Metal Uptake and Thiol Production in Spirodela polyrhiza (L.) SP20

• JOUR.AL OF •

J Plant Physiol Vol. 154. pp. 634-640 (1999)

Plani Pllys~oiOIJ

http://www. urbanfischer.de/journals/jpp

© 1999 URBAN & FISCHER

Metal Uptake and Thiol Production in Spirode/a polyrhiza (L.) SP2o SANJULA PANDEY 1, SINGH 1

*

R. K. AsTHANA\ ARviND M.

KAYASTHA2 , NEETU SINGH\

1

Centre of Advanced Study in Botany,

2

School of Biotechnology, Banaras Hindu University, Varanasi-221 005, India

and S. P.

Received June 2, 1998 ·Accepted October 26, 1998

Summary

Spirodela polyrhiza (L.) SP20 was hypersensitive to Cu (1-100 J.Lmol!L) compared with Ni (25500 J.Lmol/L). Lower Cu concentration (lJ.Lmol/L) or Ni (25J.Lmol/L) stimulated general growth of the test organism compared with toxicity at elevated concentrations of both of the cations. Cu uptake was very fast during the first 10 min and remained stable irrespective of the extension of incubation. Ni uptake also showed concentration- and time-dependence, with saturation only at 40 h. If compared at equimolar concentration (100 J.Lmol/L, each), Cu accumulated to the extent of 2.7 mgg-1 dw, a value in close proximity with that ofNi (2.84 mgg-1 dw). At lower concentrations, both of the cations showed high accumulation in root; however, the root/top ratio for either Ni or Cu decreased at elevated concentrations, indicating that the metal(s) could be translocated only after a threshold limit of accumulation in the root. The hypertoxic Cu was also the effective inducer of thiol biosynthesis in the test plant as 10 J.Lmol/L Cu increased it to 15J.Lmol thiol g-1 dw (10 h) over the metal-less control (10.26J.Lmol). A 10-fold higher Ni concentration (100 J.Lmol/L) was, however, needed to achieve the same level of thiol. Thiol biosynthesis was also correlated with the intracellular buildup of Cu or Ni.

Key words: Spirodela polyrhiza, Cu, Ni toxicity, uptake, metal compartmentalization, thiol Abbreviations: dw = dry weight; fw = fresh weight; Chl = chlorophyll. Introduction

The inherent capacity of duckweeds to selectively accumulate certain chemicals has been variously exploited to deploy such aquatic plants as biological monitors (Ray and White, 1976; Nasu and Kugimoto, 1981; Klaine, 1985; Wang and Williams, 1990). Such an aspect also created interest in investigating the plant response to various heavy metals in terms of general growth, differentiation, metal accumulation and its translocation and partitioning into different plant parts (Wolverton and McDonald, 1978; Taylor and Crowder, 1983; Schreinemakers and Dorhout, 1985; Xylander et al., 1993).

* Corresponding author.

Whereas metal-induction of phytochelatin, histidine or oxalate production has been documented in terrestrial plants (Steffens, 1990; Reddy and Prasad, 1990; Kramer et al., 1996; Ma et al., 1997), aquatic plants seem to be little studied with the exception of the report on Cd-binding proteins or Cd-thioneins in water hyacinth (Fujita, 1985) or phytic acid binding with Zn in one of the duckweeds (Van Steveninck, 1990). As cellular thiols are the building blocks of most of the complexing ligands, there is need to monitor the extent of their synthesis in plants against heavy metal stress and to elucidate whether such precursor molecules can also act as parameters in the assessment of metal toxicity/accumulation. This paper reports on the toxicity/uptake of Cu and Ni, metal translocation in different plant parts and the level of intracellular thiol as affected by the two metal cations. 0176-1617/99/154/634 $ 12.00/0

Metal Uptake and Thiol Production in Spirodela

Materials and Methods Plant material Stock cultures of locally isolated Spirodela polyrhiza (L.) SPzo were maintained under continuous white light (14.4 Wm- 2 , fluorescent) at 25 ± 1 oC in half strength Bonner and Devirian medium (1939): 0.085gL-1 KN0 3, 0.242gL-1 Ca(N03)z-4H 2 0, 0.02gL-1 KH 2 P04, 0.042 g L- 1 MgS04 · 7H 2 0, 0.061 g L- 1 KCl, 1 mg L- 1 H 3B03, 1 mg L- 1 ZnS04 · 7H 2 0, 0.1 mS L- 1 MnS04 · H20, 0.03 mS L-1 CuS04 · 5H 20, 0.025 mg L- Na2Mo04 · 2Hz0, 4 mg L- Fe(C6Hs0 7) • 3H2 0. The nutrient medium was supplemented with sucrose (1 %).

