Plant Science 128 (1997) 131 – 140
Influence of cadmium on soluble carbohydrates, free amino
acids, protein content of in vitro cultured Lupinus albus
Guy Costa *, Emmanuelle Spitz
Laboratoire de Biologie Cellulaire Végétale et Valorisation des Espèces Ligneuses, Faculté des Sciences, 123,
A6enue Albert Thomas, F-87060 Limoges, France
Received 19 August 1996; received in revised form 6 June 1997; accepted 6 June 1997
Abstract
The objective of this study was to evaluate the consequences of Cd stress on soluble carbohydrates, free amino
acids, proteins, CO2 fixation, stomatal conductance, intercellular CO2 and water potential of white lupin (Lupinus
albus L. cv. lublanc) in vitro cultures. Plants were grown for 15 days in axenic condition in medium containing
variable CdNO3 concentrations (0, 0.01, 0.1, 1, 10, 100 mM Cd). Cd treatment did not cause significant dry weight
differences between Cd-treated and control plants except above 0.1 mM Cd. At lower Cd concentrations, total soluble
carbohydrate and total free amino acid of Cd-treated plants increased, but decreased above 0.1 mM Cd. In contrast
raffinose, mannose and hydroxyproline levels increased with Cd concentration. © 1997 Elsevier Science Ireland Ltd.
Keywords: White lupin; Photosynthesis; Raffinose; Hydroxylisine; Proline; Mannose
1. Introduction
Effects of toxic trace metals on vegetation have
been reported in regions having high emissions,
like in mining areas, around industrialised regions
Abbre6iations: A, net CO2 assimilation rate; Cd, cadmium;
Ci, intercellular CO2 partial pressure; DW, dry weight; Gs,
stomatal conductance.
* Corresponding author. Tel.: + 33 555457481; fax: + 33
555457386; e-mail: gcosta@unilim.fr
and in agricultural soils contaminated by phosphoric fertilizers and/or sewage sludge [1,2]. It is
known that Cd2 + ions can be readily absorbed by
roots and translocated into the leaves of several
species [3]. High level metal accumulation causes
phytotoxicity that interferes with several physiological processes [4– 6].
One of the primary effects of toxic Cd-concentrations on plants is a reduction in growth [7].
This may be due to a reduction of cell water
content and/or on cell wall elasticity [8,9,6]. In
0168-9452/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 1 4 8 - 9
132
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
this condition and in comparison with other
stressors, Cd adapted plant cells exhibit a number of physiological changes including considerable osmotic adjustment which involves
accumulation of ions and organic solutes [9,10],
vacuole compartmentalisation of ions [10] and
Cd sequestration by specific molecules such as
phytochelatins and/or metallothioneins [11– 15].
Inhibition of photosynthesis by high Cd-concentrations is well documented [16– 18], but there is
little information on the effects of low-Cd concentrations on photosynthesis, water relations
and primary plant metabolism of compounds
such as carbohydrates, amino acids and proteins.
Changes in carbohydrate and sugar levels could
be associated with osmotic regulation of guard
cells that control stomatal movement and regulate plant water flux [19]. Moreover, carbohydrate accumulation or deprivation have been
directly correlated with modifications of photosynthetic processes. As indicated by Moya et al.
[20] little is known about the effects of Cd on
carbohydrate and sugar content. Cadmium stress
could be equally manifested as protein degradation, via amino acid catabolism resulting from a
general reduction of plant development. In this
regard, osmotic adjustment has been associated
with increase in proline, and polyamine contents
of plant cells [21,22]. Except for the work of
Kastori et al. [23], no published study has
demonstrated effects of Cd-stress on amino acid
content and more particularly on proline accumulation after Cd exposure. Here we show that
plant adaptation to heavy metals results in an
increase in low molecular compounds.
In this study, we used young white lupin
seedlings for analysing the effects of various Cd
concentrations on several physiological characteristics and on accumulation of low molecular
weight soluble compounds. Lupin seedlings were
used because of their adaptability to adverse environments and their high Cd tolerance [24,25].
