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Plant Biosystems - An InternationalJournal Dealing with all Aspects ofPlant BiologyOfficial Journal of the Societa Botanica ItalianaPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713737104

Nickel distribution in the hyperaccumulator Alyssumserpyllifolium Desf. spp. from the Iberian Peninsula

To cite this Article: de la Fuente, V., Rodríguez, N., Díez-Garretas, B., Rufo, L.,Asensi, A. and Amils, R. , 'Nickel distribution in the hyperaccumulator Alyssumserpyllifolium Desf. spp. from the Iberian Peninsula', Plant Biosystems - AnInternational Journal Dealing with all Aspects of Plant Biology, 141:2, 170 - 180To link to this article: DOI: 10.1080/11263500701401422

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Nickel distribution in the hyperaccumulator Alyssum serpyllifolium

Desf. spp. from the Iberian Peninsula

V. DE LA FUENTE1, N. RODRIGUEZ2, B. DIEZ-GARRETAS3, L. RUFO1, A. ASENSI3 &

R. AMILS2,4

1Departamento de Biologıa, Universidad Autonoma, Madrid, Spain, 2Centro de Astrobiologıa (INTA-CSIC), Torrejon de

Ardoz, Madrid, Spain, 3Departmento de Biologıa Vegetal, Universidad de Malaga, Malaga, Spain, and 4Centro de Biologıa

Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Madrid, Spain

AbstractThe relative concentration and distribution of nickel (Ni) in vegetative tissues (leaves, stems and trichomes) andreproductive organs (seeds) was studied using energy-dispersive X-ray microanalysis (EDXS) and scanning electronmicroscopy (SEM) in two previously studied Ni-hyperaccumulator subspecies of Alyssum serpyllifolium Desf. growingnaturally in ultramafic soils of the Iberian Peninsula: A. serpyllifolium ssp. lusitanicum Dudley & P. Silva and A. serpyllifoliumssp. malacitanum Rivas Goday ex G. Lopez. Both taxa showed that Ni accumulates preferentially in the leaves, exhibiting ahomogeneous distribution on both epidermis surfaces. The highest Ni concentrations were found inside the epidermal cellsand at the base of trichome stalks. Ni accumulation in seeds was lower than in the vegetative organs. The location of Ni inthese plants allows us to postulate that its accumulation is a protection mechanism against external stress.

Key words: Alyssum serpyllifolium, EDXS, elemental mapping, hyperaccumulation, Iberian Peninsula, nickel

Introduction

The genus Alyssum L. of the Brassicaceae has a large

number of Ni hyperaccumulator taxa. Jaffre et al.

(1976) coined the term ‘hyperaccumulator’ for such

plants, and Brooks with his collaborators extensively

surveyed metal hyperaccumulating plants, especially

for Ni (Brooks et al., 1977). Ni hyperaccumulator

plants represent more than three-quarters of all metal

hyperaccumulators. Forty-eight species have been

reported in ultramafic soils from southwest Europe

to Asia Minor (Brooks, 1998). Ultramafic soils

contain relatively high amounts of Ni that are

potentially toxic to plants, yet they support a highly

specialized flora rich in endemics and apparently

adapted to this condition (Brooks, 1998). Some

species growing in these soils show high concentra-

tions of Ni in the plant tissues, often exceeding those

in the soils (Brooks, 1998).

Exhaustive analytical studies of the flora of many

parts of the world where ultramafic rocks are

significant (Cuba, Caribbean countries, NW

USA, Newfoundland, Brazil, New Caledonia,

Mediterranean Europe, Turkey, central and eastern

Africa, islands of Indonesia and the Philippines, etc.)

have led to the specific identification of more than

300 Ni hyperaccumulating species to date (Baker

et al., 2000; Reeves & Baker, 2000). These have been

reported and discussed elsewhere (Reeves, 1992,

2003; Medina et al., 1994; Reeves et al., 1996, 1999;

Brooks, 1998). However, the mechanism of Ni

hyperaccumulation in plants is still not well under-

stood, in spite of recent advances in the field. As has

been shown for other elements, Ni and the means of

its hyperaccumulation have been described for

epidermal cells in several species, mainly belonging

to the Brassicaceae (Streptanthus Nutt., Alyssum L.

