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Copper-binding protein and copper tolerance in Agrostis gigantea
Plant Science Letters, 33 (1984) 239--247 Elsevier Scientific Publishers Ireland Ltd.
239
COPPER-BINDING PROTEIN A N D COPPER TOLERANCE IN A GR OS TIS G I G A N T E A
W I L F R I E D E. R A U S E R Department of Botany and Genetics, Universityof Guelph, Guelph, Ontario, N I G 2W1 (Canada) (Received June 17th, 1983) (Revision received August 22nd, 1983) (Accepted August 31st, 1983)
SUMMARY
Cu-binding protein was measured in roots of a Cu-tolerant and t w o nontolerant ecotypes of Agrostis gigantea. Cu-binding protein was present in all three ecotypes and was induced to increase by application of Cu. In the Cu-tolerant e c o t y p e Cu-binding protein increased within 1 day of growing the plants in various concentrations of excess Cu. These roots continued to elongate. Under the same conditions the roots of the non-tolerant ecotypes grew little and their Cu-binding protein contents rose only after 2 or 3 days. It is proposed that the rapid production of Cu-binding protein is essential for the expression of Cu-tolerance in A. gigantea.
Key words: Agrostis gigantea -- Copper -- Cu-binding protein -- Coppertolerance -- Metallothionein
INTRODUCTION
Metal tolerance occurs in certain ecotypes of vascular plants originating from softs with high concentrations o f metals [1--3]. The mechanism of metal tolerance remains unclear. Consideration has been given to the exclusion of metal from the plant, adsorption of metal by cell walls, compartmentation of metal intraceUularly and enzymic adaptations. In the case of metal tolerant grasses exclusion of Cu or Zn from the roots and shoots seems not to be part of the tolerance mechanism [4]. The degree to which the other mechanisms complement each other is unclear and there is evidence that a
0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
240
number of mechanisms are responsible for metal tolerance in different plants [4]. In a Cu-tolerant ecotype of A. gigantea R o t h more Cu was bound to root cell walls and more Cu was translocated to the shoots than in a non-tolerant ecotype, y e t the absorption of Cu by the roots was the same for both ecotypes [ 5]. From these circumstances it was suggested that complexation of Cu intracellularly in an innocuous form was important for metal tolerance. The role of organic acids in complexing Cu in vacuoles, as is known for Zn [6,7], has not been investigated in our ecotypes. We have emphasized an alternative sequestering agent, the protein metallothionein. Such a low molecular weight Cu-binding protein was reported for the roots of the Cu-tolerant ecotype of A. gigantea [8]. Metallothioneins have been implicated in metal detoxification in animals [9]. Metals induce such proteins in animals [10] and in plants challenged with Cd [11--13]. This communication describes the relationship between the Cu-binding protein c o n t e n t of roots and root elongation growth in Cu-tolerant and nontolerant ecotypes of A. gigantea exposed to various concentrations of Cu. The findings are a first step in the evaluation of the hypothesis that a metallothionein-like protein is involved in Cu-tolerance. MATERIALS AND METHODS
The three ecotypes of A. gigantea used were the Cu-tolerant clone 4 and the non-tolerant clones 6 and 14 grown in nutrient solution in a growth room [ 2]. Tests were conducted with healthy rootless tillers bearing 3--4 leaves which grew as laterals in the stock cultures. The tillers were wedged near their bases into slits cut on one edge o f 2 cm thick 27 X 6 cm pieces of styrene foam. Two such units with a third separating blank were floated on 5 1 of aerated nutrient solution in polyethylene tanks. Uniform tillers were selected after 6--7 days of rooting and transferred to clean styrene foam holders. A m a x i m u m of 24 tillers occupied one culture tank. In metal tolerance experiments the root systems were photographed in water at the beginning of the monitoring of root growth. Tillers were then returned to their culture tanks containing nutrient solution. After 3 days of growth the root systems were photographed again. The tillers were then placed in either fresh nutrient solution or solutions amended with extra CuSO4 as listed in Table I. Four days later the root systems were photographed a third time. At harvest the roots of individual tillers were rinsed briefly in water, blotted, excised, weighed and frozen for later use. In time course experiments all root systems were photographed prior to treatment and just before harvest. In one test the tillers were in nutrient solution with basal Cu (0.25 pM) or 16 pM Cu for up to 7 days. Solutions were replaced every other day. In a second test the tillers were in 0.25, 8, 12 or 16 uM Cu for up to 3 days. Solutions were replaced daily. The lengths o f all adventitious roots on a tiller were measured by project-
241