In order to simulate how Cd affects agricultural
crops and natural plant communities, experiments were conducted at concentrations of available Cd that may occur in field soil solutions as
suggested by Cataldo et al. [26].
2. Materials and methods
2.1. Plant culture
Seeds of Lupinus albus L. cv lublanc were rinsed
with distilled water and washed with 95% ethanol
for 5 min. Then seeds were surface sterilized with
0.7% Ca(ClO)2 for 20 min and rinsed three times
with sterile distilled water. Germination was performed in the dark at 25°C on sterile water solidified with 1% agar for 12 h. Germinated
seedlings were transfered to the sterile nutrient
medium containing 4 mM of Ca(NO3)2, 2 mM of
KCl, 0.5 mM of MgSO4, 0.5 mM of KH2PO4, 3
mM of Mo + , 80 mM of B3 + , 9 mM of Mn2 + , 10
mM of Cu2 + , 3.5 mM of Zn2 + and 10.3 mM of
Fe (as Fe-EDTA). Nutrient solution was supplemented with variable Cd concentrations. Cadmium was added, as CdNO3, at concentrations
0 – 100 mM. The Cd solution was not autoclaved,
but rather filtered (0.45 mm pore diameter), to
avoid any precipitation due to pH changes during
autoclaving. The pH of the media was adjusted to
5.8 and solidified with 0.2% phytagel (Sigma).
Plants were placed in growth chamber (16 h light,
350 mE m − 2 s − 1, 25°C, 75% relative humidity)
and grown for 15– 17 days.
2.2. Cadmium analysis
The dry weight of roots and shoots was measured after 48 h desiccation at 70°C. Cd concentrations are presented as the mean of three plant
determinations per treatment. Dry roots were
washed at 450°C for 5 h in a furnace. Ashes
were dissolved in 14N HNO3 and evaporated at
60°C. The pellets were redisolved in HNO3 5%
and analysed by atomic absorption spectrophotometry (Perkin-Elmer model 503 with a
graphite furnace HGA 74).
2.3. Net photosynthesis
The rate of net photosynthesis (A) and stomatal conductance (Gs) of the intact leaf were determined using a Li-cor 6200. CO2 uptake and
transpiration were measured at 900 mmol m − 2
s − 1 light flux 1 h after plant acclimation in open
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
flask. The initial CO2 concentration was approximately 400 vpm. Relative humidity and room
temperature were constant for all measurements
(75% relative humidity, 25°C). Gas exchange was
measured on three plants per treatment. Water
relations were determined using a pressure chamber as described by Schoelander et al. [27].
2.4. Soluble carbohydrate and sugar analysis
Soluble carbohydrates and sugar were extracted
from lyophilized tissue at 80°C for 20 min using
80% ethanol containing mannitol as an internal
standard. The extract was centrifugated at
12 000×g for 15 min. The supernatant was
lyophilized (70 mTor, 6 h) and the pellet was
133
solubilized in 400 ml distilled water then filtered
(0.2 mm pore diameter, Sartorius Minisart RC4).
Carbohydrates were separated by HPLC (Beckman, System Gold) with a refractive index detector (Beckman, Refractive Index Detector 156).
Carbohydrates were purified by passage through
anionic and cationic pre-columns (Merck, France)
and were then separated with a polyspher CHPB
column (Merck, Pre-packed column RT300-7,8).
Starch was hydrolysed to glucose by amyloglucosidase at pH 4.6 (Sigma, France) and the glucose quantified by HPLC.
2.5. Amino acid analysis
Lyophilized tissues were homogenised at 4°C
with 70% methanol and the homogenate centrifuged at 12 000×g for 20 min. The supernatant
was filtered (0.2 mm pore diameter, Sartorius
Minisart RC4) and lyophilized (70 mTor, 30 min).
