ZINC TOLERANCE IN BETULA SPP. I. EFFECT OF EXTERNAL CONCENTRATION OF ZINC ON GROWTH AND UPTAKE BY HILARY J. DENNY AND D. A.

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1 New Phytol. (1987) 106, ZINC TOLERANCE IN BETULA SPP. I. EFFECT OF EXTERNAL CONCENTRATION OF ZINC ON GROWTH AND UPTAKE BY HILARY J. DENNY AND D. A. WILKINS Department of Plant Biology, University of Birmingham, Birmingham B15 2TT, UK {Accepted 19 February 1987) SUMMARY Clones of Betula pendula Roth, and B. pubesce Ehrh., collected as seed from zinc-contaminated mine tailings, were grown aseptically in a liquid/perlite medium. Three zinc-tolerant and four non-tolerant genotypes were compared with respect to exteion growth and zinc uptake over a range of external concentratio of zinc. The effect of increasing zinc concentration on total root length and root cell length was also examined. Exteion growth declined rectilinearly as the external zinc concentration increased, but the relatiohip between uptake and external concentration was not rectilinear. After an initial proportional increase, concentratio of zinc in tissues remained cotant over much of the range of external concentratio at which some growth was possible. Fresh weight concentratio of zinc in tolerant and non-tolerant genotypes were similar at similar external concentratio. However, at a characteristic threshold external concentration, control of uptake broke down and plants were inundated with zinc. The decrease in exteion growth was found to be due mainly to a progressive inhibition of cell elongation. Zinc tolerance in Betula lies in an ability to maintain proportionally more exteion growth at similar internal and external zinc concentratio. Neither a tolerance mechanism involving internal detoxification, nor binding of zinc io to electronegative sites in the cell walls, are coistent with the results. However, these findings are compatible with a tolerance mechanism involving control of uptake operating at the endodermis. Key words: Betula, zinc tolerance, zinc uptake, threshold concentration. INTRODUCTION Soils containing potentially toxic concentratio of metals such as lead, zinc and copper are found on ore outcrops, or at sites where the ores have been mined, smelted or otherwise processed. Many plant species are known to have evolved metal tolerance in respoe to metalliferous soils (Antonovics, Bradshaw & Turner, 1971). In temperate zones, most species which colonize these sites are herbaceous, and much of the physiological investigation of the mechanisms of tolerance has been carried out on such plants. However, Betula pendula Roth, and B. pubesce Ehrh. are two essentially pioneer species of open habitats that are common throughout Britain, and they are found growing in lead- and zinccontaminated mine tailings, of moderate calcium content, in Shropshire and mid-wales. Brown & Wilki (1985) took seed samples of Betula spp. from zinccontaminated and control sites. These were cultured aseptically on agar. Work on clonal material revealed difterences of tolerance between genotypes. Chemical analysis of leaf material showed there was reduced uptake of zinc in tolerant plants. The aim of the present experiments was to gain a better understanding of the X/87/ $03.00/ The New Phytologist ANP 106