and Thlaspi L. sp. pl.) (Brooks et al., 1981a,b; Reeves

et al., 1981; Reeves, 1992; Heath et al., 1997;

Kramer et al., 1997; Boyd & Moar, 1999; Psaras

et al., 2000; Davis & Boyd, 2000; Kupper et al.,

2001; Asensi et al., 2004). Recent works describing

Ni localization in Alyssum (A. murale Waldst. & Kit.,

A. corsicum Duby, A. pterocarpum Dudley) evidenced

Correspondence: Vicenta de la Fuente, Departamento de Biologıa, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, E-28049 Madrid,

Spain. Tel.: þ34 91 4978100. Fax: þ34 91 4978300. E-mail: vicenta.fuente@uam.es

Plant Biosystems, Vol. 141, No. 2, July 2007, pp. 170 – 180

ISSN 1126-3504 print/ISSN 1724-5575 online ª 2007 Societa Botanica Italiana

DOI: 10.1080/11263500701401422

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the existence of high amounts of Ni in epidermal

tissues and emphasized the presence of this element,

together with Mn, in leaf trichomes (Broadhurst

et al., 2004a,b; Mcnear et al., 2005). The study of Ni

metal localization in vegetative and reproductive

organs of other hyperaccumulator plants such as

Berkheya coddii Roessler (Asteraceae) (Robinson

et al., 2003), Senecio coronatus (Thunb.) Harv.

(Asteraceae) (Przybylowicz et al., 1995), Senecio

anomalochrous Hilliard (Mesjasz-Przybylowicz et al.,

2001), Stackhousia tryonii Bailey (Stackhousiaceae)

(Bhatia et al., 2003), and Sebertia acuminata Pierre ex

Baill. (Sapotaceae) (Perrier et al., 2004), has recently

begun.

There are two well-known Ni hyperaccumulators

from the Brassicaceae that grow naturally on ultra-

mafic soils of the Iberian Peninsula: Alyssum serpylli-

folium ssp. lusitanicum Dudley & P. Silva and

A. serpyllifolium ssp. malacitanum Rivas Goday ex G.

Lopez (Menezes de Sequeira, 1969; Brooks &

Radford, 1978; Brooks et al., 1981a, Asensi et al.,

2004). A. serpyllifolium ssp. malacitanum is a facul-

tative serpentine taxon related to secondary scrub

communities growing on soils of dolomite and

peridotite ultramafic rocks of the Betic Province

(Rivas-Martınez et al., 1991). This taxon is repre-

sentative of the ultramafic chamaephyte vegetation of

southern Spain (Malaga) that belongs to the endemic

alliance Staehelino-Ulicion baetici. It is located in the

western Betic Cordillera of Malaga (Sierra Bermeja,

Palmitera, Alpujata and Carratraca), one of the

largest areas (more than 300 km2) of ultramafic rocks

in the Iberian Peninsula (Asensi et al., 2004). It

constitutes an extensively serpentinized area with

abundant iron oxides produced by hydrothermal

activity (Brooks et al., 1981a). Total Ni concentra-

tions of between 1,532 and 4,254 mg kg71 have been

obtained for soils from these areas (Rufo et al., 2005).

The second subspecies, A. serpyllifolium ssp. lusitani-

cum, belongs to the dwarf scrub serpentine vegetation

growing on the highly Mg-rich ultramafic outcrops

(80 km2) in north-east Portugal (Braganca – Vinhais,

Macedo de Cavaleiros – Mogadoauro) (Asensi et al.,

2004; Sequeira & Pinto da Silva, 1992). Total soil Ni

concentrations between 851 and 2962 mg kg71 have

been reported in these areas (Peterson et al., 2003).

The bioclimatic characterization of both areas corre-

sponds to the Mediterranean macrobioclimate, with a

marked summer drought (Rivas-Martınez et al.,

2002), an extensive dry – subhumid thermomediter-

ranean to dry mesomediterranean level in southern

Spain, and a subhumid supramediterranean to dry

mesomediterranean level in Portugal.