ing 3 5 - m m negatives onto a 50.8~m square digitizing tablet (Model DTR2020 Datatizer, G T C O Corp., MD). The X-Y coordinates from the cursor used to trace the adventitious roots were analyzed by a microcomputer to give total lengths of roots per tiller.In the metal tolerance experiments the following growth index was calculated: Growth index =
growth (cm/day) in presence of Cu growth (cm/day) in basal nutrient solution
The root extension growth determined in this way included the elongation resulting from those adventitious roots initiated during the time between taking photographs. Cu-binding protein was estimated in roots of individual tillers according to Rauser [ 14]. Briefly, roots were homogenized in a solution containing 20 mM Tris--HC1 (pH 8.0) and 20 uM KCN, transferred to centrifuge tubes and the homogenate heated quickly to, and held at, 60°C for 3 min. The material was cooled, centrifuged and the supernatant fluid was passed through a 0.3-ml column bed (7.5 mm i.d. × 7 mm) of QAE-Sephadex A-25 equilibrated with 5 mM Tris--HC1 (pH 8.0). The loaded column was washed with 30 ml of equilibration buffer and the desired fraction eluted with 5 ml 400 mM NaC1 in buffer. Protein in the salt eluates was quantitated by differential pulse polarography [ 15], except that the reactions were performed at 10°C with a Model 303 static mercury drop electrode controlled by a Model 384 polarographic analyzer (Princeton Applied Research Corp., NJ). The amount of protein was expressed as ~A g-' from the current generated by the protein during differential pulse polarography. Cu in salt eluates was determined by atomic absorption spectroscopy. RESULTS
The results of a standard tolerance test are presented in Table I. The-deviations of growth indices from 1.00 in basal nutrient solution (0.25 uM Cu) are attributed to the extra day available for root initiation and elonga~tion in the second 4-day period as compared to the initial 3-day period of monitoring root growth. Clone 4 was tolerant of Cu compared to clones 6 and 14. For instance, at 20 pM Cu the growth index for clone 4 was high whereas it was low in both clones 6 and 14. R o o t extension growth during the 4 days of exposure to 20 pM Cu was decreased by 20% in clone 4 and by 97% and 98% of controls in clones 6 and 14, respectively. Exposure of clone 4 to increasing concentrations of Cu resulted in higher protein contents to a maximum at 40 #M external Cu and a plateau thereafter. Cu was associated with the protein and followed the same pattern (r between protein and Cu was 0.941). In the non-tolerant clones the protein in the roots increased only after the plants were exposed to 14 and 8 ~M Cu,
242 TABLE I THE R E L A T I O N S H I P B E T W E E N R O O T G R O W T H A N D T H E A M O U N T O F Cu-BINDING P R O T E I N IN R O O T S A F T E R 4 D A Y S O F E X P O S I N G T H R E E C L O N E S O F A. G I G A N T E A TO E X C E S S Cu Means ± S.E. o f 6 replicates w i t h p r o t e i n d e t e r m i n e d in d u p l i c a t e for each sample. Clones
External Cu (~M)
Growth index
Growtha (era)
Protein 0z A g-i )
Cu (~g g-i )
Clone 4
0.25 20 30 40 50
1.02 +-0.07 0.97 + 0.05 0.44 + 0 . 0 7 c 0.20_+0.03 c 0.09 _+0.02 c
90.12 _+9.01 72.53 +- 6.65 b 32.95 + 4 . 3 7 c 14.12_+1.87 c 6.63 +_ 1 . 1 9 c
8.30 +-0.52 12.81 +_0.74c 17.39+0.88c 24.73 + 1.12c 24.22 +- 1.14c
4.47 +-0.40 12.69 +- 1.14 16.20+-1.67 23.75+-1.73 23.31 + 0.96
Clone 6
0.25 3 8 14 20
1.08+-0.11 1.21+-0.18 0.83+-0.09 0.04-4-0.01 c 0.03 +-0.01 c
30.33+-3.24 37.42+-5.31 27.77+-3.02 1.15+0.15 c 0.82 _+0.29 c
17.79+-1.77 23.03+_3.41 26.37+-2.71 35.92+_3.36 c 40.68 +_5.03 c
22.16+-1.33 21.97+-3.26 29.87+-4.10 42.56+-4.75 50.79 +-5.10
Clone 14
0.25 3 8 14 20
1.13+_0.07 0.87+_0.03 c 1.19 +- 0.09 0.15 +_0.03 c 0.06 +- 0.01 c
35.97+-4.46 35.34+_7.06 45.30 -4- 3.39 4.91 + 0 . 4 1 c 1.64 +- 0.34 c
19.43+-1.74 19.17+-1.31 29.93 +- 1.43 c 37.74 _+2.87 c 39.44 +_ 4.07 c
21.88+5.54 21.32+-2.86 29.13 +- 1.61 40.59 +-4.10 52.11 _+ 4.01
a R o o t g r o w t h b e t w e e n days 3 and 7 during e x p o s u r e to excess Cu. b Significantly d i f f e r e n t f r o m t h e c o n t r o l at t h e 0.05 level. c Significantly d i f f e r e n t f r o m t h e c o n t r o l at t h e 0.01 level.
respectively. A similar pattern occurred with the Cu associated with the protein (r between protein and Cu was 0.808 for clone 6 and 0.721 for clone 14). At 20 pM Cu the Cu-binding protein c o n t e n t in the non-tolerant ecotypes had at least doubled over the controls while a 1.5-fold increase occurred in the Cu-tolerant clone. These increases in Cu-binding protein were-associated with little root extension growth in the non-tolerant clones but with continued root growth in clone 4. From the tolerance test it was n o t clear whether the tolerant and nontolerant clones were different with regard to the time of increase in Cubinding protein after exposure to excess Cu, or to the concentration of Cu. Two sets of time course experiments were conducted to explore these possibilities. In the first test, the tillers were exposed to 0.25 or 16 uM Cu -{Table II). Cu-binding protein c o n t e n t increased significantly within one day of exposing the Cu-tolerant clone 4 to 16 ~M Cu. This concentration of Cu inhibited root elongation growth by 25%. Cu-binding protein in the non-tolerant clones increased significantly only after 2 and 3 days o f exposure to 16 ~M Cu and their root growth was severely inhibited from day 1 onward. Cu was associated with the protein fractions and generally
