determine statistically significant effects of different treatments on plants, Duncan’s multiple comparison test was used. All statistical analyses were performed using SPSS software (version 13). 84 Northeastern Naturalist Vol. 16, Special Issue 5 Results Elemental analysis of field-collected plants and trichomes Elemental analysis of plants collected from the natural habitat showed that the range of accumulated Ni in the shoot (in leaves and adjacent stems) was between 800 and 3100 μg g-1, with a mean of 2100 μg g-1. The concentrations of different elements in trichomes and also leaf/stem samples are presented in Table 1. Nickel concentration of trichomes was almost in the range of shoot Ni concentration; therefore, in natural conditions, Ni was not preferentially concentrated in the trichomes when compared to the concentration of Ni in the leaf/stem samples. Calcium in trichomes had the highest concentration among the measured elements. Concentration of Ca in trichomes was much higher than the concentration of Ca in the shoot (mean in the shoot was 42,400 μg g-1). This result showed that trichomes are a depository for high Ca accumulation in the leaves. Potassium concentration of trichomes was lower than the concentration of K in the shoot (mean in the shoot was 41,200 μg g-1). Some other elements, such as S and P, had lower concentrations in trichomes relative to the shoot. Effects of different concentrations of nickel on growth and accumulation of nickel in the shoot of A. inflatum To determine tolerance of A. inflatum to high concentrations of Ni in the growth medium, seeds were sown on medium containing different concentrations of Ni (Fig. 1). Results showed that plants were tolerant of up to 300 μM Ni in the medium, as this was the highest Ni level for which biomass production was not significantly decreased. The Ni concentration of 350 μM caused a significant decrease in biomass production, showing that 350 μM Ni in the medium was a toxic concentration for plants. For this concentration of Ni in the medium, concentration of Ni in the shoot increased to more than 10,000 μg g-1 (Fig. 2). Another visible symptom of Ni toxicity at 350 μM Ni in the medium was interveinal chlorosis of leaves. Element measurement of shoots of plants treated with different concentrations of Ni showed that increased concentration of Ni in the growth medium was accompanied by an increase in shoot Ni concentration (Fig. 2). Significant differences occurred between all treatments except treatments of 200 to 300 μM Ni in the growth medium. Table 1. Concentrations of elements (μg g-1, mean ± SD) of trichomes and leaf-stem samples of A. inflatum collected from its natural habitat on serpentine soils. Element Leaf/stem Trichome Element Leaf/stem Trichome Ca 42,400 ± 4500 87,200 ± 2603 Mg 9890 ± 1100 7402 ± 621 Cd 1.36 ± 0.4 0.87 ± 0.2 Mn 148 ± 21 85 ± 7.9 Co 10.7 ± 2.1 11 ± 1.3 Ni 2103 ± 903 1672 ± 39.6 Cu 10.4 ± 3.5 221 ± 28 P 3758 ± 733 1307 ± 352 Fe 379 ± 118 1352 ± 265 S 8535 ± 1871 2345 ± 91 K 41,200 ± 3320 3909 ± 134 Zn 317 ± 68 36.5 ± 16.9 2009 R. Ghasemi, S.M. Ghaderian, and U. Krämer 85 Figure 1. Effect of different concentrations of nickel on shoot biomass production of Alyssum inflatum. 350 μM nickel in the growth medium was toxic for this plant and a statistically significant decrease (P < 0.05) in biomass production occurred at this concentration. Columns indicate means ± SD. Different letters indicate significant differences between treatments based on Duncan’s multiple comparison test. Figure 2. Effect of nickel concentration in the growth medium on accumulated nickel levels in shoots of A. inflatum. Columns indicate means ± SD. Different letters indicate statistically significant differences (P < 0.05) based on Duncan’s multiple comparison test. 86 Northeastern Naturalist Vol. 16, Special Issue 5 Staining of trichomes with DMG Within a single leaf, trichomes stained deep purple at the midrib of the base of the leaf. Trichomes in the central parts of leaves usually had less staining. For high concentrations of Ni in the medium (350 μM), staining was higher at the base, tip, and margins of the leaf and sometimes extended to the central parts. Figure 3 shows the patterns of staining of trichomes with DMG. With no Ni in