Annals of Botany 86: 73±78, 2000doi:10.1006/anbo.2000.1161, available online at http://www.idealibrary.com on

Relative Abundance of Nickel in the Leaf Epidermis of Eight Hyperaccumulators: Evidencethat the Metal is Excluded from both Guard Cells and Trichomes

G. K. PSARAS*{, TH. CONSTANTINIDIS{, B. COTSOPOULOS{ and Y. MANETAS{

{Section of Plant Biology, Department of Biology, School of Sciences, University of Patras, Patras 265 00, Greeceand {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

been even

0305-7364/0

* For corrupatras.gr

Scanning electron microscopy combined with X-ray microanalysis was used to localize the sites of nickel accumula-tion on the leaf epidermis of eight nickel accumulators grown on ultrama®c soils in Greece. In all species, nickel wasexcluded from guard cells, and in species possessing hairy leaves (seven out of eight) nickel was excluded from thehairs. In some species the metal was present in subsidiary cells, yet at low levels, while the sites of higher accumulationwere epidermal cells away from stomata. Results indicate that nickel is not compatible with the functions anddevelopment of certain epidermal cell types. # 2000 Annals of Botany Company

Key words: Brassicaceae, epidermis, metal hyperaccumulators, microanalysis, serpentine plants.

grown on ultrama®c soils in Greece.

INTRODUCTION

Although some heavy metals are essential trace elements forplant life, at relatively high concentrations they are toxicsince they interfere with enzyme function (De Vos et al.,1989; Krupa et al., 1993). Accordingly, ultrama®c soils richin such metals are considered to be hostile, and they oftensupport a specialized ¯ora (Brooks, 1998a) of metalresistant plant species (metallophytes). Although, in manycases, resistance is due to exclusion of the metal from theprotoplast (Ernst, 1976), some plants actively take upmetals leading to accumulation at extremely high levels, farexceeding those in the soil (Brooks, 1998b).

Hyperaccumulation raises interesting biological ques-tions such as the mechanisms by which toxicity is avoidedand the possible adaptive signi®cance of such high levels ofheavy metals. With regards to nickel hyperaccumulators, ithas been reported that the absorbed metal is renderedinactive by complexing with histidine (KraÈ mer et al., 1996),other amino acids and carboxylic acids including malate(Brooks, 1998a). Regarding the adaptive signi®cance of theaccumulation trait, an antiherbivore defensive function hasbeen proposed (Boyd, 1998). Relative to the above is thequestion 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; KraÈ mer et al., 1997b); inthe three cases where inter-tissue distribution within a leafwas studied, nickel was found to be deposited in theepidermis and its appendages (Severne, 1974; Mesjasz-Przybylowicz et al., 1994; KraÈ mer et al., 1997a). There have

fewer investigations in which the intercellular

0/070073+06 $35.00/00

espondence. Fax �3 061 997411, e-mail g.k.psaras@

distribution in the epidermis has been addressed (Heathet al., 1997).

In the present investigation, a combination of scanningelectron microscopy and X-ray microanalysis was used tostudy the relative abundance of nickel in various epidermalcell types of eight hyperaccumulating Brassicaceae species

MATERIALS AND METHODS

Plant material

The work presented here was carried out using dry leavesfrom plant material kept in the herbarium at the Universityof Patras, Greece. Ten species belonging to the familyBrassicaceae grown on ultrama®c soils in Greece werechosen for the study. Eight of these are known to be metalhyperaccumulators (Brooks, 1998b), while the other twonon-accumulating species from the serpentine ¯ora wereincluded for comparison. Plant material was taken from thefollowing specimens.

Nickel accumulators:Alyssum euboeumHala csy (388460N,238190E, Phitos & Kamari 20241), Alyssum heldreichiiHausskn. (398470N, 218130E, Charpin et al. AC 11124),Alyssum lesbiacum (P. Candargy) Rech. ®l. (398020N,268180E, Strid et al. 26184), Alyssum smolikanum Nya ra dy(408020N, 218050E, Phitos et al. 25666), Bornmuellerabaldacii (Degen) Heywood ssp. baldacii (408060N, 208590E,Hartvig & Seberg 4407), Bornmuellera tymphaea (Hausskn.)Hausskn. (398490N, 218230E, Constantinidis 8007), Lepto-plax emarginata (Boiss.) O. E. Schulz (398490N, 218240E,Constantinidis 8049) and Thlaspi pindicum Hausskn.

(398070N, 228120E, Constantinidis 7394).

# 2000 Annals of Botany Company

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Non-accumulators:Aubrieta glabrescens Turrill (408050N,208540E, Phitos et al. 25579) and Erysimum microstylumHausskn. (398090N, 228040E, Constantinidis & Iliadis 7812).To ascertain whether the drying procedure involved in

the preparation of the herbarium specimens could havecaused redistribution of the element, fresh material from theaccumulators T. pindicum and L. emarginata growing wildon ultrama®c soil (398060N, 228180E and 398080N, 228110E,respectively) was also examined. Mature leaves wereharvested, stored in air-tight plastic bags and immediately

74 Psaras et al.ÐNickel Localizat

transferred to the laboratory for microanalysis.