243 TABLE II CHANGES IN R O O T G R O W T H AND Cu-BINDING P R O T E I N C O N T E N T WITH TIME OF E X P O S U R E OF T H R E E CLONES OF A. GIGANTEA TO 0.25 A N D 16 uM Cu Means ~ S.E. of 5 replicates with protein determined in duplicate for each sample. Clones Clone 4
Cu (uM) 0.25
16
Clone 6
0.25
16
Clone 14
0.25
16
Day
Growth a (cm)
Protein (uA g-l)
0 1 2 4 7
8.03 28.36 105.59 141.41
7.79 8.48 6.84 8.09 7.64
1 2 4 7
5.79 4-- 1.69 2 2 . 7 1 + 4.51 53.83 -4-_ 8.48 c 105.92 4--14.79 c
0 1 2 4
7.59 4-- 1.81 11.824-, 3.83 43.86 4-- 6.51
1 2 3 4 0 1 2 4 1 2 3 4
4-_ 2.01 4-- 5.48 4-- 3.37 4--22.93
1.68 4-0.624-0.63 4-1.29 4--
1.25 0.20 c 0.35 c 0.60 c
2.91 4-- 0.31 6.20 4-- 1.22 29.92 4-- 3.61 0.81 +_ 0.67 4-0.83 4-0.464--
0.18 0.19 b 0.20 c 0.10 c
12.92 13.45 14.95 21.71
Cu (ug g-l)
4--0.46 4--0.55 +- 0.87 4--0.53 4-,0.21
10.79 12.55 5.30 2.87 2.52
4-4-4-4-+
1.83 3.27 1.07 0.28 0.26
+0.53 c +0.81 c _+0.94 c 4-- 1.21 c
21.29 20.16 21.99 26.22
4-4-4-_+
1.61 1.74 1.51 0.95
11.45 4-,2.61 7.81 4--0.27 10.834-,2.10 8.52 +_0.42
27.24 4-- 6.04 11.43 4-- 0.84 34.144-,14.62 13.02_+ 1.93
11.46 4--1.32 14.10_+1.44 22.37+_2.66 c 29.87 _+4.97 c
41.24 4-- 5.56 50.304-- 9.19 78.87 +_17.81 67.71 4-,15.15
10.03 11.60 8.93 10.41
53.62 27.88 37.53 15.32
4--0.72 4--1.20 4--1.31 4--0.96
12.23 4-_1.54 19.93 +_2.57 32.86 4--4.58 c 33.78+_5.42 c
4--10.33 4-- 4.34 4-- 5.77 4-- 3.94
49.30 4-- 4.28 94.56 4-,16.65 166.07 4--22.26 134.834-,18.50
a R o o t growth from day 0 to harvest. b Significantly different from the control of the day at the 0.05 level c Significantly different from the control of the day at the 0.01 level increased with time of exposure to excess Cu. The declines in Cu in the controls with time are attributed to the small quantities of Cu in these roots. At the earlier harvests the masses of roots were small (35-60 mg fresh wt./tiller) which brought the precision of the analysis into question. In the second time course experiment clones 4 and 6 were exposed to three concentrations of excess Cu (Table III). The roots of the Cu-tolerant clone 4 contained significantly more Cu-binding protein within 1 day of exposure to excess Cu. By days 2 and 3 the protein content had increased further, particularly after exposure to 12 and 16 ~M Cu. Root growth was
244 T A B L E III C H A N G E S IN R O O T G R O W T H A N D Cu-BINDING P R O T E I N C O N T E N T O V E R 3 D A Y S O F E X P O S I N G TWO C L O N E S O F A. G I G A N T E A TO F O U R C O N C E N T R A T I O N S O F Cu Means +_ S.E. o f 5 replicates w i t h p r o t e i n d e t e r m i n e d in duplicate for each sample. Clones
Cu (#M)
Growth a (cm)
Protein (~A g-~)
Cu (ug g - l )
0 1 2 3
10.31 _+1.47 22.26_+2.96 20.63 _+2.12
10.18 _+0.53 9.44 _+0.49 8.98+0.59 12.30 _+ 1.23
10.65 _+0.76 11.52 _+0.25 9.69_+1.02 10.02 + 1.24
1 2 3
8.90 _+1.75 19.38 _+ 1.44 26.28 -+4.15
13.80 + 0 . 9 2 c 14.98 _+0.69 c 16.55 -+ 1.01 c
15.43 -+1.61 19.47 _+ 1.84 20.72 _+2.97
12
1 2 3
11.35_+1.18 15.19 + 3 . 0 6 b 37.70 + 1 . 1 6 c
11.66+_0.61 b 16.38 -+ 0.74 c 18.00 _+1.22 c
21.37_+1.40 20.85 -+3.00 18.25 -+1.73
16
1 2 3
7.01 _+ 0.73 14.06 _+ 1.55 b 20.74-+2.60
14.31 _+ 0.90 c 18.82 _+0.46 c 22.58+1.64 c
20.45 _+ 1.82 20.91 _+ 3.23 24.79-+4.61
0 1 2 3
4.66_+1.21 18.51_+4.49 26.26 _+ 3.24
10.90_+0.61 9.23 _+0.45 9.03-+0.67 10.00 _+ 1.24
28.13_+10.15 21.71_+ 2.37 14.64_+ 3.82 18.91 _+ 9.58
Clone 4
Clone 6
0.25
0.25
Day
1 2 3
5.52_+1.50 3.37_+0.57 c 4.99 _+ 1.09 c
11.23_+0.71 16.03-+1.37 b 21.29 _+ 1.29 c
1 3 . 6 5 + 1.62 40.61-+ 7.49 39.21 _+ 7.95
12
1 2 3
2.79 + 1.20 1.70 _+0.29 c 1.13+0.29 c
11.22 -+ 0.65 15.51-+1.08 b 24.33-+3.35 c
23.39 -+ 2.97 39.47-+ 5.92 33.13_+ 5.20
16
1 2 3
0.96 _+0.17 1.19+0.50 c 1.39 _+0.76 c
10.02 _+0.33 16.86_+1.25 23.90+0.87 c
21.64_+ 37.01+_ 32.92_+
2.15 7.62 1.82
a R o o t g r o w t h f r o m day 0 t o harvest. b Significantly d i f f e r e n t f r o m t h e c o n t r o l o f t h e d a y at t h e 0.05 level. c Significantly d i f f e r e n t f r o m t h e c o n t r o l o f t h e day at t h e 0.01 level.
inhibited somewhat by excess Cu. In clone 6 the Cu-binding protein c o n t e n t increased significantly over controls only after 2 days o f exposure to excess Cu, root elongation growth was severely inhibited after 1 day. The Cu-binding protein contents in clone 6 at any one time were the same for the three concentrations of excess Cu.