the medium, there was no staining in leaves (Fig. 3a). With an increase in the concentration of Ni in the medium and a consequent increase of concentration of Ni in the leaves, staining of trichomes was observed. For 50 and 100 μM Ni in the medium, staining of trichomes was very low, although a stained background was seen in leaves because of accumulation of Ni in the leaves (Fig. 3b). With increased Ni concentration, trichomes stained with greater intensity. In moderate concentrations of Ni, the stained areas were mostly in the base and central part of trichomes but not in the radial branches (Fig. 3c and f). In the higher concentration for which plants were still healthy (300 μM Ni in the growth medium), staining extended into Figure 3. Staining of trichomes with DMG. Trichomes of the first mature leaf pairs from tip of stem of plants were treated with different concentrations of nickel. (a) No nickel in the growth medium, (b) 100 μM nickel, (c) 200 μM nickel, (d) 300 μM nickel, (e) 350 μM nickel, (f) side view of trichome of plant treated with 200 μM nickel showing accumulation of nickel inside of the base of trichome, (g) side view of a trichome of plant treated with 350 μM nickel showing accumulation of nickel in all parts of trichome, and (h) staining of trichomes on stem of plant treated with 300 μM nickel. Arrows in (b) and (c) indicate stained location in trichome. Scale bars = 100 μm. 2009 R. Ghasemi, S.M. Ghaderian, and U. Krämer 87 the radial branches (Fig. 3d). At the concentration of Ni for which toxicity was observed (350 μM Ni in the growth medium), all parts of trichomes, including the cell wall and the nodules on the outer surface of trichomes, were stained (Fig. 3e and g). Trichomes of stems also showed accumulation of Ni (Fig. 3h). Patterns of staining of stem trichomes were similar to those of leaf trichomes, but were less intense. Discussion Trichomes are specialized cells of plant epidermis and are classified into several types (Fahn 1990). As trichomes are specialized cells, a different elemental composition is expected for them compared to other epidermal cells. Elemental analysis of Alyssum inflatum trichomes showed that they are rich in Ca. In agreement with this result, several reports have mentioned that the surface of trichomes of several Alyssum species is covered with Ca-rich crystallites (Broadhurst et al. 2004a, Küpper et al. 2001, Psaras et al. 2000). Also, relatively high concentrations of Ca in the shoot of Alyssum species (Broadley et al. 2003, Ghaderian et al. 2007) can be explained by the presence of Ca-rich trichomes on the surface of leaves and stems. Elemental analysis of trichomes also showed that K concentration is lower than in shoots. One possible reason is the low ratio of cytoplast volume, which is the main compartment of K in trichome cells (Marschner 1995), to the entire cell volume of trichomes. Another possibility is that K in the vacuole has been replaced with other cations such as Ca (Marschner 1995). Measured concentrations of Ni in field-collected and growth chamber- grown A. inflatum were not high when compared to some other Ni hyperaccumulators of the genus Alyssum (Baker and Brooks 1989, Broadhurst et al. 2004a). The results were, however, in agreement with results reported by Ghaderian et al. (2007) for A. inflatum. Concentrations of 350 μM Ni in the growth medium, which were accompanied by about 10000 μg g-1 Ni in the shoot, were toxic for this plant and resulted in decreased biomass production. This result suggests that the capacity of A. inflatum for detoxification of Ni in the shoot is not as high as for other Ni hyperaccumulators, such as A. murale and A. lesbiacum. Accumulation of Ni in excess of the detoxification capacity of the plant causes Ni toxicity in the shoot; although other mechanisms may also cause Ni toxicity in plants (for review see Krämer and Clemens 2006, Seregin and Kozhevnikova 2006). We have no evidence that Ni is sequestered in the trichomes: the concentration of Ni in the trichomes of plants from the natural habitat was almost in the same range as that of the shoot. The similar concentration of Ni in trichomes and shoots shows that, in natural conditions, trichomes are not locations for high accumulation of Ni. Collected