Thlaspi pindicum leaf is shown in Fig. 1A.

G, H).

Microscopy and microanalysis

In preliminary trials, fresh plant material from Thlaspipindicum and Leptoplax emarginata was mounted on thestage of a scanning electron microscope (Jeol 6300 SEM,Tokyo), immersed in liquid nitrogen within a cryo-transferunit (CT 1500 Oxford Instruments, Oxford, UK) andtransferred to the SEM. The plant material was observed atlow voltage before it was slightly etched for a few seconds atÿ908C, moved back to the cryo-preparation chamber andcoated with gold. The specimens were examined at 20 kV.Dry leaves from the same species kept in the University ofPatras Herbarium were examined for comparison. The dryplant material was mounted on stubs with double-sidedadhesive tape, sputter coated with gold and observed atroom temperature. X-ray maps and energy dispersivespectra (EDS) derived from both fresh and dry materialshowed a similar relative distribution of Ni between thevarious epidermal cell types, indicating that the dryingprocedure did not cause substantial element redistribution.Therefore, further work was carried out using dryherbarium material. Four to ®ve leaves from di�erentindividuals of each species were sampled.

Dry plant material was mounted on stubs with double-sided carbon adhesive tape and sputter coated with gold.The samples were examined with a Jeol 6300 scanningelectron microscope connected to a Link Pentafet (model6699) system (Oxford Instruments) for microanalysis. Thesystem is equipped with a Si(Li) detector and a thin window(Be-window) for elements from B to U. The acceleratingvoltage was 20 kV, the working distance 15 mm and theprobe current 15 nA. During microanalysis the specimentemperature was about 208C and the column vacuum3 � 10ÿ6 torr. Quantitative calibration was made by meansof standard specimens from MAC (Microanalysis Consul-tants Ltd). Element mapping, composition and energydispersive spectra were processed using a Link ISIS soft-ware (series 300, revision 3.2, Oxford), enabling the systemto give directly the elemental composition percentage foreach selected element. Although it is preferable to usealuminium coating for quantitative estimation of elementconcentrations (McCully et al., 1998), quantitative data onnickel abundance given here are relative, thus are nota�ected by the gold coating; elemental concentrations wereestimated excluding gold from the normalized percentages.The signal for each element (including Ni) results from thecorresponding `Hall voltage' level created from the colli-

sions 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 avery high number of point microanalyses made severaltimes per second. The grid for the points of microanalysiswas set at ultra-®ne resolution. Micrographs and X-raymaps made on the computer monitor were taken in digital

n in Metal Hyperaccumulators

RESULTS

Whole leaf

Examination of leaf transverse sections under the analyticalSEM revealed that nickel accumulates in the epidermis ofboth leaf surfaces. An X-ray map from a cross-sectioned

Trichomes

Seven of the eight nickel hyperaccumulators studied herepossess either isolated non-glandular hairs or densetrichomes on their leaves. Only one species, namely Thlaspipindicum, does not possess trichomes (Fig. 1A, B). Lepto-plax emarginata leaves possess isolated T-shaped hairs(Fig. 1C) on their lower surface only. Bornmuellera baldaciissp. baldacii exhibits several asymmetrical stellate hairs(Fig. 1E) also on its lower leaf surface. Both surfaces ofBornmuellera tymphaea leaves are covered by a dense layerof T-shaped hairs (Fig. 1G). All Alyssum species examinedhere possess dense trichomes consisting of stellate hairs onboth 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 sevenhairy-leaved metal accumulators, nickel seems to beexcluded from the trichomes (Figs 1C, E, H, 2B, C±E,

Other epidermal cell types

As shown in X-ray maps of the epidermis, nickel seems tobe excluded from the guard cells and a more-or-less cleargradient in its relative abundance is established, peakingaway from stomata (Figs 1, 2). This is especially evident inthe 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 inlarge epidermal cells between stomatal complexes. Thus, thelevels of nickel in the subsidiary cells were intermediate,while in the case of T. pindicum (Fig. 1B), nickel seems to beexcluded from the subsidiary cells as well.

Besides the X-ray maps, energy dispersive X-ray spectrawere also taken. The case of Leptoplax emarginata is shownin Fig. 3. Figure 3A refers to the whole area shown in themicrograph of Fig. 1C. Figure 3B±D refers to the smallareas on the T-shaped hair, the epidermal cell and thestoma, respectively, indicated by the small circles in Fig. 1C.The inserted numbers indicate the percent contribution ofnickel in the elemental composition in each case. Asexpected, this contribution was low in the guard cell

(0.54%) and the trichome (0.47%), increasing almost

FIG. 1. SEM micrographs combined with X-ray maps from leaves of four nickel-accumulating species. Cross section (A) and surface view (B) of aThlaspi pindicum leaf. Leptoplax emarginata leaf under low (C) and high (D) magni®cation. Low (E) and high (F) magni®cation of a Bornmuellerabaldacii 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.

