Download RELATIONS OF HEAVY METAL SEQUESTRATION AND

RELATIONS OF HEAVY METAL SEQUESTRATION AND PRODUCTION OF METAL ION
LIGANDS IN PLANTS UNDER DIFFERENT ENVIRONMENTAL CONDITIONS
Teresa W-M Fan1, Fabienne Baraud2, Andrew N. Lane3, and Richard M. Higashi4
ABSTRACT: Root exudation of metal ion ligands (MIL) is vital to nutritional acquisition of Fe and Zn,
and may also be important to mobilization of metal contaminants by plants. We have developed a
multidimensional NMR and GC-MS approach for broad profiling of exudate and tissue components
including MIL. Amino and organic acids plus mugineic acid (MA) phytosiderophores were identified
and quantified. SH-rich peptides were also analyzed using fluorescent probe and SDS-PAGE. The
MIL profile differed among plants and genotypes. MA exudation by wheat and barley roots was
induced by Fe deficiency, which is consistent with MA’s role in Fe acquisition. Cd treatments of
wheat greatly reduced the exudation of MA and other MIL; yet transition metal sequestration into
roots increased substantially. This suggests that metal uptake may be mediated by a different
mechanism in Cd-contaminated rhizosphere. SH-rich peptides (i.e., phytochelatins) and other MIL
accumulated greatly in Cd-treated wheat tissues, possibly related to the intracellular immobilization of
Cd and transition metals. Moreover, co-treatment with soil humic substance (HS, an important extant
rhizosphere ligand) alleviated Cd-induced loss of wheat biomass and root exudation, while causing a
higher sequestration of Cd and transition metals into roots. This is contrary to HS role as a
competitive chelator.
Vascular plants are known to utilize two different strategies to mobilize Fe(III) from soils (Marschner,
1991). The “Strategy II” plants (graminaceous monocots) release powerful iron chelators into root
exudates to complex Fe(III) for uptake. The iron chelators produced are termed phytosiderophores
(PS), which consist mainly of mugineic acid and its derivatives, 2’-deoxymugineic acid (2’-DMA) and
3-epi-hydroxymugineic acid (3-epi-OHMA) (Kawai et al., 1988). In addition to Fe(III), PS also
complex with Zn(II) and Cu(II) to facilitate their uptake by plants (Römheld, 1991; Hopkins et al.,
1998) However, it is unclear whether PS are involved in the acquisition of heavy metal ions (e.g.
Cd(II), Pb(II), Sr(II), Cs(II)) commonly found in soils contaminated via a variety of industrial, military
and urban activities. Also uncertain is the involvement of exudate components other than PS in metal
ion mobilization, primarily for lack of a comprehensive knowledge of root exudate composition.
Moreover, once absorbed, it is largely unknown how these metal ions (usually toxic to plants) are
translocated to shoots or sequestered inside root cells. Insights into these questions are crucial to the
design and implementation of plant-based remediation of metal contamination from soils and sediments.
Without prior knowledge, analysis of crude plant root exudates requires a profiling approach, for which
we developed a combined nuclear magnetic resonance (NMR) spectroscopy and gas chromatographymass spectrometry (GC-MS) method. We have used this approach for a simultaneous determination of
known, unexpected, and even unknown metal ion ligands (MIL), directly from crude exudates (Fan et
al., 1997 & in press). With this tool in hand, it is now possible to examine various environmental and
genetic factors that influence intra- and extra-cellular MIL profiles, and their role in metal ion
acquisition.
1
Dept. of Land, Air & Water Resources, Univ. of California, One Shields Ave., Davis, CA 95616, USA
ERPCB, Campus II - Sciences 2, Boulevard du Maréchal Juin, 14032 Caen Cedex, France
3
National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
4
Crocker Nuclear Lab., Univ. of California, One Shields, Ave., Davis, CA 95616, USA
2
1
In this report, we analyzed the root exudation profile of several crop species as shown in Figure 1. It is
clear that the exudation profile differed among plant species and genotypes. The barley exudate had 3epi-OHMA and MA as the dominant PS while wheat exudates were rich in 2’-DMA. The relative
quantity of glycinebetaine (GB, an osmolyte), amino acids, and organic acids also differed between the
two species. Rice exudate was very low in PS, which may account for its lower tolerance to Fe
deficiency than barley and wheat. Since many of these exudate components can influence microbial
activities, how species or genotypic differences in root exudate composition affect plant-microbe
interactions and modulate metal sequestration is under study in our laboratory.