trichomes were a mix of trichomes from stems and leaves and it is possible that they were not equal in their accumulation of Ni. In experimental conditions, we observed that, at higher concentrations of Ni, trichomes of stems can also accumulate Ni. Results of this study suggest that the concentration of Ni in trichomes is correlated to the concentration of Ni in the shoot. It is possible that low 88 Northeastern Naturalist Vol. 16, Special Issue 5 concentrations of Ni in the shoot under natural conditions resulted in the low Ni concentration of trichomes of plants from the natural habitat. We conclude that in A. inflatum under natural conditions, trichomes do not have an important role in hyperaccumulation of Ni. There is general agreement that concentration of Ni in the leaves of Ni hyperaccumulators increases from central mesophyll cells toward epidermal cells and that epidermal cells have the highest concentration of Ni in leaves (Asemaneh et al. 2007, Bidwell et al. 2004, Broadhurst et al. 2004a, de la Fuente et al. 2007, Mesjasz-Przbylowicz et al. 2001). Therefore, under natural conditions, other compartments, including apoplast and vacuoles of epidermal cells, are more important in compartmentation of Ni. In this study, seeds were directly sowed on the treatment medium to be certain Ni was always available to plants during development. We followed this protocol because nonglandular trichomes are physiologically active in early stages of leaf development and, after that, most of them are inactive and do not have connections to other cells (Fahn 1986, Uphof 1962). In low concentrations of Ni in the leaves, which can be achieved by low concentrations of Ni in the growth medium, staining of trichomes was very low. The concentration of Ni in the shoot in this situation was more than the threshold used to define Ni hyperaccumulation (>1000 μg g-1 Ni in the shoot; Baker and Brooks 1989). Therefore, in low concentrations of Ni, other compartments such as cell walls and vacuoles of epidermal cells seem to be more important than trichomes in accumulation of Ni. Krämer et al. (2000) determined that in the Ni hyperaccumulator Thlaspi goesingense Halac, the apoplast of the leaf is a major location of accumulated Ni. They suggested that the high Ni-binding capacity of the apoplast in Ni hyperaccumulators is a reason for higher Ni tolerance in these plants. Indeed, under lower concentrations of Ni in leaves, less accumulation of Ni in trichomes occurs, and this result may be due to the ability of the leaf apoplast to bind most of the Ni. By increasing the Ni concentration in leaves and occupying all of the Ni-binding sites of the apoplast, the role of intracellular mechanisms and trichomes in compartmentation of Ni is more obvious, as we observed by the greater staining of trichomes under higher concentrations of Ni. A question of interest is which compartment in trichomes is responsible for accumulation of Ni. As trichomes are specialized epidermal cells, they may be similar to other epidermal cells, which primarily accumulate Ni in vacuoles. Both vacuolar sequestration of Ni and compartmentation in the apoplast have been proposed as key tolerance mechanisms in hyperaccumulator plants (Hall 2002). These mechanisms result in less interaction of Ni with cytoplasmic components. Küpper et al. (2001) reported a preferential accumulation of Ni in intracellular compartments of epidermal cells, most likely in the vacuoles, of A. bertolonii Desv., A. lesbiacum (candargy) Rech. f., and Thlaspi goesingense. Asemaneh et al. (2006) also reported similar results for A. murale Waldst. & Kit. and A. bracteatum Boissier & Buhse. Accumulation of Ni in the vacuoles of leaf epidermal cells of Hybanthus floribundus (Lindl.) f. Muell., a Ni hyperaccumulator, has also been reported (Bidwell et al. 2004). It has been reported that the base of trichomes 2009 R. Ghasemi, S.M. Ghaderian, and U. Krämer 89 in different plants are the main location for accumulation of heavy metals such as Ni, Zn, and Mn (Broadhurst et al. 2004a, 2004b, 2009; de la Fuente et al. 2007; Küpper et al. 2000, 2001; Marmiroli et al. 2002; Zhao et al. 2000). Indeed, it is possible that vacuolar sequestration of Ni also occurs in the base of trichomes. Our results are in agreement