Metal sensitivity test Two frond colonies of S. polyrhiza (6-8 days old) were transferred to 1/10 strength of growth medium (pH 6.2) containing either Cu (l-100Jlmoi!L) or Ni (25-500Jlmoi!L) at a common biomass of 2 g (fresh wt) in 100 mL. Dilution of the growth medium was to avoid metal complexation at elevated levels of medium ingredients in view of the previous report (Laddaga and Silver, 1985). Mter 24 h of metal exposure, plants were repeatedly washed with sterile double distilled water, reinoculated into fresh (half strength) growth medium (metal-free) and frond number and root length determined after 6 days.

Metal uptake Equal biomass (2 g each) of S. polyrhiza was added to 100 mL medium (1/10 strength) containing Cu (1-100 Jlmoi!L) or Ni (25500 Jlmoi!L) separately, and samples taken out at selected intervals with respect to the rwo metals. Distribution of Cu and Ni in root and top fractions was monitored in the excised roots and top from the intact plants exposed to the respective metal. Plants or plant parts were rinsed of the sorbed or exchangeable cations by washing with 10 Jlmoi!L EDTA before acid digestion with HN03: HCl04 mixture (10: 1, v/v) for metal quantification. Metals (Cu and Ni) were estimated by atomic absorption spectrophotometry (Perkin Elmer model 2380, USA) at 232 nm for Ni and 325 nm for Cu, respectively. Metal content is expressed as mg g- 1 dw.

Dry weight determination Intact plants or the separated top as well as roots were incubated at 60 oc until the weight remained constant.

Chlorophyll determination Chlorophyll in the photosynthetic portion (top) of S. polyrhiza was extracted in 80 % acetone after 6 day exposure of plants to graded concentrations of either Cu or Ni in growth medium (1/10 strength). The total chlorophyll (a+ b) content was calculated according to the formula of Witham et a!. (1971) and expressed as mg Chlg-1 dw.

Thiol determination S. polyrhiza plants exposed to graded concentrations of either Cu (1-100 Jlmoi!L) or Ni (25-500 Jlmoi!L) in 1/10 strength growth medium were harvested during 5-40 h of incubation. Thiol was assayed in the Spirodela extract supernatant using Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid); DTNB), Sigma, USA (Ellman, 1959). DTNB (6mmoi!L in 0.1 moi!L phosphate buffer, pH

635

7.5) was allowed to react with the thiol present in the supernatant procured by grinding plants with mortar and pestle, followed by cold centrifugation (10,000 gi" 10 min). The yellow colour formation due to 2-nitro-5-mercaptobenzoate was measured spectrophotometrically (412 nm) on a UNICAM UV2 spectrophotometer. The extinction coefficient (e) at 412 nm is reported to be 1.36x 104 mol! L- 1 em - 1• Thiol is expressed as Jlmol g- 1 dw.

Statistical analysis All experiments were carried out in triplicate with standard errors represented as bars wherever necessary. Least significant difference (LSD) = to.os ~2s 2I r to.os =Tabular value for degree of freedom for error i = Mean square for error r = Number of replicates