Free amino acids were determined as their stable
PITC derivatives [28,29]. Separation utilized a C18
column (Merck, EC240-4, 250 mm, 5 mm pore
diameter) and detection was at 269 nm (Beckman,
Detector module 168). Amino acids were
quantified by comparison with a solution containing free amino acids standard (Sigma, physiological basic, acid and neutral amino acid)
supplemented with 500 pM glutamic (Sigma).
2.6. Protein analysis
Fig. 1. Biomass production (A) and Cd content (B) of Lupinus
albus as a function of Cd concentration after 15 – 17 days of
Cd-treatments. () root dry weight, () shoot dry weight.
Each point represents the mean of three replicates; bars = S.D.
Samples were powdered in liquid N2 with a
mortar and pestle. The powder was homogenized
in
cold
acetone
(−20°C)
containing
trichloroacetic (TCA) (10%, w/v) and b-mercaptoethanol (0.07%, v/v), and proteins were precipitated at − 20°C for 45 min. After centrifugation
at 10 000×g for 30 min, supernatants were discarded, and pellets were washed with 1 ml of cold
acetone (− 20°C) containing b-mercaptoethanol
(0.07%, v/v). After a second centrifugation
(10 000× g for 30 min), the solution was discarded and pellets were dried over night under
vacuum.
Pellets were resuspended in Laemmli [30] buffer
and boiled for 5 min to solubilize the proteins.
After centrifugation at 10 000×g for 3 min,
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
134
Table 1
Net photosynthesis, stomatal conductance (Gs), internal CO2 concentration (Ci) and water potentiel (y) in Lupinus albus after 15
days exposure to different levels of Cd
Cd concentration (mM)
0
16.2 91.1
Net photosynthesis (mmol CO2
m−2 s−1)
Stomatal conductance (mmol H2O 0.384 9 0.11
m−2 s−1)
424915
Intercellular CO2 partial pressure
(ml l−1)
Water potential (MPa)
−0.6 9 0.2
0.01
0.1
1
10
100
17.3 9 0.5
16.4 9 0.8
13.1 9 0.4
0.451 9 0.2
0.4059 0.09
0.298 9 0.15
0.1759 0.1
0.042 9 0.01
4219 25
378 9 21
3189 20
241 9 17
105 9 9
−0.6 9 0.2
−1 9 0.3
−1.56 9 0.2
−2.5 9 0.4
−4.29 0.4
9.8 9 0.2
2.41 90.1
Values expressed as 9S.E.M. based on five estimations.
protein contents were estimated by the Bradford
procedure (Bio-Rad, Protein Kit) [31] with bovine
serum albumin serving as standard.
2.7. Data analysis
Each experiment was conducted three times.
The data presented are from a representative experiment. Statistical calculations were performed
with SYSTAT Macintosh computer program [32].
nificantly lower than in control at the highest Cd
concentration (100 mM Cd). Stomatal conductance (Gs) increased with Cd concentrations up to
0.1 mM Cd, but decreased above this concentration (Table 1). For 100 mM Cd, Gs was about 10%
of the control level. We also observed a decrease
in internal CO2 concentration (Ci) (Table 1).
Water potential decreased continously with an
elevated Cd concentration above 0.01 mM (Table
1).
3.3. Soluble carbohydrate content
3. Results
3.1. Plant growth and Cd content
Biomass production of Cd-treated plants decreased above 1 mM Cd. Cadmium effects on dry
matter were more pronounced for shoots than for
roots (Fig. 1A). Cadmium accumulation increased
with increased Cd concentration in the medium
and reached a maximum level in 100 mM Cd
exposed seedling. Root Cd contents were higher
than that of shoots (Fig. 1B).