Figure 1 – Root exudation profiles
of barley, wheat, and rice analyzed
by 1H NMR. Two wheat genotypes
differing in salt tolerance were
surveyed: salt-sensitive Chinese
spring, CS and
salt-tolerant
amphiploid of CS and European
saltgrass, AgCS.
Tissue metabolite profiles were also analyzed by a combination of 1H NMR, GC-MS, and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Example analysis of thiol-rich
peptides in both plant and animal tissues is shown in Figure 2, where thiol-rich peptides were
derivatized by fluorescent probe bromobimane (BrB) and separated by SDS-PAGE. Fluorescent bands
of < 3.5 kDa were prominent in Cd-treated plant tissues (lanes 6, 8) while a fluorescent band of 17.7
kDa was related to a higher Cd content in the clam tissues (lanes 3, 4, 5).
kDa
25.6
17.7
11.5
6.5
3.5
Figure 2 – SDS-PAGE of bromobimane-tagged SH-rich
peptides and proteins from wheat, rice, and clam tissues.
Lanes 1, MW standards; 2, rabbit metallothionein (MT); 35, clam MT extracts; 6, Cd-treated and 7, control wheat root
extracts; 8, Cd-treated rice shoots.
<3.5
Moreover, we examined the interactive effect of Cd(II) and
soil humate (an important extant rhizosphere MIL) on root
8 7 6 5 4 3 2 1
exudation and internal metabolism in relation to metal
acquisition by CS wheat. Wheat roots responded to Cd treatment by a large accumulation of Cd as
well as decrease in growth (data not shown) and in major exudate components while transition metals
accumulated to a higher level than under control conditions (e.g. Fig. 3). Thus, it is clear that these low
molecular weight MIL including 2’-DMA were not responsible for the enhanced metal acquisition.
This does not rule out the role of macromolecular exudate components (e.g. extracellular
polysaccharides) in facilitating metal uptake. Coupling Cd with humic treatment partially alleviated Cd
inhibition of root growth (data not shown) and exudation of 2’-DMA, Ala, and acetate while
2
R = OH, MA
R = H, 2'-DMA
1
R = R'= OH,
3-epi-OH-MA
acetate
glycinebetaine
COOH- H COO-H H COO1'
1"
epi-OHMA
MAs
MES
succinate
Ala
Gly
lactate
malate
2'-DMA
GAB
MA
CS Wheat Exudate
Barley Exudate
C
C
R'
2
3 C
N
C 3' NH
C 3" OH
H
C
H 2' R
H 2" H
H
H
4
MAs
malate
2'-DMA
Thr
Val
GAB
malate
Rice Exudate
Gly
glycinebetaine
2'-DMA
succinate
lactate
Ala
2'-DMA
2'-DMA
Leu
Val Ile
Glu
AgCS Wheat Exudate
MES
lactate
Unknown
polyol
acetate
Unknown
2'-DMA
2'-DMA
5.0
4.5
4.0
3.5
1H
3.0
2.5
Chemical Shift (ppm)
2.0
1.5
1.0
5.0
2'-DMA
4.5
4.0
3.5
1H
3.0
2.5
Chemical Shift (ppm)
2.0
1.5
1.0
accumulation of Zn, Ni and Cd in roots was slightly enhanced (Fig. 3). These results suggest that soil
humate can modulate both root exudation and internal metabolism.