Psaras et al.ÐNickel Localization in Metal Hyperaccumulators 75

FIG. 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 mapsindicates 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.

76 Psaras et al.ÐNickel Localization in Metal Hyperaccumulators

A

C D

B

C

O

Au

Ni

Caβ

K

ClNi Mg

Caα

Ni: 1.09Ni: 0.47

Ni: 0.54Ni: 4.42

C

O

MgNi

KCl

Ni

Au

Caβ

Caα

C

O

MgNi KCl Ni

Au

Caβ

Caα

C

OMgNi

K

Cl

Ni

Au

Caβ

Caα

0 2 4 6 8 keV 0 2 4 6 8 keV

FIG. 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

lu

Psaras et al.ÐNickel Localization in Metal Hyperaccumulators 77

nine-fold in the epidermal cell (4.42%). An intermediatevalue (1.09%) was evident for the larger area of Fig. 1C(i.e. EDS in Fig. 3A), embracing all cell types. Figure 3 alsoshows that trichomes are an e�cient sink for extra calcium(see De Silva et al., 1996). Nickel was not detected by X-raymicroanalysis in the non-metal accumulators (i.e. Aubrieta

percent contribution of nickel (gold exc

glabrescens and Erysimum microstylum; results not shown).

DISCUSSION

The results clearly show a consistent exclusion of nickelfrom guard cells in all eight nickel accumulators studied. Asimilar result was found for Thlaspi montanum examined byHeath et al. (1997). Therefore, we may assume that nickelinterferes with normal stomatal function and, accordingly,guard cells must be protected from high concentrations ofthe element. The nature of this interference can be deducedby considering the mechanisms through which an absorbedtoxic metal is rendered inactive, in relation to the speci®cfunctions of guard cells. Successful detoxi®cation probablyrequires the formation of a stable organometallic complexand a physiologically inert cell compartment for permanentstorage (see Brooks, 1998a and literature therein). Accord-ing to the ligand-based classi®cation of Nieboer andRichardson (1980), nickel falls within the border linebetween classes A and B of metals, forming complexes

with ligands containing both carboxyl and sulphydryl

groups. Indeed, complexes of nickel with citrate and malatehave been proposed as mechanisms for nickel detoxi®cation(Brooks 1998a). With regards storage, a possible candidatesite could be the vacuole, as shown for cadmium (VoÈ geli-Lange and Wagner, 1990) and zinc (Va zquez et al., 1992).However, vacuoles of guard cells are not physiologicallyinert since they are engaged in a complex ion tra�c throughthe tonoplast membrane, leading to the osmotically drivenchanges 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 atransient presence of nickel in guard cells could immobilizemalate, whose migration between the cytoplasm andvacuole is essential. In addition, ion movements across theplasma and vacuolar membranes in guard cells requireparticular pumping properties, which could be perturbedby the presence of nickel. Indeed, it has been shown that thein vitro activity of the plasma membrane ATPase wasseverely inhibited by nickel (Ros and Picazo, 1990). TheATP needed to drive the ion movements may result fromphotosynthetic activity of guard cells (Willmer and Fricker,1996). However, nickel is known to suppress photosyntheticelectron ¯ow (Sheoran et al., 1990) and to impair photo-synthetic activity by substituting magnesium in the chloro-phyll molecule (KuÈ pper et al., 1996). Thus, we may assumethat nickel is incompatible not only with the unique ion-

ded from the normalized percentage).

movements characterizing the guard cells, but with their

io

ability to photosynthesize as well. Hence, its epidermallocalization should separate it spatially from these func-tions. We may add here that the absence of Ni from themesophyll cells (Severne, 1974; Mesjasz-Przybylowicz et al.,1994; KraÈ mer et al., 1997b; and results from the presentinvestigation, e.g. Fig. 1A) can be correlated with itsdeleterious e�ects on photosynthesis.

The absence of nickel from the trichomes was ratherunexpected in view of the fact that KraÈ mer et al. (1997a),working with Alyssum lesbiacum (which was also tested inthe present study) found a preferential sequestration of themetal within epidermal trichomes. The reasons for thisdiscrepancy are not known at present. We may argue,however, that in all the species studied here, the trichomesare non-glandular, probably serving an antitranspirantfunction, as judged by their density. Such trichomes losetheir protoplasm and die very early during leaf development(Uphof, 1962) and in certain cases the walls of the lowestcells are cutinized to prevent apoplasmic water movement(Fahn, 1986). Apparently, such structures lack the bio-chemical and mechanical attributes to accumulate nickel,unless they do so during the earliest phases of leaf develop-ment. Further research is needed to elucidate possiblechanges in nickel accumulating capacity between variouscell types during leaf development.

We may conclude that, in the epidermis of hyperaccu-mulators, nickel is sequestered in physiologically moreinert, yet living, cells. The preferential exclusion from guardcells and trichomes can be explained on the basis of the

78 Psaras et al.ÐNickel Localizat

constraints imposed by their function and development.

ACKNOWLEDGEMENTS

Th.C. is a recipient of a Post-Doctoral Scholarship from the

State Scholarship Foundation.

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