Results

Metal sensitivity and plant growth S. polyrhiza plants starting with an initial two fronds finally reached the four frond stage during 6 days in the growth medium (Table 1). The subsequent data pertain to target plants pre-exposed to Cu (1-100 J.tmol/L) or Ni (25500 Jlmol/L) for 24 h and resuspended in metal-free medium. The frond number did not increase beyond 3 for plants exposed to increasing Cu concentrations (5-50 Jlmol!L), and the highest concentration (100 J.tmol/L) resulted in yellowing of fronds. Although the metal-less control and 1J.tmol/L sets had a common number of four fronds per plant, one was always smaller in size in the former case. The lowest Ni concentration (25J.tmol/L) and control shared a common number of four fronds, but in the higher range (50- 300 Jlmol/L) the frond number did not increase beyond three, and interestingly one was smaller than the rest. Still higher concentrations (400 and 500 Jlmol Ni/L) caused yellowing of fronds. Root development was stimulated at lower Cu doses (120 Jlmol/L); however, 25 J.tmol and control sets compared well with each other. Root growth decreased by 20 % in 50 Jlmol Cu/L and by 90 % in 100 Jlmol/L. Likewise, Ni at lower doses also favoured root development, as evident from an increase (10 %) in 25J.tmol!L although it declined slightly for 50 Jlmol/L, a value comparable to the metal-less control. Root development decreased by 20% in 100 J.tmol Ni/L but there was only 60 % reduction in the higher range (200500 Jlmol/L). Cu (1J.tmol!L) showed stimulated chlorophyll biosynthesis (1.1-fold) over the metal-less control sets followed by an insignificant decline between the 5-20 J.tmol Cu/L range, which increased to an average 12 % for 25 and 50 J.tmol/L Cu. The yellowing of S. polyrhiza fronds in 100 J.tmol/L Cu corresponded with the 29 % decrease in chlorophyll amount (0.043 mg Chi g-1 dw). The lowest Ni concentration (25 Jlmol/L) also stimulated chlorophyll biosynthesis (1.3fold) but was little altered between 50-400 J.tmol/L. The yellowing of the fronds in 400 and 500 J.tmol/L Ni also corresponded with the 15% and 43.33% decrease in chlorophyll amount, respectively.

636

SANJULA PANDEY, R. K. AsTHANA, ARvrND M. KAYASTHA, NEETU SrNGH, and S. P. SrNGH

Table 1: A comparison of frond and root development in 5. po!yrhiza pre-exposed to varying concentrations of Cu or Ni (24 h). Cu (J.Lmol!L)

Frond number

Root length (em)

Total Chi** (a+b) (mg Chi g- 1 dw)

Ni (J.Lmol/L)

Frond number

Root length (em)

Total Chi** (a+b) (mg Chi g- 1 dw)

Control

3+ 1.00* ±0.15 4.00 ±0.12 3.00 ±0.12 3.00 ±0.15 3.00 ±0.15 3.00 ±0.12 3.00 ±0.09 2.00 ±0.08

1.00 ±0.04 1.80 ±0.05 1.30 ±0.04 1.30 ±0.04 1.10 ±0.03 1.00 ±0.02 0.80 ±0.04 0.10 ±0.005

0.060 ±0.003 0.066 ±0.002 0.060 ±0.003 0.057 ±0.002 0.057 ±0.002 0.053 ±0.002 0.053 ±0.002 0.043 ±0.002

Control

3+ 1.00* ±0.15 4.00 ±0.08 2+ 1.00* ±0.06 2+ 1.00* ±0.06 2+ 1.00* ±0.08 2+ 1.00* ±0.06 2.00 ±0.06 2.00 ±0.10

1.00 ±0.04 1.10 ±0.06 1.00 ±0.05 0.80 ±0.03 0.40 ±0.01 0.40 ±0.01 0.40 ±0.02 0.40 ±O.Dl

0.060 ±0.002 0.079 ±0.002 0.055 ±0.001 0.054 ±0.002 0.052 ±0.002 0.051 ±0.001 0.051 ±0.002 0.034 ±0.001

5 10 20 25 50 100

25 50 100 200 300 400 500

* Frond smaller in size. ** Total chlorophyll was estimated following 6-day growth of plants in varying concentrations of Cu or Ni.