3.2. Net photosynthesis, stomatal conductance and
water potential
Net photosynthesis was constant for Cd concentrations lower than 0.1 mM Cd, but declined
with higher Cd concentrations in nutrient solution
(Table 1). The rate of CO2 assimilation was sig-
The concentration of fructose, glucose, sucrose,
starch, mannose and raffinose in 15– 17 days old
roots and shoots are show in Table 2. Starch in
shoots and sucrose in roots represented the most
abundant carbohydrates in control plants. ManTable 2
Soluble carbohydrate content of Lupinus albus for control
plant
Soluble carbohydrate
Roots (mg g−1 DW) Shoots (mg g−1 DW)
Starch
Fructose
Glucose
Sucrose
Mannose
Raffinose
109 2.1
39 1.2
159 3
249 4.1
29 0.7
0.4 9 0.1
21 9 1.8
891
14 9 2.7
18 9 3.8
1 9 0.5
0.4 9 0.1
DW, dry weight. Values expressed as 9 S.E.M.
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
135
Fig. 2. Soluble carbohydrate contents of Lupinus albus as a function of Cd concentration in the medium after 15 – 17 days of
Cd-treatment. (A) raffinose, (B) mannose, (C) fructose, (D) glucose, (E) starch and (F) sucrose content in () roots and () shoots
of Cd-treated plants. Each point represents the mean of three replicates; bars = S.D.
nose and raffinose were present at lower levels in
both organs.
The addition of Cd to the nutrient solution
decreased fructose, glucose, sucrose and especially
shoot starch content with in a Cd concentration
dependent manner (Fig. 2C, D, E, F). At 0.1 mM
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
136
Cd, shoot starch content was significantly higher
than that in control plants (P B0.05) and represented a 170-fold increase (Fig. 2E). Raffinose
(Fig. 2A) and mannose (Fig. 2B) increased with
increasing Cd concentrations. The effects of Cd
on raffinose and mannose contents were similar
for both organs (root and shoot).
3.4. Free amino acid content
Soluble amino acids of control roots and shoots
are shown in Table 3. The most abundant amino
acids in roots and shoots were glutamate, glutamine, alanine, arginine, tyrosine, valine, methionine, cysteine, isoleucine and leucine.
Tyrosine, valine, methionine alanine and arginine
represented aproximately 72% of the total soluble
amino acid. Alanine and arginine, but not glutamine were more abundant in roots than in
shoots.
Table 3
Amino acid content of Lupinus albus for control plants
The addition of Cd to the medium induced
modifications in the amino acid composition. The
influence of Cd on amino acids in roots and
shoots can be divided into three groups. One
group represented amino acids which increased
with Cd, the second group of amino acids which
decreased and the third group which was not
affected by increased Cd concentrations. Amino
acid which were the most affected by elevated Cd
concentrations are shown in Fig. 3. These six
amino acids can be devided into two sub-groups.
The first sub-group contained glutamate, cysteine
and glycine. Their levels increased for Cd exposures lower than 1 mM Cd. Higher Cd concentrations caused decreased levels. Amino acids of the
second sub-group, consisting of hydroxylysine,
proline and asparagine, increased with elevated
Cd concentrations. Maximum concentrations
were found for 100 mM Cd-treatment. In the case
of proline, the root level was not affected by
elevated Cd in the medium.
3.5. Protein content and composition
Amino acid
Roots (nmol g−1 DW)
Asp
Glu
HPro
Gln
Ser
Asn
Gly
His
Cit
Thr
Ala
Arg
Pro
Tyr
Val
Met
Cys
Ile
Leu
HLys
Phe
Orn
Try
Lys
224.1 9 31
394.5 9 41
29 9 10
3.19 1
26.69 5
115.49 19
77.39 12
232.4 9 31
11 9 1
45.8 9 9
707.5 9 79
638.2 9 81
131.3 9 34
981.7 9 106
305.4 9 49
930.9 9 124
530.4 9 112
246.9 9 56
269.1 9 87
34.2 9 10
268.4 9 68
37.1 9 8
34.4 9 3
32.7 9 8
Values are expressed as 9 S.E.M.