245 DISCUSSION Metallothionein is an attractive agent for intracellullar sequestration of metal because it is a metal inducible protein which itself binds Cu, Zn and Cd [9]. Exclusive measurement of this non-enzymic protein is possible b y immunological assays [ 16]. Unfortunately antibodies to the plant protein are not y e t available and the plant protein isolated so far does not cros~ react with rabbit antibodies to rat Cu-metallothionein (J.S. Garvey, pers. comm.). Quantitation of metallothioneins b y high performance liquid chromatography [17] remains to be evaluated for plant extracts. The work presented here relies on the selective purification and enrichment of metal-binding protein by anion exchange chromatography of heat denatured r o o t extracts [14]. The procedure used exceeded the precautions taken to remove interfering substances in a study of the dynamics o f metallothionein changes in mice [ 18]. Metallothionein is rich in thiol groups [8,9] and consequently is reactive at a dropping mercury electrode during differential pulse polarography [19]. The electrochemical reactivity of animal metallothioneins is validated [15]. The protein fractions analyzed for Agrostis roots gave clear wave patterns typical of thiol groups during differential pulse polarography. Cu was associated with the isolated protein (Tables I, II and III) and served as a confirmatory second measure of Cu-binding protein. The fraction of Cu measured here was from 58 to 76% of the buffer-soluble Cu of roots [14]. The measurements are referred to as Cu-binding protein pending further specific characterization and are given as ~Ag -1 for lack of a suitable plant calibration protein. These protein contents are taken as reasonable and useful first estimates in evaluating the relationship between metallothionein-like Cu-binding protein and Cutolerance in A. gigantea. Cu-binding protein is present in the three clones o f the grass and is induced to increase b y exposing the plants to increased Cu. Metallothioneinlike protein appears in t o m a t o [ 11 ], soybean [ 12], and cabbage and tobacco [13] exposed to Cd. This study was begun with the metal tolerance experiment to test the general idea that tolerance depends on the a m o u n t of metal sequestering agent in roots. One expectation was that Cu-binding protein would be high in a tolerant plant and low in a non-tolerant plant when exposed to excess Cu. The data in Tables I, II and III clearly show that for equivalent treatments the Cu-tolerant clone 4 contained less Cu-binding protein than did the non-tolerant clones on an absolute basis or relative to the controls. The higher Cu-binding protein contents in non-tolerant clones were, however, associated with little r o o t growth. These data on Cu-binding protein c o n t e n t are contrary to expectations. One possibility is that the protein measured b y differential pulse polarography is a poor estimate of metal sequestering protein, or that Cu-binding protein is not involved in Cutolerance.
246 An alternative view stems from the two time course experiments (Tables II and III) which clearly showed that the Cu-tolerant clone 4 contained more Cu-binding protein than controls within one day of exposure to excess Cu. These roots continued to elongate. The non-tolerant clones had higher Cu-binding protein contents only after 2 or 3 days, their r o o t elongation was severely inhibited from day 1 onward. These findings show that Cu-tolerance in A. gigantea is associated with a capacity to make Cu-binding protein rapidly in response to elevated Cu. It is proposed that a rapid increase in Cu-binding protein would effectively enhance intracellular metal sequestration. If dividing cells and their progeny were to respond by making Cu-binding protein rapidly, they themselves would experience low free Cu allowing for division and enlargement. Repeated acquisition of Cu-binding protein rapidly upon further entry of Cu into the cells would result in a r o o t that continues to elongate in the presence of excess Cu, as shown by the Cu-tolerant clone 4 (Tables I, II and III}. Such a scheme would bear some cost as amino acids and metabolic energy are diverted to the synthesis of Cu-binding protein. Perhaps the decreased r o o t elongation in the Cu-tolerant e c o t y p e exposed to increasing external Cu (Tables I, II and III) is an expression of such a biological cost. Work is in progress to determine what proportion of the intracellular Cu is associated with Cu-binding protein and whether the changes in protein predominate in the r o o t tips or elsewhere. The view that metal tolerance depends on the capacity to make Cubinding protein rapidly is based on the response of one clone of A. gigantea. It is n o w necessary to evaluate this model for a population of Cu-tolerant clones o f A . gigantea, extend it to other species and perhaps to the metals Zn and Cd. Experiments along these lines are in progress. The sudden exposure of roots to excess Cu and our use of nutrient solutions are completely artificial compared with field conditions. Perhaps allowing roots to initiate and grow in 0.5--1 gM Cu would precondition them so that they could better deal with subsequent increases in Cu, a situation known for Holcus lanatus pretreated with Cd [20]. However, both in our experimental situation and in Cu-rich soil, a Cu-tolerant plant such as clone 4 would be producing new r o o t tip cells which experience excess Cu during formation and enlargement. Nevertheless, the experiments reported here have clearly identified an association between the capacity to produce metallothionein-like Cu-binding protein rapidly and the expression of Cu-tolerance as r o o t elongation growth. ACKNOWLEDGEMENTS This work was supported b y Operating Grant A4921 and Strategic Grant G0529 from the Natural Sciences and Engineering Research Council of Canada. I thank Miss Linda Elder and Mr. Peter Taylor for their excellent technical support.