Metal uptake The pattern of Cu uptake in a wide range of concentrations (1-100 11mol/L) indicates that the process was concentration- and time-dependent (Fig. 1). It is apparent that a major part of Cu uptake is over within 1 min for all of the concentrations, with saturation at 10 min. The two lowest Cu concentrations (1 and 5 11mol/L) showed uptake by S. folyrhiza corresponding to 0.025 and 0.08 mg Cu2 + g- dw, respectively. An increase to 10 11mol CulL also raised the uptake to almost 10-fold (0.22 mg Cu 2 + g-1 dw). Likewise, higher uptake was also evident for 20 or 25!lmol!L, and the 3.0

~

'0

"101

"'E z

+

N

5

0

30 MINUTES

T

-"l

~ 1.2 "0

~

60 5

; ;

40

80

HOURS

Fig. 2: Ni uptake in 5. polyrhiza in 25 J.Lmol!L (•--•), 50 J.Lmol! L (x--x), 100 J.Lmol/L (\7--\7), 200 J.Lmol!L (~--~), 300 J.Lmol!L (0--0), 400 J.Lmol!L (e--e) and 500 J.Lmol!L (.6-.6)Ni.

OOI 01

i'

E

"':::~

u

T

0.6

0

0.5

'1---'

HOURS

24

Fig. I: Cu uptake in 5. po!yrhiza in 1 J.Lmol/L (0--0), 5 J.Lmol!L (.6-.6), 10 J.Lmol!L ( • - • ) , 20 J.Lmol!L (0-0), 25 J.Lmol!L (•--•), 50 J.Lmol!L (x--x) and 100 J.Lmol!L (\7-V)Cu.

values showed close proximity (0.47 and 0.63 mgCu 2 + g - 1 dw, respectively). A 50-fold increase in Cu concentration (50 11mol/L) under comparable duration (10 min) raised the uptake to 44-fold (1.1 mg Cu 2 + g-1 dw). A proportionate increase in Cu uptake (2.68 mg g - 1 dw) was also observed for the highest Cu concentration (100 !lmol!L). As all of the Cu uptake values did not fluctuate beyond 10 min, it is suggested to indicate the saturation time irrespective of the concentration applied. Ni uptake by S. polyrhiza was also concentration- and timedependent (Fig. 2), and 1 min was crucial as in the case of Cu. Compared with Cu, Ni uptake continued until saturation at 40 h. The two lower concentrations (25 and 50 11mol/ L) showed low uptake rates at 20 min, with an average of

637

Metal Uptake and Thiol Production in Spirodela

0.04 mg Ni 2 + g- 1 dw min - 1 while for 100 and 200 fmol/L Ni it reached an average of 0.11 mg N?+ g- 1 dw min- . The concentration-dependent nature of uptake was also evident as for higher concentrations (300 and 400 ~mol/L) during comparable time ~20 min), th~ ~ptake rat~ reache~ an average_ of 0.25 mg N12+ g- 1 dw mm . The h1ghest N1 concentration (500 ~mol!L) at the same duration raised the uptake rate to 0.34mg Ni2 + g-1 dwmin-1• Ni uptake, if compared with that of Cu at a common concentration (100 ~mol/L) and duration (1 h), indicated that the former followed a course of steady rise to reach 2.14 mg Ni2 + g- 1 dw, a much lower value compared with the latter (2.7mgCu2 +g-1 dw, Fig. 1), thus indicating that hypertoxic Cu was also characterized by its higher transport/accumulation in the test organism. Such observations do indicate that Ni uptake also follows saturation kinetics as applicable to other cations; however, the initiation of the second phase of uptake points towards the possible remobilisation of Ni 2 + subsequent to its uptake in the first phase.

Metal compartmentalization in plant parts (root and top) The data in Fig. 3 A are based on the relative Cu accumulation by the photosynthetic portion (top) and the roots subsequent to 1 h of uptake at different concentrations (1100 ~mol/L). A constant root/top ratio (10: 1) of Cu uptake for 1 and 5 ~mol/L indicates that at lower concentrations, a major fraction of the cation accumulates in the roots. However, such was not the case for elevated Cu concentrations as the ratio declined to 4: 1 in the case of 10 ~mol!L, thus indicating that a major fraction of Cu in the roots is readily translocated to the top. The ratio declined further (3.5: 1, average) for still higher Cu concentrations (20, 25 and 50 Jlmol/L). The maximum decline in the root/top ratio (2.15: 1) was achieved for 100 ~mol/L Cu. The root/top ratio with regard to Ni2+ distribution has been compared in Fig. 3 B subsequent to 1 h of uptake at dif4

ferent concentrations (25-500~mol/L). As already indicated, Ni2+ uptake remained invariably low compared with Cu (Figs. 1, 2); nevertheless, the root/top ratio ofNi2 + distribution followed almost the same sequence as applicable to Cu2+ (Fig. 3A). In other words, it can be suggested that although both of the cations have different uptake/saturation maxima, their ultimate partitioning in S. polyrhiza followed the same course.