2 5i8 H. J. DENNY AND D. A. W i L K i N S mechanism of zinc tolerance. A more detailed comparison of the effects of zinc on growth and zinc uptake by Betula clones of different tolerance was therefore undertaken. M A T E R I A L S AND M E T H O D S Culture of Betula Seed was collected in autumn from Betula spp. trees growing on metalliferous soils at the same Shropshire sites listed by Brown & Wilki (1985). Seed lots B64 and B109 came from Gravels (grid ref. SJ334002), and B112 from Snailbeach (SJ374021). Seeds were surface-sterilized and germinated on water agar. Seedlings were traferred to glass boiling tubes containing 15 g acid-washed perlite and 10 cm^ Ingestad standard start solution (Ingestad, 1971). This was sufficient to thoroughly wet the perlite without causing it to float. Perlite at the top of the medium was well aerated, that at the bottom was waterlogged. Each tube had a vented, tralucent plastic cap and was autoclaved before use. Perlite was found to produce better plant growth and survival than agar. The seedlings were grown for 8 weeks and were then cloned by placing single node cuttings on nutrient agar supplemented with sucrose and IAA. After rooting, the cuttings were traferred to growth tubes for a further 8 weeks. Clones established in this way were screened for zinc tolerance, using the tolerance ratio method of Wilki (1957). Three tolerant genotypes, B64.6, B64.5, B112.8, and four non-tolerant genotypes, B64.1, B109.4, B109.6, B64.3, were selected for further investigation. The experimental treatments were obtained by adding different amounts of zinc sulphate to the nutrient medium. Perlite adsorbs substantial amounts of zinc io in applied solutio. Therefore, zinc concentratio were coiderably higher than those used elsewhere in studies employing culture solutio. Control treatments always contained 0-75 fimol dm"^ zinc sulphate as a micronutrient. All experiments were carried out in a controlled environment at 20 C, with continuous light from fluorescent tubes at an irradiance of 7-8 W Growth and zinc uptake Plants were harvested after 8 weeks. Lengths of shoots and roots were measured, and tissues were dried and weighed. Zinc contents of roots and shoots were assessed by atomic absorption spectrophotometry after digestion in concentrated nitric acid. An analysis of variance was conducted on all results. Root cell length A length of root of approximately 5 cm, which had grown during the experiment, was removed from each plant. The root tips, including the meristems and cells that were not fully elongated were cut off. The remaining pieces were placed in small glass vials containing 1 cm^ macerating solution (5 % chromic acid in 2 % HCl), at 70 C for 2 h. After separating the cells by shaking, one drop of cell suspeion was examined by light microscopy. The length of 24 cells from each of 20 plants was measured at x 400, under phase contrast. Experimental design Experiment 1. Cuttings of six genotypes, B64.6, B64.5, B64.1, B64.3, B109.6, B109.4, were divided between five zinc concentratio (0*00075, 0-8, 1-5, 2-3 and 3-1 mmol zinc dm"^) in a fully randomized design. All of the B64 clones had five replicates at each concentration, the B109 and B112 clones had three. (This was due to a lack of suitable material at this early stage in the work.)

3 Zinc tolerance in Betula spp. I 519 Experiment 2. Cuttings of five genotypes, B64.6, B64.5, B64.1, B112.8 and B109.6, were divided between five zinc treatments (22, 3-2, 3-9, 4-6 and 7'0 mmol dm~^) with four replicates at each concentration, in a fully randomized design. Experiment 3. Five replicates of the genotypes, B64.6, B64.5, B112.8, B64.1, B109.6 and B109.4, were grown at 2 mmol dm"^ for 4 weeks. The plants were then harvested, shoots were weighed, then dried, reweighed and analyzed for zinc. Experiment 4. Rooted cuttings of a single clone of intermediate zinc tolerance were grown at , 0-8, 15 and 24 mmol dm"^, with five replicates at each concentration. The tubes were fully randomized. The length of the longest root, and the length of 24 fully elongated cortical cells, without root hairs, on each plant was measured. RESULTS Experiment 1 Exteion growth of stems and roots declined rectilinearly with increasing zinc concentration in the medium [Fig. l(a), (b)]. Very highly significant, straight line regressio could be calculated for these growth characters (Table 1). Shoot and root dry weights were far less severely affected by zinc [Fig. l(c), (d)], and the straight line regressio calculated from these data from each genotype were less significant. This was particularly true for root dry weights. The stem and root length regressio were compared. It was found that, in both cases, the lines for the tolerent genotypes cut the jc-axis at higher zinc concentratio than those for the non-tolerant genotypes. These differences in ^-intercept values between tolerant and non-tolerant genotypes were statistically significant when tested by the method of Zar (1984). The jc-intercept was taken as indicating the zinc concentration at which growth would just cease. Therefore, this result implied that the tolerant genotypes could survive to higher zinc concentratio than non-tolerant genotypes. The values of zinc concentration in shoot and root of genotypes within the tolerant and non-tolerant classes were combined and compared (Fig. 2). The pattern of uptake did not appear to be rectilinear. At both the low and high extremes of zinc concentratio, the concentration of zinc in the plant parts was proportional to the concentration of zinc in the medium, but between 0-8 and 2*3 mmol dm~^ zinc in the medium, the zinc concentration in the plant changed little. Up to and including 2-3 mmol dm"^ zinc, the differences in internal zinc concentratio between tolerant and non-tolerant genotypes were not significant, but at 3-1 mmol dm"^ the zinc concentration in non-tolerant genotypes rose suddenly to become significantly higher than that in tolerant plants. This pattern of uptake was particularly striking in the shoots. It would appear that low-level increases in the external zinc concentration lead to an increase in internal zinc concentratio that is tolerated by Betula spp. At a certain concentration uptake becomes restricted. The internal concentration of zinc is then maintained more or less cotant until at a threshold external zinc concentration, which is lower in less tolerant genotypes, there is total failure in the control of uptake. At this point, the plant is lethally inundated with zinc. 18-2

