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Annals of Botany 86: 73±78, 2000 doi:10.1006/anbo.2000.1161, available online at http://www.idealibrary.com on Relative Abundance of Nickel in the Leaf Epidermis of Eight Hyperaccumulators: Evidence that the Metal is Excluded from both Guard Cells and Trichomes G . K . P S A R A S *{, T H . CO N S TA N T I NI DIS{, B. COT S O PO U LO S{ and Y. M A N E TA S { {Section of Plant Biology, Department of Biology, School of Sciences, University of Patras, Patras 265 00, Greece and {Laboratory of Electron Microscopy and Microanalysis, School of Sciences, University of Patras, Patras 265 00, Greece Received: 15 November 1999 Returned for revision: 2 February 2000 Accepted: 16 March 2000 Scanning electron microscopy combined with X-ray microanalysis was used to localize the sites of nickel accumulation on the leaf epidermis of eight nickel accumulators grown on ultrama®c soils in Greece. In all species, nickel was excluded from guard cells, and in species possessing hairy leaves (seven out of eight) nickel was excluded from the hairs. In some species the metal was present in subsidiary cells, yet at low levels, while the sites of higher accumulation were epidermal cells away from stomata. Results indicate that nickel is not compatible with the functions and # 2000 Annals of Botany Company development of certain epidermal cell types. Key words: Brassicaceae, epidermis, metal hyperaccumulators, microanalysis, serpentine plants. I N T RO D U C T I O N Although some heavy metals are essential trace elements for plant life, at relatively high concentrations they are toxic since they interfere with enzyme function (De Vos et al., 1989; Krupa et al., 1993). Accordingly, ultrama®c soils rich in such metals are considered to be hostile, and they often support a specialized ¯ora (Brooks, 1998a) of metal resistant plant species (metallophytes). Although, in many cases, resistance is due to exclusion of the metal from the protoplast (Ernst, 1976), some plants actively take up metals leading to accumulation at extremely high levels, far exceeding those in the soil (Brooks, 1998b). Hyperaccumulation raises interesting biological questions such as the mechanisms by which toxicity is avoided and the possible adaptive signi®cance of such high levels of heavy metals. With regards to nickel hyperaccumulators, it has been reported that the absorbed metal is rendered inactive by complexing with histidine (KraÈmer et al., 1996), other amino acids and carboxylic acids including malate (Brooks, 1998a). Regarding the adaptive signi®cance of the accumulation trait, an antiherbivore defensive function has been proposed (Boyd, 1998). Relative to the above is the question of the distribution of nickel within the plant body. Leaves seem to be the main sinks of nickel (Severne, 1974; Mesjasz-Przybylowicz et al., 1994; KraÈmer et al., 1997b); in the three cases where inter-tissue distribution within a leaf was studied, nickel was found to be deposited in the epidermis and its appendages (Severne, 1974; MesjaszPrzybylowicz et al., 1994; KraÈmer et al., 1997a). There have been even fewer investigations in which the intercellular * For correspondence. Fax 3 061 997411, e-mail g.k.psaras@ upatras.gr 0305-7364/00/070073+06 $35.00/00 distribution in the epidermis has been addressed (Heath et al., 1997). In the present investigation, a combination of scanning electron microscopy and X-ray microanalysis was used to study the relative abundance of nickel in various epidermal cell types of eight hyperaccumulating Brassicaceae species grown on ultrama®c soils in Greece. M AT E R I A L S A N D M E T H O D S Plant material The work presented here was carried out using dry leaves from plant material kept in the herbarium at the University of Patras, Greece. Ten species belonging to the family Brassicaceae grown on ultrama®c soils in Greece were chosen for the study. Eight of these are known to be metal hyperaccumulators (Brooks, 1998b), while the other two non-accumulating species from the serpentine ¯ora were included for comparison. Plant material was taken from the following specimens. Nickel accumulators: Alyssum euboeum HalaÂcsy (388460 N, 238190 E, Phitos & Kamari 20241), Alyssum heldreichii Hausskn. (398470 N, 218130 E, Charpin et al. AC 11124), Alyssum lesbiacum (P. Candargy) Rech. ®l. (398020 N, 268180 E, Strid et al. 26184), Alyssum smolikanum NyaÂraÂdy (408020 N, 218050 E, Phitos et al. 25666), Bornmuellera baldacii (Degen) Heywood ssp. baldacii (408060 N, 208590 E, Hartvig & Seberg 4407), Bornmuellera tymphaea (Hausskn.) Hausskn. (398490 N, 218230 E, Constantinidis 8007), Leptoplax emarginata (Boiss.) O. E. Schulz (398490 N, 218240 E, Constantinidis 8049) and Thlaspi pindicum Hausskn. (398070 N, 228120 E, Constantinidis 7394). # 2000 Annals of Botany Company 74 Psaras et al.