Metal vs. thiol content The data in Table 2 display thiol content in S. polyrhiza in response to different Cu concentrations (1-100 ~mol/L) and incubation time (5, 10, 20 and 40 h). The salient observations that emerged out of such comparisons are: (a) low Cu concentrations (1 and 5Jlmol/L) were insignificant inducers of thiol even in prolonged exposure compared with the control set, (b) a significant thiol content could be achieved in 10 h only for a restricted range of Cu (10, 20 and 25 ~mol/L), and (c) the higher Cu range (50 or 100 ~mol/L) invariably reduced the thiol content. For Ni also, there was an insignificant increase in thiol content in 25 and 50 ~mol/L. A further increase in Ni concentration to 100 and 200 ~mol/L significantly increased thiol content to 15.94 and 17.04~molg- 1 dw, respectively, at 10h. During the comparable time (10h), there was a maximum increase in thiol content at elevated Ni concentrations (300- 500 ~mol/L). The latter concentration range also showed a significant increment in thiol content even at 5h. Table 2: Thiol content in S. polyrhiza exposed to graded concentrations of Cu or Ni. Cu

Incubation time

(~tmol/L)

Control

50

±0.42 10.80 ±0.32 11.0 ±0.33 1o.43 ±0.31 10.50 ±0.32 10.20

100

±0.30 9.80

~

"0

Cl

10.26 ±0.41 10.60

10

E

10h

20

2

25

+

"':::~

()

±0.39

10.26 ±0.41 10.70

so

Cu(!Jm)

100 0

250

Ni (!Jm)

500

Fig. 3: A: Cu content in root (0--0) and top (e--e) of S. polyrhiza vs. different external Cu concentrations (after 1 h). B: Ni content in root (0--0) and top (e--e) of S. polyrhiza vs. different external Ni concentrations (after 1 h).

Ni 40h (~tmol!L)

Incubation time

p <0.05

10h

5h

20h 40h

(~tmol thiol g- 1 dw)

10.26

10.26 Control

10.26

10.26

10.26

10.26

±0.41 10.92

±0.41 10.71

±0.41 11.30

±0.41 ±0.41 11.32 11.29 ±0.34 ±0.45 11.36 11.20

±0.41 11.20

25 ±0.32 ±0.33 ±0.43 11.0 11.24 10.90 50 ±0.33 ±0.34 ±0.33 15.0 11.14 10.43 100 ±0.45 ±0.33 ±0.31 12.21 10.29 10.07 200 ±0.48 ±0.31 ±0.30 13.25 7.88 7.01 300 ±0.40 ±0.24 ±0.21 7.00 400 9.50 7.25 ±0.29 ±0.22 ±0.21 8.04 6.74 6.00 500 ±0.24 ±0.20 ±0.18

LSD (Cu) = 2.28

0

20h

(~tmol thiol g- 1 dw)

A 'ICI

5h

±0.45 11.33 ±0.34 12.73 ±0.38 13.61 ±0.41 14.46

±0.34 ±0.34 15.94 11.30 ±0.48 ±0.34 17.05 11.37 ±0.51 ±0.23 19.25 13.15 ±0.58 ±0.40 20.00 14.15

±0.45 10.12 ±0.30 10.67 ±0.32 10.94 ±0.33 12.94

±0.43 14.57 ±0.44 ±0.60 15.00 20.00

±0.42 14.50

±0.39 12.07 ±0.36 10.10

±0.45

±0.29

±0.30

±0.60

LSD (Ni) = 4.35 p <0.05

Discussion

Duckweeds are highly sensitive to many surrounding factors and are used as indicators of water pollution (Hillman