Previous studies tested the Ni tolerance of both

subspecies (Brooks et al., 1981a) in comparison to

the non-accumulator A. serpyllifolium Desf. ssp.

serpyllifolium. They also investigated the chemical

form of Ni in the leaves, establishing a relation

between Ni and malic and malonic acids (Brooks

et al., 1981b), in an attempt to explain the

physiological function of Ni in these taxa. The Ni-

hyperaccumulating capacity of these taxa has been

recently analysed and compared by Asensi et al.,

(2004), who established that the A. serpyllifolium ssp.

malacitanum population of Sierra Bermeja reaches

higher Ni accumulation levels (1,880 – 2,810 mg Ni

kg71 DW) than the Portuguese A. serpyllifolium ssp.

lusitanicum population (1,990 mg Ni kg71 DW).

These Ni concentrations contrast with the 2 mg

kg71 DW Ni found in the non-accumulator

A. serpyllifolium ssp. serpyllifolium from the dolomitic

area of Sierra de Cazulas (Malaga, Spain).

Having established the total concentrations of Ni

in the Iberian subspecies of A. serpyllifolium, the aim

of this work was to determine the comparative

distribution of this element in different plant tissues

using energy-dispersive X-ray microanalysis coupled

to SEM.

Materials and methods

Plant samples

All plant samples are from the natural environment

of ultramafic areas from Sierra Bermeja (Malaga,

Spain) and Braganca (Portugal). Branches, leaves

and fruits from three specimens of A. serpyllifolium

ssp. malacitanum Rivas Goday ex G. Lopez and three

of A. serpyllifolium ssp. lusitanicum Dudley & P. Silva

from Braganca were collected directly in the field,

from a shrub community growing in rocky areas.

Herbarium voucher specimens are preserved in the

MAF and MGC Herbarium (Faculty of Pharmacy,

Universidad Complutense in Madrid, Spain) and in

personal collections. The material from A. serpyllifo-

lium ssp. serpyllifolium was obtained from specimens

preserved in the MAF Herbarium (MAF 82506)

collected in the dolomitic area of Sierra de Cazulas.

Sample preparation for SEM

Micromorphological analysis of leaves, stems and

seeds using SEM – EDXS were performed on the

Iberian subspecies of A. serpyllifolium. There are

epidermal elements in these plants, such as tri-

chomes, which massively cover leaves, cuticles, fruits

and to a lesser degree the stems, and for this reason

we treated trichomes as an independent unit.

It has been suggested that cryogenic SEM-EDXS

techniques (Kupper et al., 2001; Broadhurst et al.,

2004a) coupled with the use of frozen bulk samples

may be ideal to study Ni accumulation since there is

minimal processing of the samples and structural

features can be easily identified. However, according

Nickel distribution in A. serpyllifolium 171

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to Psaras et al. (2000) no significant changes in Ni

distribution can be observed when dry instead of

fresh and/or frozen material is used if care is taken to

avoid sample embedding. We chose the Psaras et al.

(2000) method because it allowed us to use material

preserved in herbarium collections.

To obtain micro-morphological SEM images of

leaf and stem cross sections, samples were treated in

order to enhance the visualization of the different

plant tissues. Dry leaf and stem samples were fixed

in situ with formyl acetic alcohol (FAA). After

washing with a 0.1 M phosphate buffer (pH 7.4)

they were dehydrated through a graded ethanol

series. Then, they were cut with a sharp blade and

mounted onto stubs.

The following tissues were used for the semi-

quantitative SEM-EDX analysis: (i) from leaves,

upper and lower epidermal cell walls and cell

content, mesophyll, and vascular bundles; (ii) from

stems, epidermis, cortex, central cylinder and pith;

and (iii) from seeds, endosperm and testa. Tri-

chomes, which massively cover leaves, fruits and to a

lesser degree stems, were treated as an independent

unit. Sample preparation was done following the

methodology described by Psaras et al., (2000). For

each type of tissue four to five samples were analysed

from each species. Cross-sections of leaf and stem

were cut with a sharp blade and mounted flat on the

surfaces of conductive graphite stubs and sputters,

and then gold-coated in a BIO-RAD SC 502

apparatus for electrical conductivity in order to

prevent charging under the electron beam. Samples

were examined with a Hitachi S-3000N (Japan)

SEM using an acceleration voltage of 20 kV and a

working distance for analysis of 15 mm. During

analysis the sample stage was at room temperature.