247
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11 12 13 14 15 16 17 18 19 20
J. Antonovics, A.D. Bradshaw and R.G. Turner, Adv. Ecol. Res., 7 (1971) 1. G.D. Hogan and W.E. Rauser, N e w Phytol., 83 (1979) 665. R.M. Cox and T.C. Hutchinson, N e w Phytol., 84 (1980) 631. D.A. Thurman, Mechanism of metal tolerance in plants, in: N.W. Lepp (Ed.), Effect of Heavy Metal Pollution on Plants, Vol 2, Applied Science Pub., London,, 1981, p. 239. G.D. Hogan and W.E. Rauser, J. Exp. Bot., 32 (1981) 27. A. Brookes, J.C. Collins and D.A. Thurman, J. Plant Nutr., 3 (1981) 695. D.A. Thurman, J.A. Rankin, J.C. Collins and D. Godbold, Organic acids and metal tolerance in Desehampsia caespitosa, in: Int. Conf. Heavy Metals in the Environment, C E P Consultants Ltd., Edinburgh, 1981, p. 277, W.E. Rauser avd N.R. Curvetto, Nature, 287 (1980) 563. J.H.R. K~igi,Y. Kojima, M.M. Kissling and K. Lerch, Metallothionein: an exceptional metal thiolate protein, in: Sulphur in Biology Ciba Foundation Syrup., Vol. 72, Excerpta Medica, 1980, p. 223. M. Nordberg and Y. Kojima (Eds,), Report of the first international meeting on metaUothionein and other low molecular weight metal-binding proteins, in: J.H.R. K~'gi and M. Nordberg (Eds.), Metallothionein, Birkh~iuser,Basel, 1979, p. 41. M. Bartolf, E. Brennan and C.A. Price, Plant Physiol., 66 (1980) 438. J.L. Casterline Jr. and N.M. Barnett, Plant Physiol., 69 (1982) 1004. G.J. Wagner and M.M. Trotter, Plant Physiol., 69 (1982) 804. W.E. Rauser, Z. Pflanzenphysiol., 112 (1983) 69. R.W. Olafson and R.G. Sire, Anal. Biochem., 100 (1979) 343. R.J. Vander Mallie and J.S. Garvey, Anal. Biochem., 98 (1979) 89. S. Klauser, J.H.R. K~igiand K.J. Wilson, Biochem., J. 209 (1983) 71. R.W. Olafson, J. Biol. Chem., 256 (1981) 1263. E. Pale~ek and Z. Pechan, Anal Biochem., 42 (1971) 59. H. Brown and M.H. Martin, N e w Phytol., 89 (1981) 621.
239
COPPER-BINDING PROTEIN A N D COPPER TOLERANCE IN A GR OS TIS G I G A N T E A
W I L F R I E D E. R A U S E R Department of Botany and Genetics, Universityof Guelph, Guelph, Ontario, N I G 2W1 (Canada) (Received June 17th, 1983) (Revision received August 22nd, 1983) (Accepted August 31st, 1983)
SUMMARY
Cu-binding protein was measured in roots of a Cu-tolerant and t w o nontolerant ecotypes of Agrostis gigantea. Cu-binding protein was present in all three ecotypes and was induced to increase by application of Cu. In the Cu-tolerant e c o t y p e Cu-binding protein increased within 1 day of growing the plants in various concentrations of excess Cu. These roots continued to elongate. Under the same conditions the roots of the non-tolerant ecotypes grew little and their Cu-binding protein contents rose only after 2 or 3 days. It is proposed that the rapid production of Cu-binding protein is essential for the expression of Cu-tolerance in A. gigantea.
Key words: Agrostis gigantea -- Copper -- Cu-binding protein -- Coppertolerance -- Metallothionein
INTRODUCTION
Metal tolerance occurs in certain ecotypes of vascular plants originating from softs with high concentrations o f metals [1--3]. The mechanism of metal tolerance remains unclear. Consideration has been given to the exclusion of metal from the plant, adsorption of metal by cell walls, compartmentation of metal intraceUularly and enzymic adaptations. In the case of metal tolerant grasses exclusion of Cu or Zn from the roots and shoots seems not to be part of the tolerance mechanism [4]. The degree to which the other mechanisms complement each other is unclear and there is evidence that a
0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
240
number of mechanisms are responsible for metal tolerance in different plants [4]. In a Cu-tolerant ecotype of A. gigantea R o t h more Cu was bound to root cell walls and more Cu was translocated to the shoots than in a non-tolerant ecotype, y e t the absorption of Cu by the roots was the same for both ecotypes [ 5]. From these circumstances it was suggested that complexation of Cu intracellularly in an innocuous form was important for metal tolerance. The role of organic acids in complexing Cu in vacuoles, as is known for Zn [6,7], has not been investigated in our ecotypes. We have emphasized an alternative sequestering agent, the protein metallothionein. Such a low molecular weight Cu-binding protein was reported for the roots of the Cu-tolerant ecotype of A. gigantea [8]. Metallothioneins have been implicated in metal detoxification in animals [9]. Metals induce such proteins in animals [10] and in plants challenged with Cd [11--13]. This communication describes the relationship between the Cu-binding protein c o n t e n t of roots and root elongation growth in Cu-tolerant and nontolerant ecotypes of A. gigantea exposed to various concentrations of Cu. The findings are a first step in the evaluation of the hypothesis that a metallothionein-like protein is involved in Cu-tolerance. MATERIALS AND METHODS