with other reports about accumulation of Ni in the basal compartment of trichomes. We observed extensions of stained regions into the trichome rays in high but non-toxic concentrations of Ni in the leaves. These extensions are not trichome cell walls or nodules on the surface of rays; rather it seems that the purple-coloured elongations are extensions of cytoplasm or vacuole into the rays. At very high and toxic concentrations of Ni in the shoots, which probably exceeded the detoxification capacities of the plant, the behavior of trichomes was different. In A. inflatum, we observed that, under toxic concentrations, Ni can be placed into the outer cell wall of trichomes that are rich in Ca. This finding suggests that Ca can be replaced by Ni if excess Ni concentrations are available during developing stages of trichomes. In these situations, it seems that all parts of trichomes are filled with Ni. Smart et al. (2007) reported that peripheral regions and rays of trichomes of Alyssum lesbiacum contain high concentrations of Ni. Contrarily, Broadhurst et al. (2004a) discussed that, in A. murale, even under toxic Ni levels, trichome rays were not preferred Ni compartments. Differences between those and our results could be due to different growth conditions and species-specific traits. We suggest that presence of Ni in the trichome rays is not a physiological response of plants to toxic concentrations of Ni. Deposition of Ni in the rays may be an inactive process; when Ni concentration is too high during development of trichome rays, Ni may penetrate to developing rays and incorporate into the structure of different compounds. We conclude that trichomes are a destination for Ni accumulation in Ni hyperaccumulator A. inflatum. Accumulation of Ni in leaf trichomes seems to be a function of the concentration of Ni in the leaf. Therefore all factors which can affect the concentration of Ni in the leaves can affect Ni accumulation in trichomes. These factors include concentration of Ni in the medium, interactions with other elements such as Ca, plant-growth condition, and developmental stage of plant, leaf, and trichomes at the time of sampling. Time and duration of exposure to Ni are other factors that may affect accumulation levels of Ni. Also, it is important to consider which section of leaf is used to determine Ni accumulation in trichomes, because staining of trichomes was not even in all parts of a single leaf. It must also be noted that staining with DMG is generally not used for determining the localization of Ni in tissues. It is believed that artifacts appear due to redistribution of Ni during sample preparation and due to the solvents that are used (Bhatia et al. 2004, Seregin et al. 2003). Formation of crystals is another problem that occurs during the use of DMG (Bhatia et al. 2004). Further, DMG is not able to penetrate into the cells and cell walls containing hydrophobic materials such as wax and suberin (Smart et al. 2007). In consideration of these limitations, a semi-quantitative DMG method has been recently developed to determine the microscopic distribution of Ni at the tissue level in Ni hyperaccumulating plants (Gramlich 90 Northeastern Naturalist Vol. 16, Special Issue 5 2008). However, our findings showed that using DMG for staining of trichomes can be accurate since the results were quite repeatable and in agreement with other reports (Broadhurst et al. 2004a, 2004b, 2009; de la Fuente et al. 2007; Küpper et al. 2001; Tappero et al., 2007) on the accumulation of Ni in the body of trichomes. In addition, none of the noted artifacts were observed in our efforts of staining trichomes with DMG. Acknowledgments We gratefully acknowledge a scholarship to R. Ghasemi from the Ministry of Science, Research and Technology of Iran (MSRT) and University of Isfahan. Special thanks to Naser Karimi for his assistance in collecting seed and to two anonymous reviewers for useful comments. Literature Cited Asemaneh, T., S.M. Ghaderian, S.A. Crawford, A.T. Marshall, and A.J.M. Baker. 2006. Cellular and subcellular compartmentation of Ni in the Eurasian serpentine plants Alyssum bracteatum, Alyssum murale (Brassicaceae), and Cleome heratensis (Capparaceae). 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