The qualitative element composition of samples was

determined by energy dispersive X-Ray microanaly-

sis using an INCAx-sight with a Si-Li Detector

(Oxford, UK), with a detection limit of 10% of the

main element. This instrument is able to detect the

lighter elements, C, O and N, and the quantitative

numerical data of the obtained spectra are referenced

as default to the highest peak obtained in each

spectrum, which in our case generally corresponded

to C. Mapping was displayed using the Microanalysis

Suite, INCA Suite version 3.04 (Oxford, UK)

software. The grid of points for microanalysis was

set at ultra-fine resolution.

Data analysis

Quantitative numerical data were extracted directly

from spectra in % weight. Data analysis was carried

out using Statistica release 6.0 (Statsoft Inc., Tulsa,

USA). Means, standard deviations and standard

errors were calculated. Data were log-transformed

after being tested for normality with the Shapiro Wilk

test (p4 0.05) and for homogeneity of variance with

the Levene test (p4 0.05). In order to test possible

differences between adaxial and abaxial leaf epider-

mis and cuticle metal concentrations we used the

Student’s t-test. Measures from different tissues were

compared by one-way analysis of variance. Least

significant differences (LSD) between means were

calculated only if an F – test was significant at the

0.05 level of probability.

Results

Leaf

The leaf components analysed by SEM – EDXS

were: epidermal cell walls, the interior of epidermal

cells, mesophyll tissue, and vascular bundles (Figure

1). Micro-morphological SEM analyses showed the

presence of trichomes on both surfaces in the two

subspecies analysed. Transverse leaf sections showed

a dominance of palisade parenchyma on both leaf

sides (Figure 1). Stomata were only present on leaf

abaxial surfaces.

No significant differences in Ni, Ca and Mg

concentrations between adaxial and abaxial epider-

mal cell walls and epidermal cells were detected; thus

the two surfaces were treated as a whole.

There were significant differences between Ni

concentration of leaf tissues in the two subspecies

(Table I). The highest relative concentration of Ni

was found inside epidermal cells with 5.75+ 0.97%

(mean+SE) for A. serpyllifolium ssp. malacitanum,

and 4.94+ 0.34% for A. serpyllifolium ssp. lusitani-

cum. Epidermal cell walls, mesophyll tissue and

vascular bundles had significantly lower Ni concen-

trations than the inside of epidermal cells (Table I).

Values obtained for these tissues oscillated around

0.92+ 0.12%, but A. serpyllifolium ssp. malacitanum

they were below the level of instrument detection with

the following Ni distribution pattern: inside epider-

mal cell4 epidermal cell wall – mesophyll (Figure

2A). In the case of A. serpyllifolium ssp. lusitanicum Ni

concentration in epidermal cell walls, and mesophyll

and vascular bundle tissues varied between 0.25+0.13 and 2.14+ 0.37%. Thus, its Ni distribution

pattern was: inside epidermal cell4 epidermal cell

wall4mesophyll – vascular bundle (Figure 2A).

Ca showed a homogeneous distribution in leaf

tissues of both taxa (Figure 2B). As shown in Table I,

only slight differences were found only for the Ca

contained inside epidermal cells of A. serpyllifolium

ssp. lusitanicum, which is higher than the Ca content

in the rest of the leaf tissues of this taxon. Con-

centrations of this element acquired values between

0.33+ 0.06 and 1.12+ 0.31% for A. serpyllifolium

ssp. malacitanum, and 0.18+ 0.05 – 0.65+ 0.08%

172 V. de la Fuente et al.

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for A. serpyllifolium ssp. lusitanicum. Often Ca was

present as cystoliths and crystals inside vascular

bundle cells.

Mg concentrations were significantly different in

different leaf tissues of both taxa (Table I), with a

higher concentration inside epidermal cells (Figure

2C). Mg content of leaf tissues of A. serpyllifolium ssp.

lusitanicum were more homogeneous (0.35+ 0.09 –

0.69+ 0.06%) than those of A. serpyllifolium ssp.

malacitanum (0.22+ 0.04 – 1.53+ 0.22%).

Elemental mapping of abaxial leaf surfaces was

carried out to complete the leaf study. Figure 3

shows the distribution of Ni, Ca and Mg in the

different cells of the leaf surface. These results clearly

show that the presence of Ni is homogeneous at the

cellular level, while Mg and Ca are almost absent.