638

SANJULA PANDEY, R. K. AsTHANA, ARVIND M. KAYASTHA, NEETU SiNGH, and S. P. SiNGH

and Culley, 1978; Nasu and Kugimoto, 1981). Cations like Cd and Cu reduced frond number in Spirodela and Lemna (Srivastava and Jaiswal, 1989; Nasu and Kugimoto, 1984). In contrast to inhibitions at high Cu concentration, low Cu concentration O!lmol/L) stimulated frond multiplication in S. polyrhiza (Table 1), an observation similar to stimulation of general growth of the Caryophyllaceae member, Silene cucubalus (Lolkema et a!., 1984). Excessive Cu resulted in chlorosis, root growth inhibition and alterations in plasma membrane permeability, leading to ion efflux (DeVos eta!., 1991). Also, the increase in chlorophyll content of S. polyrhiza by 1!-lmol/L Cu is similar to stimulation in Alyssum montanum at 4 and 8!-lmol/L (Ouzounidou, 1994). The stimulation of root growth by Cu, however, presented a wide range (1-20 !lmol/L) compared with the parameters just described. Root development as a parameter to assess plant tolerance to heavy metals has been adopted by many investigators (Baker and Walker, 1989), and the process was sensitive to metals (Punz and Sieghardt, 1993). Ouzounidou (1994) observed that only high Cu concentrations affected root growth in Alyssum. S. polyrhiza root growth showed a positive response towards a wide Cu range (1-20 !lmol/L) with the increase corresponding to 80% in 1!-lmol/L but limited to 10 % in 20 !lmol/L. The root growth, however, remained unaffected at 25!-lmol Cu/L followed by inhibition at 50 and 100 !lmol/L. The data on Ni effect revealed that the cation was hypotoxic compared with Cu and the simulation of general growth including chlorophyll levels showed a range 25-fold over that of Cu. Xylander et a!. (1993) observed inhibitory effects of Ni on the vegetative growth, turion formation and germination in S. polyrhiza. Interestingly, the retardation of growth and carbohydrate content in Ni-exposed plants had the inverse correlation. Such plants also showed growth stimulation at 1!-lmo!Ni/L: no significant change in 10-30!-lmol/ L and inhibition at 60 11mol Ni/L. The presently used plant showed stimulation of general growth and chlorophyll content only at 25!-lmol Ni/L followed by inhibition at 50 !lmol! L onward. Ni sensitivity of chlorophylls has also been observed in Salvinia natans, and the loss in pigment content accompanied a decline in levels of free amino acids, plant dry wt and the induction of scenescence (Sen and Bhattacharyya, 1994). Cu uptake in S. polyrhiza shows parity with the observations in Lemna (Tanaka et a!., 1982) and Salvinia (Sen and Monda!, 1990), with the maximum accumulation (2.7 mg Cu2 + g-1 dw) within the first 10 min of uptake at 100 11mol Cu/L external concentration (Fig. 1). Such shortterm metal uptake studies present a contrast as the latter investigatiors followed Cu uptake during prolonged metal exposure (days). A5 the followup of uptake up to 24 h did not show an increase of intracellular Cu in S. polyrhiza, it did not seem reasonable to extend metal uptake duration. If compared in terms of initial rate or total accumulation, Ni uptake was slower than that of Cu and required more time (40 h) to attain saturation (Fig. 2). Ni uptake duration extended up to 80 h did not reveal any rise in Ni intake by plants. The metal removal efficiency of S. polyrhiza reached 60% for 500 !lmol! L (8.84mgNi 2 + g-1 dw). Ni uptake studies extended up to 25 days in S. polyrhiza in other cases indicated that the intracel-