The three ecotypes of A. gigantea used were the Cu-tolerant clone 4 and the non-tolerant clones 6 and 14 grown in nutrient solution in a growth room [ 2]. Tests were conducted with healthy rootless tillers bearing 3--4 leaves which grew as laterals in the stock cultures. The tillers were wedged near their bases into slits cut on one edge o f 2 cm thick 27 X 6 cm pieces of styrene foam. Two such units with a third separating blank were floated on 5 1 of aerated nutrient solution in polyethylene tanks. Uniform tillers were selected after 6--7 days of rooting and transferred to clean styrene foam holders. A m a x i m u m of 24 tillers occupied one culture tank. In metal tolerance experiments the root systems were photographed in water at the beginning of the monitoring of root growth. Tillers were then returned to their culture tanks containing nutrient solution. After 3 days of growth the root systems were photographed again. The tillers were then placed in either fresh nutrient solution or solutions amended with extra CuSO4 as listed in Table I. Four days later the root systems were photographed a third time. At harvest the roots of individual tillers were rinsed briefly in water, blotted, excised, weighed and frozen for later use. In time course experiments all root systems were photographed prior to treatment and just before harvest. In one test the tillers were in nutrient solution with basal Cu (0.25 pM) or 16 pM Cu for up to 7 days. Solutions were replaced every other day. In a second test the tillers were in 0.25, 8, 12 or 16 uM Cu for up to 3 days. Solutions were replaced daily. The lengths o f all adventitious roots on a tiller were measured by project-
241
ing 3 5 - m m negatives onto a 50.8~m square digitizing tablet (Model DTR2020 Datatizer, G T C O Corp., MD). The X-Y coordinates from the cursor used to trace the adventitious roots were analyzed by a microcomputer to give total lengths of roots per tiller.In the metal tolerance experiments the following growth index was calculated: Growth index =
growth (cm/day) in presence of Cu growth (cm/day) in basal nutrient solution
The root extension growth determined in this way included the elongation resulting from those adventitious roots initiated during the time between taking photographs. Cu-binding protein was estimated in roots of individual tillers according to Rauser [ 14]. Briefly, roots were homogenized in a solution containing 20 mM Tris--HC1 (pH 8.0) and 20 uM KCN, transferred to centrifuge tubes and the homogenate heated quickly to, and held at, 60°C for 3 min. The material was cooled, centrifuged and the supernatant fluid was passed through a 0.3-ml column bed (7.5 mm i.d. × 7 mm) of QAE-Sephadex A-25 equilibrated with 5 mM Tris--HC1 (pH 8.0). The loaded column was washed with 30 ml of equilibration buffer and the desired fraction eluted with 5 ml 400 mM NaC1 in buffer. Protein in the salt eluates was quantitated by differential pulse polarography [ 15], except that the reactions were performed at 10°C with a Model 303 static mercury drop electrode controlled by a Model 384 polarographic analyzer (Princeton Applied Research Corp., NJ). The amount of protein was expressed as ~A g-' from the current generated by the protein during differential pulse polarography. Cu in salt eluates was determined by atomic absorption spectroscopy. RESULTS
The results of a standard tolerance test are presented in Table I. The-deviations of growth indices from 1.00 in basal nutrient solution (0.25 uM Cu) are attributed to the extra day available for root initiation and elonga~tion in the second 4-day period as compared to the initial 3-day period of monitoring root growth. Clone 4 was tolerant of Cu compared to clones 6 and 14. For instance, at 20 pM Cu the growth index for clone 4 was high whereas it was low in both clones 6 and 14. R o o t extension growth during the 4 days of exposure to 20 pM Cu was decreased by 20% in clone 4 and by 97% and 98% of controls in clones 6 and 14, respectively. Exposure of clone 4 to increasing concentrations of Cu resulted in higher protein contents to a maximum at 40 #M external Cu and a plateau thereafter. Cu was associated with the protein and followed the same pattern (r between protein and Cu was 0.941). In the non-tolerant clones the protein in the roots increased only after the plants were exposed to 14 and 8 ~M Cu,
242 TABLE I THE R E L A T I O N S H I P B E T W E E N R O O T G R O W T H A N D T H E A M O U N T O F Cu-BINDING P R O T E I N IN R O O T S A F T E R 4 D A Y S O F E X P O S I N G T H R E E C L O N E S O F A. G I G A N T E A TO E X C E S S Cu Means ± S.E. o f 6 replicates w i t h p r o t e i n d e t e r m i n e d in d u p l i c a t e for each sample. Clones
External Cu (~M)
Growth index
Growtha (era)
Protein 0z A g-i )
Cu (~g g-i )
Clone 4
0.25 20 30 40 50
1.02 +-0.07 0.97 + 0.05 0.44 + 0 . 0 7 c 0.20_+0.03 c 0.09 _+0.02 c
90.12 _+9.01 72.53 +- 6.65 b 32.95 + 4 . 3 7 c 14.12_+1.87 c 6.63 +_ 1 . 1 9 c
8.30 +-0.52 12.81 +_0.74c 17.39+0.88c 24.73 + 1.12c 24.22 +- 1.14c
4.47 +-0.40 12.69 +- 1.14 16.20+-1.67 23.75+-1.73 23.31 + 0.96
Clone 6
0.25 3 8 14 20
1.08+-0.11 1.21+-0.18 0.83+-0.09 0.04-4-0.01 c 0.03 +-0.01 c
30.33+-3.24 37.42+-5.31 27.77+-3.02 1.15+0.15 c 0.82 _+0.29 c
17.79+-1.77 23.03+_3.41 26.37+-2.71 35.92+_3.36 c 40.68 +_5.03 c
22.16+-1.33 21.97+-3.26 29.87+-4.10 42.56+-4.75 50.79 +-5.10
Clone 14
0.25 3 8 14 20
1.13+_0.07 0.87+_0.03 c 1.19 +- 0.09 0.15 +_0.03 c 0.06 +- 0.01 c
35.97+-4.46 35.34+_7.06 45.30 -4- 3.39 4.91 + 0 . 4 1 c 1.64 +- 0.34 c
19.43+-1.74 19.17+-1.31 29.93 +- 1.43 c 37.74 _+2.87 c 39.44 +_ 4.07 c
21.88+5.54 21.32+-2.86 29.13 +- 1.61 40.59 +-4.10 52.11 _+ 4.01
a R o o t g r o w t h b e t w e e n days 3 and 7 during e x p o s u r e to excess Cu. b Significantly d i f f e r e n t f r o m t h e c o n t r o l at t h e 0.05 level. c Significantly d i f f e r e n t f r o m t h e c o n t r o l at t h e 0.01 level.