Stem

The epidermis, cortex, central cylinder and pith were

the stem tissues analysed by SEM – EDXS (Figure 4).

Both taxa showed a similar Ni distribution pattern

between the different stem tissues (Figure 2D). The

highest Ni concentrations were found in epidermis

and cortex tissues (0.50+ 0.11 – 0.80+ 0.23%),

with no significant differences between their means

(Table II). Central cylinder and pith Ni concentra-

tions are clearly lower than those of epidermis and

cortex, and ranged from 0.09+ 0.02% to below

detection limits.

Ca distribution in stem tissues of both taxa was

similar (Figure 2E). As shown in Table II, the

highest concentrations of this element were found in

epidermis and cortex tissues (0.83+ 0.18 –

1.69+ 1.01%), while the central cylinder and pith

had lower quantities of Ca (0.10+ 0.03 –

0.60+ 0.18%). Ca crystals were common in pith,

cortex and epidermis of both Alyssum subspecies.

Mg concentrations were significantly different

in different stem tissues of both taxa (Table II). In

both cases, a higher Mg concentration occurred

in epidermis and cortex tissues (Figure 2F). Mg

values ranged from 0.22+ 0.07 – 0.65+ 0.27% for

A. serpyllifolium ssp. malacitanum, and 0.02+ 0.01 –

0.66+ 0.15% for A. serpyllifolium ssp. lusitanicum.

Trichomes

Both Alyssum subspecies have many trichomes on leaf

and stem surfaces, which give a tomentose-whitish

Figure 1. SEM micrographs of a cross section of an Alyssum serpyllifolium ssp. lusitanicum leaf (A, B). Energy-dispersive X-ray microanalysis

of IE (C) and VB (D) of Alyssum serpyllifolium ssp. lusitanicum. Abbreviations: E: epidermal cell walls; IE: inside epidermal cells; M: inside

mesophyll cells; VB: vascular bundle cells; T: trichome.

Nickel distribution in A. serpyllifolium 173

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appearance to the plant. Trichomes consisting of

stellate and shortly stalked hairs have a variable

number of rays with thick walls and many thickenings

on external surfaces. Sometimes stellate hairs are

superimposed on each other.

EDX analyses showed that the external surface of

trichomes was mainly composed of Ca usually

together with Mg. Similar Ca concentrations were

obtained for the two taxa (Student’s t-value¼ 1.02,

p4 0.05) with 26.43+ 4.65% for A. serpyllifolium

ssp. malacitanum, and 21.13+ 1.60% for A. serpyl-

lifolium ssp. lusitanicum. Mg concentrations with

values of 1.39+ 0.08% for A. serpyllifolium ssp.

malacitanum and 3.88+ 0.51% for A. serpyllifolium

ssp. lusitanicum were also noted.

In contrast, the base of trichome stalks showed a

more variable composition which included Ni

together with Mg and Ca. Ca concentrations in the

base of stalks were lower than those of trichome

surfaces (Table III) with values of 9.65+ 1.58% for

A. serpyllifolium ssp. malacitanum and 9.99+ 2.39%

for A. serpyllifolium ssp. lusitanicum. In the case of Mg

the pedicle concentration was lower than that of

the trichome surface only for A. serpyllifolium ssp.

lusitanicum, with no significant differences in

A. serpyllifolium ssp. malacitanum (Table III). Map-

pings of the base of trichome stalks from both taxa

(Figure 5) revealed the presence of a considerable Ni

concentration whose mean values were 6.89+1.34% for A. serpyllifolium ssp. malacitanum, and

4.75+ 1.39% for A. serpyllifolium ssp. lusitanicum.

The Ni concentration in the bases of stalks were not

significantly different (Student’s t-value¼ 0.25,

p4 0.05).

As a complementary analysis, the trichome stalk

base of A. serpyllifolium ssp serpyllifolium, a non-Ni-

accumulator, was mapped. Results showed the

complete absence of Ni and smaller amounts of Ca

and Mg (Figure 5).

Seeds

A. serpyllifolium silicles have 1 – 2 winged seeds per

loculus. Ni concentrations were analysed in testa and

endosperm cells (Figure 6).