lular Ni could reach 1.82 mg on per gram biomass dry wt basis for 100 !lmol/L (Xylander et al., 1993). Similar longterm experiments on Salvinia showed 75 % Ni removed although the external concentration was fairly high (680 !lmol/L) (Sen and Bhattacharyya, 1994). Cu at lower concentrations (1 and 5 !lmol/L) was maximally accumulated in S. polyrhiza roots, as apparent from a root/top ratio of 10 : 1 (Fig. 3 A). However, a significant decline in the root/top ratio to 2.15 : 1 for the highest Cu concentration (100 !lmol/L) indicates that cation accumulation in roots responds positively to the external Cu concentration, although at higher concentrations it is transported to the plant top. The tendency of metal accumulation in roots agrees very well with the earlier reports on Lotus purshianus (Wu and Lin, 1990), Alyssum (Ouzounidou, 1994) and Eichhornia crassipes (Wolverton and McDonald, 1978). The relative distribution of Ni in roots and the top of S. polyrhiza followed the sequence as applicable to Cu although the total Ni accumulation in plants was lower than that of Cu (Fig. 3 B). Here also, Ni in roots was translocated to the top. The observed concentration-dependent Ni accumulation in roots of S. polyrhiza is analogous to Cu and Ni accumulation and translocation in Tjpha latifolia (Taylor and Crowder, 1983) and Salvinia (Sen and Bhattacharyya, 1994). Such metal translocation characteristics are also shared by Glycine max as 85% ofNi first accumulated in the roots and migrated to the aboveground plant parts with the passage of time (Cataldo et a!., 1978). Elements like Cd, Ni and Zn reduced root weight in Hordeum vulgare and Ni specifically, and were accumulated in the roots both at fairly low and high concentrations as well (Brune et a!., 1995). Recently, Ni 2 + and Cd2+ accumulation in the metal-sensitive and metal-resistant strain of Silene italica showed that the cations accumulated as a function of their external concentrations more in the root than the shoot (Mattioni eta!., 1997). The data on Ni intake classifY S. polyrhiza as the hyperaccumulator of Ni, as reported for other plants (Reeves and Brooks, 1983; Baker and Brooks, 1989). Plants show a rapid molecular response to changing environmental conditions (Lichtenthaler, 1996), and heavy metals in particular induce low molecular weight, cysteinerich proteins as isolated from various higher plants (Steffens, 1990). The strategy for accumulation of and tolerance to Cd in Eichhornia has been the production of a Cd-binding component resembling mammalian Cd-thionein (Fujita, 1985; Fujita and Kawanishi, 1986). Although such low molecular weight peptides are derived from thiols, the latter seem to have received little attention with regard to plant response to heavy metal stress compared with phytochelatins or the metal binding proteins (Galli eta!. 1996). Cu at very low concentrations (1 and 5 !lmol/L) failed to induce thiol biosynthesis in S. polyrhiza even during prolonged exposure (Table 2), indicating that this was the outcome oflow intracellular buildup at such concentrations (0.025 and 0.085 mg Cu 2 + g- 1 dw, respectively; 10 min, Fig. 1). Higher Cu concentrations (10, 20 and 25 !lmol/L) raised their intracellular buildup accompanied by an increase in thiol content (at 10h), indicating the requirement of a threshold intracellular metal load. Reduction in thiol content in the higher range (50 and 100 !lmol/L) reflects the after-effect of high metal load. The less toxic Ni in

Metal Uptake and Thiol Production in Spirodela such comparisons showed an insignificant effect on thiol content in the lower range (25 and 50 ~-tmol!L), with intracellular Ni buildup corresponding to 0.73 and 1.46 mg Ni2+ g-1dw, respectively, at 10 h in contrast to a significant increase in the higher range (100-500 ~-tmol!L), which yielded a maximum amount of thiol in the same duration. Noticeably, a significant increase in thiol content was recorded only in the restricted Ni range (300- 500 ~-tmol!L) even during 5 h. The interesting feature of such observations is that although S. polyrhiza could accomodate Ni or Cu to much higher levels, thiol production was only triggered by critical threshold metal concentrations, a characteristic of the cation used. The present observations on thiol induction by Ni are in contrast in those of Brune et al. (1995), where only Zn and Cd stress increased sulfhydryl content in Hordeum and not Ni and Mo stress. The latter two metals became partitioned in sectors like the vacuole or the apoplast, as an alternative to complexation with the sulfhydryl content. In addition to sulfhydryl content, the molecular response of different Alyssum species to Ni has been the proportionate increase in production of free histidine that could be coordinated with Ni in vivo (Kramer et al., 1996). The latter investigators also showed that the rise in histidine level also characterized the hyperaccumulating plants. To sum up, thiol production in S. polyrhiza can be taken as one of the molecular responses of the plant towards Ni or Cu stress, thus being metal specific. The less toxic Ni requires a greater amount to trigger thiol biosynthesis compared with the hypertoxic Cu. Acknowledgements

We gratefully acknowledge the financial assistance received from the Banaras Hindu University and the Department of Biotechnology, Govt. oflndia, New Delhi.

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