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.
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