Shoots (nmol g−1 DW)
108.2 9 21
106.3 9 45
27.8 9 10
464.4 9 49
131.8 9 34
175.6 9 19
167.5 9 12
691
709 1
77.69 9
16.69 11
21.2 9 12
1269.5 9106
460.4 949
1203.7 9124
19.8 911
371.7 956
191.1 987
47.4 910
37.9 918
0
19.6 9 3
106.9 9 8
Fig. 4 represents the variation of protein content in roots and shoots of control and Cd-treated
plants. Protein content for the both organs was
affected by Cd concentration above 1 mM Cd.
Effects of high Cd concentrations on protein content were more pronounced for roots than shoots.
4. Discussion
The overall objective of this study was to determine if Cd caused modifications on photosynthetic rate, water status, carbohydrate, amino acid
and protein contents of in vitro cultured white
lupin plants.
Our results demonstrate that lupin seedlings
growing with low-Cd concentrations tended to
increase biomass production as has been shown in
lettuce seedlings [33] suggesting a dose response
effect of Cd. This increase of biomass production
was associated to an accumulation of Cd in root
and shoot tissues but no effects on CO2 assimilations rate. At concentrations above 1 mM Cd,
biomass, photosynthesis rate, stomatal conduc-
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
137
Fig. 3. Amino acid contents of Lupinus albus as a function of Cd concentration in the medium after 15 – 17 days of Cd-treatment.
(A) glutamate, (B) hydroxylysine, (C) cysteine, (D) proline, (E) glycine and (F) asparagine content in () roots and () shoots of
Cd-treated plants. Each point represents the mean of three replicates; bars = S.D.
138
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
tance and water potential were reduced as shown
previously for soybean, bean and lettuce [6,33].
Cd also disturbed root morphology as show in
Fig. 5. At the highest Cd concentration the metal
reduced secondary root growth by a modification
of secondary root branching and root geotropic
response.
Cadmium treatment also modified soluble carbohydrate, free amino acid and protein accumulation and distribution in lupin plants. At Cd
concentrations lower than 0.1 mM, the metal increased carbohydrate and amino acid levels measured in shoots, but not in roots. At
concentrations higher than 0.1 mM, our data
showed a decrease of the amount of all primary
metabolites measured. This data corroborates the
finding of Gerger and cowokers [7,34,35] and
could be explained by the reduction of CO2 fixation in Cd-treated plants. But for lower Cd concentrations, the photosynthetic measurements
were not affected by treatment and thus could not
explain the carbohydrate increase.
More than total carbohydrate level, Cd-treated
plants exhibited marked increases in specific sugars such as raffinose, and mannose. This effect
Fig. 4. Total protein content of Lupinus albus as a function of
Cd concentration in the medium after 15 – 17 days of Cd-treatments. () roots and () shoots. Each point represents the
mean of three replicates; bars = S.D.
Fig. 5. Lupinus albus plants after 15 – 17 days culture in
absence (control) and in presence of variable Cd concentrations in the medium. Note modified root growth in 100 mM
Cd exposed seedling.
may be related to an increase of root and shoot
necrosis discharging free radicals. In fact, formation of raffinose was produced to remove toxic
galactose that may be released from galactolipids
through free radical mediated by membrane destruction that was one of the effects of Cd in
Cd-treated plant [36,37]. This hypothesis was supported by amino acids data in Cd-treated plants.
The increase of hydroxylysine and proline could
be explained by an active proteolysis of cell wall
glycoproteins [38,39].
The amino acids Q, G and C had similar evolution with the increase of Cd concentration in
solution. They exhibited the higher level at a
maximum Cd concentration where any significant
reduction of growth was demonstrated. We suggested that these amino acids participated to the
detoxification processes by themselves or by the
way of biosynthesis of chelating peptides
[11,12,15].
G. Costa, E. Spitz / Plant Science 128 (1997) 131–140
Acknowledgements
Seeds of L. albus cv. lublanc were provided by
Dr Duc, Institut National de la Recherche
Agronomique (INRA-Dijon), Department of Soil
Microbiology.
[17]
[18]
[19]
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