respectively. A similar pattern occurred with the Cu associated with the protein (r between protein and Cu was 0.808 for clone 6 and 0.721 for clone 14). At 20 pM Cu the Cu-binding protein c o n t e n t in the non-tolerant ecotypes had at least doubled over the controls while a 1.5-fold increase occurred in the Cu-tolerant clone. These increases in Cu-binding protein were-associated with little root extension growth in the non-tolerant clones but with continued root growth in clone 4. From the tolerance test it was n o t clear whether the tolerant and nontolerant clones were different with regard to the time of increase in Cubinding protein after exposure to excess Cu, or to the concentration of Cu. Two sets of time course experiments were conducted to explore these possibilities. In the first test, the tillers were exposed to 0.25 or 16 uM Cu -{Table II). Cu-binding protein c o n t e n t increased significantly within one day of exposing the Cu-tolerant clone 4 to 16 ~M Cu. This concentration of Cu inhibited root elongation growth by 25%. Cu-binding protein in the non-tolerant clones increased significantly only after 2 and 3 days o f exposure to 16 ~M Cu and their root growth was severely inhibited from day 1 onward. Cu was associated with the protein fractions and generally
243 TABLE II CHANGES IN R O O T G R O W T H AND Cu-BINDING P R O T E I N C O N T E N T WITH TIME OF E X P O S U R E OF T H R E E CLONES OF A. GIGANTEA TO 0.25 A N D 16 uM Cu Means ~ S.E. of 5 replicates with protein determined in duplicate for each sample. Clones Clone 4
Cu (uM) 0.25
16
Clone 6
0.25
16
Clone 14
0.25
16
Day
Growth a (cm)
Protein (uA g-l)
0 1 2 4 7
8.03 28.36 105.59 141.41
7.79 8.48 6.84 8.09 7.64
1 2 4 7
5.79 4-- 1.69 2 2 . 7 1 + 4.51 53.83 -4-_ 8.48 c 105.92 4--14.79 c
0 1 2 4
7.59 4-- 1.81 11.824-, 3.83 43.86 4-- 6.51
1 2 3 4 0 1 2 4 1 2 3 4
4-_ 2.01 4-- 5.48 4-- 3.37 4--22.93
1.68 4-0.624-0.63 4-1.29 4--
1.25 0.20 c 0.35 c 0.60 c
2.91 4-- 0.31 6.20 4-- 1.22 29.92 4-- 3.61 0.81 +_ 0.67 4-0.83 4-0.464--
0.18 0.19 b 0.20 c 0.10 c
12.92 13.45 14.95 21.71
Cu (ug g-l)
4--0.46 4--0.55 +- 0.87 4--0.53 4-,0.21
10.79 12.55 5.30 2.87 2.52
4-4-4-4-+
1.83 3.27 1.07 0.28 0.26
+0.53 c +0.81 c _+0.94 c 4-- 1.21 c
21.29 20.16 21.99 26.22
4-4-4-_+
1.61 1.74 1.51 0.95
11.45 4-,2.61 7.81 4--0.27 10.834-,2.10 8.52 +_0.42
27.24 4-- 6.04 11.43 4-- 0.84 34.144-,14.62 13.02_+ 1.93
11.46 4--1.32 14.10_+1.44 22.37+_2.66 c 29.87 _+4.97 c
41.24 4-- 5.56 50.304-- 9.19 78.87 +_17.81 67.71 4-,15.15
10.03 11.60 8.93 10.41
53.62 27.88 37.53 15.32
4--0.72 4--1.20 4--1.31 4--0.96
12.23 4-_1.54 19.93 +_2.57 32.86 4--4.58 c 33.78+_5.42 c
4--10.33 4-- 4.34 4-- 5.77 4-- 3.94
49.30 4-- 4.28 94.56 4-,16.65 166.07 4--22.26 134.834-,18.50
a R o o t growth from day 0 to harvest. b Significantly different from the control of the day at the 0.05 level c Significantly different from the control of the day at the 0.01 level increased with time of exposure to excess Cu. The declines in Cu in the controls with time are attributed to the small quantities of Cu in these roots. At the earlier harvests the masses of roots were small (35-60 mg fresh wt./tiller) which brought the precision of the analysis into question. In the second time course experiment clones 4 and 6 were exposed to three concentrations of excess Cu (Table III). The roots of the Cu-tolerant clone 4 contained significantly more Cu-binding protein within 1 day of exposure to excess Cu. By days 2 and 3 the protein content had increased further, particularly after exposure to 12 and 16 ~M Cu. Root growth was
244 T A B L E III C H A N G E S IN R O O T G R O W T H A N D Cu-BINDING P R O T E I N C O N T E N T O V E R 3 D A Y S O F E X P O S I N G TWO C L O N E S O F A. G I G A N T E A TO F O U R C O N C E N T R A T I O N S O F Cu Means +_ S.E. o f 5 replicates w i t h p r o t e i n d e t e r m i n e d in duplicate for each sample. Clones
Cu (#M)
Growth a (cm)
Protein (~A g-~)
Cu (ug g - l )
0 1 2 3
10.31 _+1.47 22.26_+2.96 20.63 _+2.12
10.18 _+0.53 9.44 _+0.49 8.98+0.59 12.30 _+ 1.23
10.65 _+0.76 11.52 _+0.25 9.69_+1.02 10.02 + 1.24
1 2 3
8.90 _+1.75 19.38 _+ 1.44 26.28 -+4.15
13.80 + 0 . 9 2 c 14.98 _+0.69 c 16.55 -+ 1.01 c
15.43 -+1.61 19.47 _+ 1.84 20.72 _+2.97
12
1 2 3
11.35_+1.18 15.19 + 3 . 0 6 b 37.70 + 1 . 1 6 c
11.66+_0.61 b 16.38 -+ 0.74 c 18.00 _+1.22 c
21.37_+1.40 20.85 -+3.00 18.25 -+1.73
16
1 2 3
7.01 _+ 0.73 14.06 _+ 1.55 b 20.74-+2.60
14.31 _+ 0.90 c 18.82 _+0.46 c 22.58+1.64 c
20.45 _+ 1.82 20.91 _+ 3.23 24.79-+4.61
0 1 2 3
4.66_+1.21 18.51_+4.49 26.26 _+ 3.24
10.90_+0.61 9.23 _+0.45 9.03-+0.67 10.00 _+ 1.24
28.13_+10.15 21.71_+ 2.37 14.64_+ 3.82 18.91 _+ 9.58
Clone 4
Clone 6
0.25
0.25
Day
1 2 3
5.52_+1.50 3.37_+0.57 c 4.99 _+ 1.09 c
11.23_+0.71 16.03-+1.37 b 21.29 _+ 1.29 c
1 3 . 6 5 + 1.62 40.61-+ 7.49 39.21 _+ 7.95
12
1 2 3
2.79 + 1.20 1.70 _+0.29 c 1.13+0.29 c
11.22 -+ 0.65 15.51-+1.08 b 24.33-+3.35 c
23.39 -+ 2.97 39.47-+ 5.92 33.13_+ 5.20
16
1 2 3
0.96 _+0.17 1.19+0.50 c 1.39 _+0.76 c
10.02 _+0.33 16.86_+1.25 23.90+0.87 c
21.64_+ 37.01+_ 32.92_+
2.15 7.62 1.82
a R o o t g r o w t h f r o m day 0 t o harvest. b Significantly d i f f e r e n t f r o m t h e c o n t r o l o f t h e d a y at t h e 0.05 level. c Significantly d i f f e r e n t f r o m t h e c o n t r o l o f t h e day at t h e 0.01 level.
inhibited somewhat by excess Cu. In clone 6 the Cu-binding protein c o n t e n t increased significantly over controls only after 2 days o f exposure to excess Cu, root elongation growth was severely inhibited after 1 day. The Cu-binding protein contents in clone 6 at any one time were the same for the three concentrations of excess Cu.