Ni concentrations in seeds were not high, reaching

values of 0.26+ 0.06% in A. serpyllifolium ssp.

malacitanum, and 0.18+ 0.13% in A. serpyllifolium

ssp. lusitanicum (Figure 2G). Ni concentrations in

endosperm and testa cells of both subspecies were

not significantly different, while differences for Ca

in A. serpyllifolium ssp. lusitanicum, and for Mg in

A. serpyllifolium ssp. malacitanum concentrations

were detected (Table III, Figures 2H, 2I).

Table I. ANOVA and least significant differences (LSD) between means of Ni, Mg and Ca concentrations in leaf tissues from

A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. Means were calculated only if an F – test was significant at the 0.05

level of probability.

A. serpyllifolium ssp. malacitanum A. serpyllifolium ssp. lusitanicum

ANOVA [E – EI – M – VB] ANOVA [E – EI – M – VB]

Ni: F¼28.87; p5 0.001 Ni: F¼14.52; p50.001

Mg: F¼7.93; p5 0.001 Mg: F¼3.23; p50.05

Ca: F¼ 1.64; p4 0.05 Ca: F¼2.95; p5 0.05

Element Contrasted tissue p Contrasted tissue p

Ni E – EI 50.001*** E – EI 0.0017**

E – M 0.6464 E – M 0.0021**

E – VB – E – VB 0.0208*

EI – M 50.001*** EI – M 50.001***

EI – VB – EI – VB 50.001***

M – VB – M – VB 0.5699

Mg E – EI 50.001*** E – EI 0.1880

E – M 0.8211 E – M 0.7916

E – VB 0.0442* E – VB 0.0559

EI – M 0.0182* EI – M 0.2109

EI – VB 50.001*** EI – VB 0.0035**

M – VB 0.0831 M – VB 0.2124

Ca E – EI NA E – EI 0.0410*

E – M E – M 0.3201

E – VB E – VB 0.9429

EI – M EI – M 0.0117*

EI – VB EI – VB 0.0464*

M – VB M – VB 0.3679

*p50.05; **p50.01; ***p5 0.001.

Abbreviations: E: epidermal cell wall; EI: inside epidermal cells; M: mesophyll; NA: Not applicable; VB: Vascular Bundle.

174 V. de la Fuente et al.

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Discussion

This is the first report concerning Ni localization in

populations of A. serpyllifolium ssp. lusitanicum and

A. serpyllifolium ssp. malacitanum growing naturally

on ultramafic areas of the Iberian Peninsula.

Present results show that structural features of

the different analysed tissues are preserved, and

that the distribution of Ni in these structures has a

pattern similar to that reported by Kupper et al.

(2001), Broadhurst et al. (2004a) and Mcnear

et al. (2005) in other Alyssum Ni-hyperaccumulator

species.

Both analysed taxa showed a similar Ni distribu-

tion pattern among tissues of the different vegetative

and reproductive organs selected for the study. In

both Alyssum species, the Ni abundance in leaves is

in the following order: inside epidermal cells4 epi-

dermal cell walls4mesophyll and vascular bundle

cells. The large amount of Ni found in epidermal

cells is probably located inside vacuoles, as reported

by Brooks et al., (1981b) in these two Alyssum

Iberian subspecies, and recently confirmed in other

Ni-hyperaccumulating Mediterranean Brassicaceae

(Kupper et al., 2001; Psaras et al., 2000; Broadhurst

et al., 2004a; Mcnear et al., 2005) and species from

other families (Kramer at al. 1997; Bidwell et al.,

2004). Ni was found to be homogeneously distrib-

uted on both epidermal surfaces. If was also

preferentially located in epidermis and cortex stem

tissues although in lower concentrations than in

leaves. The preferential location in external tissues

Figure 2. (A) Ni concentrations in leaf tissues of A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. (B) Ca concentrations

in leaf tissues of A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. (C) Mg concentrations of leaf tissues A. serpyllifolium

ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. (D) Ni concentrations in stem tissues of A. serpyllifolium ssp. malacitanum and

A. serpyllifolium ssp. lusitanicum. (E) Ca concentrations in stem tissues of A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp.

lusitanicum. (F) Mg concentrations of stem tissues of A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. (G) Ni

concentrations in seeds tissues of A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. (H) Ca concentrations in seeds

tissues of A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. (I) Mg concentrations in seeds tissues of A. serpyllifolium ssp.

malacitanum and A. serpyllifolium ssp. lusitanicum. Means and standard errors. All concentrations are expressed in %. Leaf: n¼ 53 – 6; Stem:

n¼34 – 11; Seeds: n¼ 12 – 3; Trichomes: n¼ 17 – 3. Abbreviations: C: cortex; CC: central cylinder; E: epidermal cell wall; EI: inside

epidermal cells; En: endosperm; M: mesophyll; P: pith; T: testa; VB: vascular bundle.