4 52O H. J. DENNY AND D. A. WILKINS 100 (d) ^- 4 - \ ^- ^"^ 2 - \ ' *... \ ' -- ^ Concentration of Zn in medium (mmol Fig. 1. Mean growth of six genotypes of Betula spp. grown aseptically for 8 weeks at a range of zinc concentratio, (a) Stem length, (b) root length, (c) shoot dry weight, (d) root dry weight. Key to genotypes: tolerant; B64,6; B64.5; B112.8: non-tolerant; - B 109.4; B109.6; B64.1, Table 1. Summary of the results of regression analysis for growth rates of six Betula genotypes as a function of zinc concentration in the growth medium Exteion growth Dry weight Genotype Stem r Root r Shoot r Root r B64.6 B64.5 B64,l B109.4 B109.6 B112.8 **# **« **# **# ##* # * *#* r = product moment correlation coefficient. Asterisks denote level of significance: * P ^ 0-05; ** P ^ 001; P ^ ;,P> Experiment 2 The results were particularly prone to experimental error because, by design, the zinc treatments were lethal or nearly so. Thus, only small amounts of tissue were available for analysis. Only data for shoots are shown because these are probably the most reliable (Fig. 3). In each case, the entire shoot had grown during the experiment, while most of the root tissue was present at the start of the experiment. Moreover, exposure of roots to zinc was patchy, while the shoots received zinc solely via the root tissues. Uptake patter in roots and shoots were similar to those in experiment 1, with

5 Zinc tolerance in Betula spp. I 521 cs Concentration of Zn in medium (mmol dm"') Fig. 2. Mean zinc concentration in the roots and shoots of three zinc-tolerant and three non-tolerant genotypes of Betula spp. grown aseptically for 8 weeks at a range of zinc concentration in the nutrient medium. Tolerant roots; non-tolerant roots; tolerant shoots; non-tolerant shoots. c 0) u o u 0 I Concentration of Zn in medium (mmol dm~ Fig. 3. Mean zinc concentration in the shoots of five genotypes oi Betula spp., differing in tolerance to zinc, grown aseptically for 8 weeks, at a range of zinc concentratio in the nutrient medium. Key to genotypes: tolerant; B112.8; B64.5; B64.6: non-tolerant; B64.1; B three distinct phases. All of the genotypes show an initial rise in concentratio of zinc in tissue with increasing external concentratio. B64.1 is the least tolerant genotype and showed a sudden increase in internal concentratio between 2-3 and 3-1 mmol dm~^; the other genotypes showed little change in the zinc concentratio in their tissues over the same external range. B109.6 is the second most non-tolerant genotype and appeared to have a threshold lethal concentration between 39 and 4-6 mmol dm~^ zinc in the medium. B64.6 is the most tolerant; it did not appear to reach a threshold concentration. The other two genotypes took intermediate positio. These results support a hypothesis that uptake is limited until an external concentration is reached at which control fails and the plant is inundated with zinc. There are clear indicatio that the more tolerant genotypes can maintain control to higher external zinc concentratio than less tolerant genotypes.