245 DISCUSSION Metallothionein is an attractive agent for intracellullar sequestration of metal because it is a metal inducible protein which itself binds Cu, Zn and Cd [9]. Exclusive measurement of this non-enzymic protein is possible b y immunological assays [ 16]. Unfortunately antibodies to the plant protein are not y e t available and the plant protein isolated so far does not cros~ react with rabbit antibodies to rat Cu-metallothionein (J.S. Garvey, pers. comm.). Quantitation of metallothioneins b y high performance liquid chromatography [17] remains to be evaluated for plant extracts. The work presented here relies on the selective purification and enrichment of metal-binding protein by anion exchange chromatography of heat denatured r o o t extracts [14]. The procedure used exceeded the precautions taken to remove interfering substances in a study of the dynamics o f metallothionein changes in mice [ 18]. Metallothionein is rich in thiol groups [8,9] and consequently is reactive at a dropping mercury electrode during differential pulse polarography [19]. The electrochemical reactivity of animal metallothioneins is validated [15]. The protein fractions analyzed for Agrostis roots gave clear wave patterns typical of thiol groups during differential pulse polarography. Cu was associated with the isolated protein (Tables I, II and III) and served as a confirmatory second measure of Cu-binding protein. The fraction of Cu measured here was from 58 to 76% of the buffer-soluble Cu of roots [14]. The measurements are referred to as Cu-binding protein pending further specific characterization and are given as ~Ag -1 for lack of a suitable plant calibration protein. These protein contents are taken as reasonable and useful first estimates in evaluating the relationship between metallothionein-like Cu-binding protein and Cutolerance in A. gigantea. Cu-binding protein is present in the three clones o f the grass and is induced to increase b y exposing the plants to increased Cu. Metallothioneinlike protein appears in t o m a t o [ 11 ], soybean [ 12], and cabbage and tobacco [13] exposed to Cd. This study was begun with the metal tolerance experiment to test the general idea that tolerance depends on the a m o u n t of metal sequestering agent in roots. One expectation was that Cu-binding protein would be high in a tolerant plant and low in a non-tolerant plant when exposed to excess Cu. The data in Tables I, II and III clearly show that for equivalent treatments the Cu-tolerant clone 4 contained less Cu-binding protein than did the non-tolerant clones on an absolute basis or relative to the controls. The higher Cu-binding protein contents in non-tolerant clones were, however, associated with little r o o t growth. These data on Cu-binding protein c o n t e n t are contrary to expectations. One possibility is that the protein measured b y differential pulse polarography is a poor estimate of metal sequestering protein, or that Cu-binding protein is not involved in Cutolerance.
246 An alternative view stems from the two time course experiments (Tables II and III) which clearly showed that the Cu-tolerant clone 4 contained more Cu-binding protein than controls within one day of exposure to excess Cu. These roots continued to elongate. The non-tolerant clones had higher Cu-binding protein contents only after 2 or 3 days, their r o o t elongation was severely inhibited from day 1 onward. These findings show that Cu-tolerance in A. gigantea is associated with a capacity to make Cu-binding protein rapidly in response to elevated Cu. It is proposed that a rapid increase in Cu-binding protein would effectively enhance intracellular metal sequestration. If dividing cells and their progeny were to respond by making Cu-binding protein rapidly, they themselves would experience low free Cu allowing for division and enlargement. Repeated acquisition of Cu-binding protein rapidly upon further entry of Cu into the cells would result in a r o o t that continues to elongate in the presence of excess Cu, as shown by the Cu-tolerant clone 4 (Tables I, II and III}. Such a scheme would bear some cost as amino acids and metabolic energy are diverted to the synthesis of Cu-binding protein. Perhaps the decreased r o o t elongation in the Cu-tolerant e c o t y p e exposed to increasing external Cu (Tables I, II and III) is an expression of such a biological cost. Work is in progress to determine what proportion of the intracellular Cu is associated with Cu-binding protein and whether the changes in protein predominate in the r o o t tips or elsewhere. The view that metal tolerance depends on the capacity to make Cubinding protein rapidly is based on the response of one clone of A. gigantea. It is n o w necessary to evaluate this model for a population of Cu-tolerant clones o f A . gigantea, extend it to other species and perhaps to the metals Zn and Cd. Experiments along these lines are in progress. The sudden exposure of roots to excess Cu and our use of nutrient solutions are completely artificial compared with field conditions. Perhaps allowing roots to initiate and grow in 0.5--1 gM Cu would precondition them so that they could better deal with subsequent increases in Cu, a situation known for Holcus lanatus pretreated with Cd [20]. However, both in our experimental situation and in Cu-rich soil, a Cu-tolerant plant such as clone 4 would be producing new r o o t tip cells which experience excess Cu during formation and enlargement. Nevertheless, the experiments reported here have clearly identified an association between the capacity to produce metallothionein-like Cu-binding protein rapidly and the expression of Cu-tolerance as r o o t elongation growth. ACKNOWLEDGEMENTS This work was supported b y Operating Grant A4921 and Strategic Grant G0529 from the Natural Sciences and Engineering Research Council of Canada. I thank Miss Linda Elder and Mr. Peter Taylor for their excellent technical support.
247
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