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may indicate the existence of efficient selective Ni

translocation mechanisms.

Previous studies have shown discrepancies con-

cerning the localization of Ni in the trichomes of

Alyssum species. Kramer et al. (1997) suggested that

Ni was preferentially accumulated in the epidermal

trichomes. Psaras et al. (2000) reported that Ni was

excluded from trichomes of various Alyssum species

from Greece. The latter observation was supported by

Kupper et al. (2001) in two Alyssum species, although

these authors pointed to the presence of Ni in the basal

part of trichomes after staining them with dimethyl-

glyoxime. Recent reports on Ni location in Alyssum

showed the existence of large amounts of Ni in

epidermal tissues, thereby emphasizing the presence

of this element, together with Mn in leaf trichomes

(Broadhurst et al., 2004a,b; Mcnear et al., 2005).

In the present study, EDX scans clearly revealed

the presence of Ni in the base of trichomes stalks. Ni

concentrates on the basal surface and in the interior

of the pedicle of trichomes. This distribution is not

easy to observe because it requires that the trichomes

be kept in an inverted position, and not in their

normal position (Psaras et al., 2000), due to the high

Ca content of the cell walls that impedes electron

beam penetration. Ca is the most abundant element

in trichomes, and Ni is distributed under the Ca,

which occupies a more external position. Ni was not

detected in the external thickening of trichome cell

walls, which have a higher Ca concentration when

compared with other morphological structures of the

plant. The existence of calcareous deposits, calcified

cells walls and trichomes are restricted to a compara-

tively small number of families, Brassicaceae

amongst them (Metcalfe, 1983). Accumulation of

other metals (Zn and Cd) in the base of trichomes

has been observed for another Brassicaceae, Arabi-

dopsis halleri L., by Kupper et al. (2000) and Zhao

et al. (2000).

Information regarding the presence of Ni in seeds

is not frequently found (Przybylowicz et al., 1995;

Psaras & Manetas, 2001; Bhatia et al., 2003). We

report here the localization of Ni in the testa and

endosperm of the Iberian Alyssum taxa studied. Ni

concentration in seeds was lower than in vegetative

organs, as previously described (Brooks, 1998;

Psaras & Manetas, 2001).

Different interpretations have been proposed for

metal hyperaccumulation in plants. Several authors

interpreted Ni accumulation as a defense mechanism

against herbivore and pathogen attacks (Boyd &

Martens, 1998). Robinson et al. (2003), among

others, disagree with this interpretation because of

Figure 4. SEM micrograph of a cross section of Alyssum

serpyllifolium ssp. lusitanicum stem. Abbreviations: C: cortex; CC:

central cylinder; E: epidermis; P: pith.

Figure 3. SEM micrograph of an abaxial surface of leaf epidermis of Alyssum serpyllifolium ssp. lusitanicum. The dot maps show the

distribution of Ni (blue), Mg (orange) and Ca (green) by EDXS analyses. For colour figures, please visit http://www.informaworld.com/

TPLB.

176 V. de la Fuente et al.

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the differences in Ni concentration found in the

upper and lower cuticle. Any role that Ni hyper-

accumulation might play in cellular osmotic adjust-

ments and resistance to drought has been ruled out

(Whiting et al., 2003).