6 522 H. J. DENNY AND D. A. WILKINS Experiment 3 Fresh weight concentratio of zinc within shoots of the six genotypes showed some variation (Fig. 4). However, analysis of variance revealed that betweengenotype variation was unlikely to be due to differences in zinc tolerance (Table 2). It would appear that concentratio of zinc in vivo are similar in the shoots of all Betula clones, whether tolerant or non-tolerant, providing that the external zinc concentration at which they are grown is not above the threshold concentration for individual genotypes. o B64-6 B64-5 BII2-8 BI09-4 BI Betula genotype Fig. 4. Mean fresh weight concentratio in the shoots of three zinc-tolerant (64.6, B64.5, B112.8) and three non-tolerant (B109.4, B109.6, B64.1) genotypes of Betula spp. grown aseptically, for 4 weeks, at an external concentration of zinc of 2-0 mmol Table 2. Analysis of variance of results of experiment 3 Due to df Sum of squares Mean squares Tolerance Genotypes within tolerance Error Total Fresh weight concentratio of zinc were measured in shoots of three zinc-tolerant and three non-tolerant genotypes of Betula after growth aseptically, for 4 weeks, at 2-0 mmol dm"^ zinc, with five replicates of each genotype Concentration of Zn in medium (mmoi Fig. 5. Mean root length (- ) and root cell length ( ) in a Betula clone after 8 weeks growth. under aseptic conditio, at four zinc concentratio, and expressed as a tolerance ratio (Wilki, 1957). 2-5

Zinc tolerance in Betula spp. I 523 Experiment 4 Mean values for root length and lengths of fully elongated cells were calculated for each zinc concentration.

7 Zinc tolerance in Betula spp. I 523 Experiment 4 Mean values for root length and lengths of fully elongated cells were calculated for each zinc concentration. These were converted to tolerance ratios so that the two types of measurement could be more easily compared. Both cell length and total root length showed a rectilinear decline with increasing external concentration of zinc (Fig. 5). DISCUSSION Zinc reduced exteion growth rates in Betula as the applied zinc concentration increased. The data do not lend themselves to regression analysis, but indicate that the zinc-induced reduction in growth rate was principally due to an inhibition of cell elongation, particularly at lower concentratio, and not of cell division, as Horst, Wagner & Marschner (1983) report for the cowpea {Vigna unguiculata) with respect to aluminium toxicity. The present conclusion is supported by studies of lead toxicity to Triticum aestivum (Lane, Martin & Garrod, 1978). Dry mass accumulation was less severely affected by exogenous zinc. Root dry weights were hardly affected at all. The retilinear decline in exteion growth with increasing zinc concentratio in the medium was not accompanied by a rectilinear increase in tissue zinc concentratio. Itead, a three-stage pattern of uptake was evident. Initially, tissue concentratio were proportional to external concentratio. Then, over much of the range of zinc concentratio at which Betula could survive, the internal zinc concentratio were kept more or less cotant. Eventually, a threshold concentration was reached at which control of uptake broke down. At this point the concentration of zinc in the plant's tissues rose very rapidly. The indicatio are that such increases are fatal. It was noted in experiment 1 that the maintenance of cotant zinc concentratio in tissues was more obvious in the shoots than the roots. This would be explained if uptake control operates at the endodermis. In this case, the great majority of the zinc in the shoot would have passed through the endodermis via the symplasm, because the Casparian strip would block to a greater or lesser extent passive movement through the apoplast to the stele. The root, on the other hand, would contain zinc in the symplast, which had been subject to active control of uptake and, in addition, zinc in those apoplastic spaces of the cortex freely accessible to the rooting medium and not subject to control. Genotypes of Betula spp. differ in their tolerance of zinc. Tolerance is manifested in an ability to maintain proportionally greater growth and to survive at higher zinc concentratio than non-tolerant plants. Just as Betula clones differ in the concentratio at which they just cease to grow, so they differ in the exogenous zinc concentratio at which their control of zinc uptake breaks down. The two types of threshold point seem to be positively linked and may be identical. There are aspects of these results which appear to be somewhat paradoxical. Whatever mechanism of tolerance is operating in Betula, it must be compatible with the following findings: there is a rectilinear decline in growth as external zinc concentratio increase, but zinc concentratio within the tissues of the plants remain roughly cotant over much of the concentration range at which Betula can survive; rates of exteion growth in tolerant clones decline less rapidly than in non-tolerant clones, despite there being similar internal zinc concentratio in both.