Exudate Ala vs Fe
1200
Exudate GAB vs Ni
150
60
100
800
Fe
Ala
80
40
400
40
0
Ctl
HS
Cd
20
0
0
Ctl
Cd+HS
Exudate Acetate vs Zn
HS
5000
Zn
acetate
120
250
200
80
Cd
Cd+HS
Exudate 2'-DMA vs Cd
160
350
300
50
Cd
2'-DMA
200
4000
150
3000
150
100
2000
100
250
40
50
1000
50
0
0
Ctl
HS
Cd
Cd+HS
Exudate Malate vs Cu
0
0
Ctl
HS
Cd
Cd+HS
µmole MIL/g root exudate dry wt
0
Figure 3 – Comparison of exudate MIL
concentration versus metal content in CS wheat
roots under a combination of soil humate (HS, 5
ppm) and Cd (5 ppm) treatments. All MIL
declined drastically in exudates while transition
metals accumulated more in roots with Cd and
Cd+HS treatments. The presence of 5 ppm Cd in
growth media resulted in a nearly 1000-fold Cd
accumulation in wheat roots.
To understand the relation of humic-Cd treatment to
intracellular MIL, we analyzed wheat tissue extracts for a
number of metabolites including phytochelatins. Due to
space limitation, only the result for phytochelatins is
shown here in Fig. 4. It is interesting to note that the
combined humic and Cd treatment led to a further increase in PC production in roots than Cd treatment
alone. This enhancement should help immobolize bioavailable Cd to alleviate its inhibitory effect on
growth, as observed in this study.
180
200
160
120
120
Cu
malate
80
60
40
0
0
Ctl
HS
Cd
Cd+HS
6000
Wheat Root
14
5000
12
4000
mg MT eq/g
µg Cd/g root
10
8
3000
6
2000
µg Cd/g root
mg MT equivalent/g root
16
4
1000
2
0
0
Ctl
0.5
Cd
HS
HS+Cd
180
Wheat Shoot
160
0.4
140
mg MT eq/g
µg Cd/g shoot
0.3
120
100
80
0.2
60
40
0.1
20
µg Cd/g shoot
mg MT equivalent/g shoot
µg Metal/g root dry wt
80
Ni
GAB
120
Figure 4 – Increase in Phytochelatin (PC) production in CS
wheat tissues in response to humic and Cd treatments. PC was
analyzed as in Fig. 2 and calibrated against rabbit
metallothionein.
Triplicate samples were analyzed per
treatment. PC accumulated dramatically in wheat roots in
response to Cd, which was accompanied by a large accumulation
of Cd.
Soil humic treatment further enhanced this PC
production and, to a less extent, Cd accumulation in wheat roots.
Both increases were proportionally much less in Cd-treated
shoot, while humic-Cd treatment did not lead to further increase
in shoot Cd. Thus, Cd was mainly sequestered in wheat roots,
mostly likely by interacting with PC, and soil humate may
enhance this process.
0
0.0
Ctl
Cd
HS
HS+Cd
In conclusion, root exudation varied with plant species, genotypes, and treatment conditions.
Exudation of PS or common organic or amino acids did not appear to be related to the acquisition of Cd
or Cd-induced acquisition of transition metals. Soil humate modulates both root exudation and internal
root metabolism to help immobolize available Cd, thereby alleviating its inhibitory effect on growth.
REFERENCES
Marschner, H. in Plant roots : the hidden half; Waisel, Y.; Eshel, A.; Kafkafi, U., Eds; M. Dekker: New York, 1991, pp
503-526.
Kawai, S.; Takagi, S.; Sato, Y. J. Plant Nutr. 1988, 11, 633-642.
Römheld, V. Plant and Soil 1991, 130, 127-134.
Hopkins, B. G.; Whitney, D. A.; Lamond, R. E.; Jolley, V. D. J. Plant Nutr. 1998, 21, 2623-2637.
Fan, T. W. M.; Lane, A. N.; Pedler, J.; Crowley, D.; Higashi, R. M. Analyt. Biochem. 1997, 251, 57-68.
Fan, T. W. M.; Lane, A.N.; Shenker, M., Bartley, J.P.; Crowley, D.; and Higashi, R.M. Phytochem., in press.
3
I
II
H+
reductant
?
PS
Soil
Fe3+–PS
?
Humic
Fe2+
?
FeR
Fe2+
Fe3+
?