The suggestion that Ni hyperaccumulation is a

protective strategy against predation or microbial

pathogenesis (Boyd & Moar, 1999) seems reason-

able. However, the high Ni concentrations found

in some plants, and their homogeneous distribution

in different tissues, offer alternative functional

interpretations, such as protection against environ-

mental stress. In the case of the Alyssum subspecies

examined here, both of which are perennial shrubs

living in extremely xeric habitats, the high concen-

tration and homogeneous distribution of Ni in their

leaves (Figure 3) suggests that it could prevent the

oxidative stress resulting from exposure of the plant

to UV radiation, as recently described for algae

growing under high concentrations of ferric iron

(Gomez et al., 2004). Low-concentration solutions

of two different Ni salts (sulphate and chloride) used

Table II. ANOVA and least significant differences (LSD) between means of Ni, Mg and Ca concentrations in stem tissues from

A. serpyllifolium ssp. malacitanum and A. serpyllifolium ssp. lusitanicum. Means were calculated only if an F – test was significant at the 0.05

level of probability.

A. serpyllifolium ssp. malacitanum A. serpyllifolium ssp. lusitanicum

Student’s t-test [E – C]{

Ni: t-value¼ 0.05; p4 0.05 ANOVA [E – C – CC – P]

ANOVA [E – C – CC – P] Ni: F¼3.23; p50.05

Mg: F¼10.32; p5 0.001 Mg: F¼20.40; p50.001

Ca: F¼ 4.25; p5 0.01 Ca: F¼19.70; p5 0.001

Element Contrasted tissue p Contrasted tissue p

Ni E – C

NA.

E – C 0.1913

E – CC E – CC –

E – P E – P 0.0143*

C – CC C – CC –

C – P C – P 50.001***

CC – P CC – P –

Mg E – C 0.3096 E – C 50.001***

E – CC 0.5922 E – CC 50.001***

E – P 50.001*** E – P 0.3853

C – CC 0.1201 C – CC 50.001***

C – P 50.001*** C – P 50.001***

CC – P 50.001*** CC – P 50.01**

Ca E – C 0.2676 E – C 0.4857

E – CC 0.1358 E – CC 50.001***

E – P 0.0513 E – P 0.4195

C – CC 0.0056** C – CC 50.001***

C – P 0.0039** C – P 0.1337

CC – P 0.3802 CC – P 50.001***

{ANOVA was not applicable in these cases.

*p50.05; **p50.01; ***p5 0.001.

Abbreviations: C: cortex; CC: central cylinder; E: epidermis; NA: Not applicable; P: pith.

Table III. Results of Student’s t-test between means Ni, Mg and Ca concentrations in trichome and seed tissues from A. serpyllifolium ssp.

malacitanum and A. serpyllifolium ssp. lusitanicum.

Trichomes A. serpyllifolium ssp. malacitanum A. serpyllifolium ssp. lusitanicum

Element Tissues t-value p Tissues t-value p

Trichomes

Ni S – B NA S – B NA

Ca S – B 3.05 50.01** S – B 4.08 50.01**

Mg S – B 1.10 40.05 S – B 5.21 50.01**

Seeds

Ni T – E 1.14 40.05 T – E 0.91 40.05

Ca T – E 1.32 40.05 T – E 3.72 50.01**

Mg T – E 8.21 50.01** T – E 0.61 40.05

*p50.05, **p50.01, ***p5 0.001.

Abbreviations: B: base of trichomes stalks; E: endosperm; NA: Not applicable; S: trichome surface; T: testa.

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Figure 5. SEM micrographs of the lower surface of a trichome of A. serpyllifolium ssp. mlacitanum (A), A. serpyllifolium ssp. lusitanicum (B),

A. serpyllifolium ssp. serpyllifolium (C). The dot maps show the distribution of Ni (blue), Mg (orange) and Ca (green) by EDXS analyses. For

colour figures, please visit http://www.informaworld.com/TPLB.

Figure 6. SEM micrographs of: (A) a seed of A. serpyllifolium ssp. lusitanicum. (B) micromorphology of seed epidermal surface of

A. serpyllifolium ssp. malacitanum. (C) cross section of A. serpyllifolium ssp. lusitanicum seed. (D) epidermal wing surface of a seed of

A. serpyllifolium ssp. lusitanicum. Abbreviations: E: endosperm; T: testa.

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to study transmittance showed a high percentage of

absorbance in specific regions of the UV range,

suggesting that an even distribution of this metal on

the leaves might have a protective effect. Work is in

progress to evaluate this additional function for Ni in

hyperaccumulator Alyssum subspecies grown in xero-

edaphic climatic conditions.

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