ÐNickel Localization in Metal Hyperaccumulators Non-accumulators: Aubrieta glabrescens Turrill (408050 N, 208540 E, Phitos et al. 25579) and Erysimum microstylum Hausskn. (398090 N, 228040 E, Constantinidis & Iliadis 7812). To ascertain whether the drying procedure involved in the preparation of the herbarium specimens could have caused redistribution of the element, fresh material from the accumulators T. pindicum and L. emarginata growing wild on ultrama®c soil (398060 N, 228180 E and 398080 N, 228110 E, respectively) was also examined. Mature leaves were harvested, stored in air-tight plastic bags and immediately transferred to the laboratory for microanalysis. Microscopy and microanalysis In preliminary trials, fresh plant material from Thlaspi pindicum and Leptoplax emarginata was mounted on the stage of a scanning electron microscope (Jeol 6300 SEM, Tokyo), immersed in liquid nitrogen within a cryo-transfer unit (CT 1500 Oxford Instruments, Oxford, UK) and transferred to the SEM. The plant material was observed at low voltage before it was slightly etched for a few seconds at ÿ908C, moved back to the cryo-preparation chamber and coated with gold. The specimens were examined at 20 kV. Dry leaves from the same species kept in the University of Patras Herbarium were examined for comparison. The dry plant material was mounted on stubs with double-sided adhesive tape, sputter coated with gold and observed at room temperature. X-ray maps and energy dispersive spectra (EDS) derived from both fresh and dry material showed a similar relative distribution of Ni between the various epidermal cell types, indicating that the drying procedure did not cause substantial element redistribution. Therefore, further work was carried out using dry herbarium material. Four to ®ve leaves from dierent individuals of each species were sampled. Dry plant material was mounted on stubs with doublesided carbon adhesive tape and sputter coated with gold. The samples were examined with a Jeol 6300 scanning electron microscope connected to a Link Pentafet (model 6699) system (Oxford Instruments) for microanalysis. The system is equipped with a Si(Li) detector and a thin window (Be-window) for elements from B to U. The accelerating voltage was 20 kV, the working distance 15 mm and the probe current 15 nA. During microanalysis the specimen temperature was about 208C and the column vacuum 3 10 ÿ6 torr. Quantitative calibration was made by means of standard specimens from MAC (Microanalysis Consultants Ltd). Element mapping, composition and energy dispersive spectra were processed using a Link ISIS software (series 300, revision 3.2, Oxford), enabling the system to give directly the elemental composition percentage for each selected element. Although it is preferable to use aluminium coating for quantitative estimation of element concentrations (McCully et al., 1998), quantitative data on nickel abundance given here are relative, thus are not aected by the gold coating; elemental concentrations were estimated excluding gold from the normalized percentages. The signal for each element (including Ni) results from the corresponding `Hall voltage' level created from the collisions of the photons on the Si(Li) detector. Mapping was displayed using the SpeedMap software, including CAMEO (Link ISIS). The colouring during mapping is achieved by a very high number of point microanalyses made several times per second. The grid for the points of microanalysis was set at ultra-®ne resolution. Micrographs and X-ray maps made on the computer monitor were taken in digital form. R E S U LT S Whole leaf Examination of leaf transverse sections under the analytical SEM revealed that nickel accumulates in the epidermis of both leaf surfaces. An X-ray map from a cross-sectioned Thlaspi pindicum leaf is shown in Fig. 1A. Trichomes Seven of the eight nickel hyperaccumulators studied here possess either isolated non-glandular hairs or dense trichomes on their leaves. Only