8 524 H. J. DENNY AND D. A. WILKINS The mechanism of tolerance is not one in which the more tolerant genotypes somehow exlude more of the metal from the symplast, thereby maintaining lower zinc concentratio within their tissues. This would seem to preclude a cell wall binding mechanism, like that proposed by Peterson (1969) and Turner (Turner, 1970; Turner & Marshall, 1972) playing an important part in tolerance. Baker (1981) identified two main groups of metal-tolerant plants, namely 'excluders' and 'accumulators'. Betula would appear to fall into the former category, given its maintenance of cotant internal concentratio until a threshold point at which control breaks down. However, this would appear to be a rather artificial classification in that Baker coidered that internal detoxification of metal io is probably central to the tolerance mechanism in both types of plant. The finding here, that tolerant plants maintain better growth than non-tolerant plants even though they have similar internal concentratio of the metal, is compatible with the operation of such a mechanism. However, this type of mechanism will not explain why exteion growth rates continue to decline with increasing zinc concentratio, despite the tissue concentratio remaining more or less cotant. Exteion growth of the shoot cannot be being affected directly by the presence of zinc in the cell wall or cytoplasm. Zinc must infiuence exteion indirectly through some unknown biochemical interaction ACKNOWLEDGEMENTS We thank Professor F. T. Last and Dr Julia Wilson of ITE, Bush for their advice and assistance, and the Science and Engineering Research Council (UK) for financial support. REFERENCES ANTONOVICS, J., BRADSHAW, A. D. & TURNER, R. G. (1971). Heavy metal tolerance in plants. Advances in Ecological Research, 7, BAKER, A. J. M. (1981). Accumulators and excluders - strategies in the respoe of plants to heavy metals. Journal of Plant Nutrition, 3, BROWN, M. T. & WILKINS, D. A. (1985). Zinc tolerance in Betula. New Phytologist, 99, HoRST, W. J., WAGNER, A. & MARSCHNER, H. (1983). Effect of aluminium on root growth, cell-division rate and mineral element contents in roots of Vigna unguiculata genotypes. Zeitschrift fiir Pfianzenphysiologie, 109, INGESTAD, T. (1971). A definition of optimum nutrient requirements in birch seedlings. II. Physiologia Plantarum, 24, LANE, S. D., MARTIN, E. S. & GARROD, J. F. (1978). Lead toxicity effects on indole-3-acetic acid induced cell elongation. Planta, 144, PETERSON, P. J. (1969). The distribution of zinc-65 in Agrostic tenius Sibth. and A. stolonifera L. tissues. Journal of Experimental Botany, 20, TURNER, R. G. (1970). Subcellular distribution of zinc and copper within roots of metal tolerant clones of Agrostis tenuis Sibth. New Phytologist, 69, TURNER, R. G. & MARSHALL, C. (1972). The accumulation of zinc by subcellular fractio of roots oi Agrostis tenuis Sibth. in relation to zinc tolerance. New Phytologist, 71, WILKINS, D. A. (1957). A technique for the measurement of lead tolerance in plants. Nature, 180, ZAR, J. H. (1984). Comparing simple linear regression equatio. In: Biostatistical Analysis, Chapter 17, 2nd Edn. Prentice-Hall, Englewood Cliffs, New Jersey.

THE RESPONSE OF ROOT ACID PHOSPHATASE ACTIVITY TO HEAVY METAL STRESS IN TOLERANT AND NON-TOLERANT CLONES OF TWO GRASS SPECIES

Netv Phytol. (\9S0) 96, 359-364 THE RESPONSE OF ROOT ACID PHOSPHATASE ACTIVITY TO HEAVY METAL STRESS IN TOLERANT AND NON-TOLERANT CLONES OF TWO GRASS SPECIES S ' ''', " ' i, \..- ' - ' ;. BY R. M. C O