one species, namely Thlaspi pindicum, does not possess trichomes (Fig. 1A, B). Leptoplax emarginata leaves possess isolated T-shaped hairs (Fig. 1C) on their lower surface only. Bornmuellera baldacii ssp. baldacii exhibits several asymmetrical stellate hairs (Fig. 1E) also on its lower leaf surface. Both surfaces of Bornmuellera tymphaea leaves are covered by a dense layer of T-shaped hairs (Fig. 1G). All Alyssum species examined here possess dense trichomes consisting of stellate hairs on both leaf surfaces (A. euboeum, Fig. 2A; A. heldreichii, Fig. 2C; A. lesbiacum, Fig. 2E; and A. smolikanun, Fig. 2G). As shown in the X-ray maps of the leaf surface of the seven hairy-leaved metal accumulators, nickel seems to be excluded from the trichomes (Figs 1C, E, H, 2B, C±E, G, H). Other epidermal cell types As shown in X-ray maps of the epidermis, nickel seems to be excluded from the guard cells and a more-or-less clear gradient in its relative abundance is established, peaking away from stomata (Figs 1, 2). This is especially evident in the cases of Thlaspi pindicum, Leptoplax emarginata, Alyssum euboeum and A. heldreichii (Figs 1B, D, 2B, F, respectively) where the highest levels of nickel were found in large epidermal cells between stomatal complexes. Thus, the levels of nickel in the subsidiary cells were intermediate, while in the case of T. pindicum (Fig. 1B), nickel seems to be excluded from the subsidiary cells as well. Besides the X-ray maps, energy dispersive X-ray spectra were also taken. The case of Leptoplax emarginata is shown in Fig. 3. Figure 3A refers to the whole area shown in the micrograph of Fig. 1C. Figure 3B±D refers to the small areas on the T-shaped hair, the epidermal cell and the stoma, respectively, indicated by the small circles in Fig. 1C. The inserted numbers indicate the percent contribution of nickel in the elemental composition in each case. As expected, this contribution was low in the guard cell (0.54 %) and the trichome (0.47 %), increasing almost Psaras et al.ÐNickel Localization in Metal Hyperaccumulators 75 F I G . 1. SEM micrographs combined with X-ray maps from leaves of four nickel-accumulating species. Cross section (A) and surface view (B) of a Thlaspi pindicum leaf. Leptoplax emarginata leaf under low (C) and high (D) magni®cation. Low (E) and high (F) magni®cation of a Bornmuellera baldacii ssp. baldacii leaf. SEM and X-ray map of Bornmuellera tymphaea leaf under low (G) and high (H) magni®cation. Green colouration in the X-ray maps indicates the presence of nickel. Note the absence of nickel from both the hairs and guard cells. Bars 50 mm. 76 Psaras et al.ÐNickel Localization in Metal Hyperaccumulators F I G . 2. SEM micrographs and X-ray maps of leaves of four nickel-accumulating Alyssum species. A, B, A. euboeum; C, D, A. heldreichii; E, F, A. lesbiacum; G, H, A. smolikanum. Low (A, C, E, G) and high (B, D, F, H) magni®cations are shown. Green colouration in the X-ray maps indicates the presence of nickel. Note the absence of nickel from both the hairs and guard cells. Bar 200 mm in G; all other bars 50 mm. Psaras et al.ÐNickel Localization in Metal Hyperaccumulators 77 B A C C Ni: 1.09 Ni: 0.47 Caα Au Au O O K Ni Mg Cl Caα Caβ K Ni Mg Ni Caβ Cl Ni C C D C Ni: 4.42 Au K Caα Au Ni Cl O 0 Ni Mg O Ni Mg Caβ 2 4 Ni: 0.54 6 8 keV 0 Cl K Caα Caβ 2 4 Ni 6 8 keV F I G . 3. Energy dispersive spectra (EDS) taken from the leaf epidermis of Leptoplax emarginata. A, From the epidermal area shown in Fig. 1C; B±D, from a hair, an epidermal cell and a guard cell as indicated by the circles in Fig. 1C. The inserted numbers indicate the relative elemental percent contribution of nickel (gold excluded from the normalized percentage). nine-fold in the epidermal cell (4.42 %). An intermediate value (1.09 %) was evident for the larger area of Fig. 1C (i.e. EDS in Fig. 3A), embracing all cell types. Figure 3 also shows that trichomes are an ecient sink for extra calcium (see De Silva et al., 1996). Nickel was not detected by X-ray microanalysis in the non-metal accumulators (i.e. Aubrieta glabrescens and Erysimum microstylum; results not shown). DISCUSSION The results clearly show a consistent exclusion of nickel from guard cells in all eight nickel accumulators studied. A similar result was found for Thlaspi montanum examined by Heath et al. (1997). Therefore, we may assume that nickel interferes with normal stomatal function and, accordingly, guard cells must be protected from high concentrations of the element. The nature of this interference can be deduced by considering the mechanisms through which an absorbed toxic metal is rendered inactive, in relation to the speci®c functions of guard cells. Successful detoxi®cation probably requires the formation of a stable organometallic complex and a physiologically inert cell compartment for permanent storage (see Brooks, 1998a and literature therein). According to the ligand-based classi®cation of Nieboer and Richardson (1980), nickel falls within the border line between classes A and B of metals, forming complexes with ligands containing both carboxyl and sulphydryl groups. Indeed, complexes of nickel with citrate and malate have been proposed as mechanisms for nickel detoxi®cation (Brooks 1998a). With regards storage, a possible candidate site could be the vacuole, as shown for cadmium (VoÈgeliLange and Wagner, 1990) and zinc (VaÂzquez et al., 1992). However, vacuoles of guard cells are not physiologically inert since they are engaged in a complex ion trac through the tonoplast membrane, leading to the osmotically driven changes in cell turgor which mediate stomatal movements (Willmer and Fricker, 1996). Among the translocated ions, malate is thought to play a crucial role. Therefore, even a transient presence of nickel in guard cells could immobilize malate, whose migration between the cytoplasm and vacuole is essential. In addition, ion movements across the plasma and vacuolar membranes in guard cells require particular pumping properties, which could be perturbed by the presence of nickel. Indeed, it has been shown that the in vitro activity of the plasma membrane ATPase was severely inhibited by nickel (Ros and Picazo, 1990). The ATP needed to drive the ion movements may result from photosynthetic activity of guard cells (Willmer and Fricker, 1996). However, nickel is known to suppress photosynthetic electron ¯ow (Sheoran et al., 1990) and to impair photosynthetic activity by substituting magnesium in the chlorophyll molecule (KuÈpper et al., 1996). Thus, we may assume that nickel is incompatible not only with the unique ionmovements characterizing the guard cells, but with their 78 Psaras et al.ÐNickel Localization in Metal Hyperaccumulators ability to photosynthesize as well. Hence, its epidermal localization should separate it spatially from these functions. We may add here that the absence of Ni from the mesophyll cells (Severne, 1974; Mesjasz-Przybylowicz et al., 1994; KraÈmer et al., 1997b; and results from the present investigation, e.g. Fig. 1A) can be correlated with its deleterious eects on photosynthesis. The absence of nickel from the trichomes was rather unexpected in view of the fact that KraÈmer et al. (1997a), working with Alyssum lesbiacum (which was also tested in the present study) found a preferential sequestration of the metal within epidermal trichomes. The reasons for this discrepancy are not known at present. We may argue, however, that in all the species studied here, the trichomes are non-glandular, probably serving an antitranspirant function, as judged by their density. Such trichomes lose their protoplasm and die very early during leaf development (Uphof, 1962) and in certain cases the walls of the lowest cells are cutinized to prevent apoplasmic water movement (Fahn, 1986). Apparently, such structures lack the biochemical and mechanical attributes to accumulate nickel, unless they do so during the earliest phases of leaf development. Further research is needed to elucidate possible changes in nickel accumulating capacity between various cell types during leaf development. We may conclude that, in the epidermis of hyperaccumulators, nickel is sequestered in physiologically more inert, yet living, cells. The preferential exclusion from guard cells and trichomes can be explained on the basis of the constraints imposed by their function and development. AC K N OW L E D G E M E N T S Th.C. is a recipient of a Post-Doctoral Scholarship from the State Scholarship Foundation. L I T E R AT U R E C I T E D Boyd RS. 1998. Hyperaccumulation as a plant defense strategy. In: Brooks RR, ed. Plants that hyperaccumulate heavy metals. New York, USA: CAB International, 181±201. Brooks RR. 1998a. Phytochemistry of hyperaccumulators. In: Brooks RR, ed. 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