Solubility of zinc and interactions between zinc and phosphorus in the hyperaccumulator Thlaspi caerulescens

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1 Plant, Cell and Environment (1998) 21, ORIGINAL ARTICLE OA 220 EN Solubility of zinc and interactions between zinc and phosphorus in the hyperaccumulator Thlaspi caerulescens F. J. ZHAO, 1 Z.G. SHEN, 2 & S. P. McGRATH 1 * 1 Soil Science Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK, and 2 Department of Agronomy, Nanjing Agricultural University, Nanjing, People s Republic of China ABSTRACT The relationship between Zn and P in the Zn hyperaccumulator Thlaspi caerulescens J. & C. Presl was investigated using hydroponic culture. Total concentrations of Zn in the shoots increased from 0 2 to 27 g kg 1 dry mass when solution Zn increased from 1 to 1000 mmol m 3. Water-soluble Zn accounted for > 80% of the total Zn in the shoots containing > 5 g Zn kg 1 dry mass. Total P was maintained at about 3 g kg 1 dry mass in the shoots containing < 20 g Zn kg 1 dry mass, but significantly decreased with higher Zn concentrations. Linear regression between insoluble P and insoluble Zn in the shoots produced a small slope, suggesting that co-precipitation of Zn and P was not an important detoxification mechanism in the shoots. In contrast, there was a strong correlation between insoluble P and insoluble Zn in the roots, with a linear slope of 0 3 close to the P:Zn ratio in Zn 3 (PO 4 ) 2. Foliar sprays of phosphate did not affect shoot dry mass significantly, but decreased root length and root dry mass significantly at Zn concentrations in solution from 10 to 3000 mmol m 3. Foliar P was translocated to roots to enhance co-precipitation of Zn and P, although this did not enhance Zn tolerance. The results suggest that T. caerulescens possesses mechanisms which allow it to accumulate and sequester huge amounts of Zn in the shoots without causing P deficiency. Key-words: Thlaspi caerulescens; zinc; hyperaccumulation; phosphorus; tolerance. INTRODUCTION Recently there has been increased interest in the phenomenon of heavy metal hyperaccumulation in certain plant species, because this property may be exploited in phytoremediation of soils contaminated with heavy metals (McGrath et al. 1993; Baker et al. 1994). Approximately 400 taxa of terrestrial plants have been identified as hyperaccumulators of various heavy metals. Eighteen are Zn hyperaccumulators, and in their natural habitat can accumulate in the above-ground parts more than 10 g Zn kg 1 on a dry-matter basis (Baker & Brooks 1989; Baker et al. 1997). Correspondence: Steve P. McGrath. Fax: ; steve.mcgrath@bbsrc.ac.uk These plant species are generally endemic to metalliferous soils enriched with Zn. Thlaspi caerulescens J. & C. Presl (Brassicaceae) is the best known example of a Zn hyperaccumulator. Compared to non-accumulating species, T. caerulescens possesses much enhanced capacity to take up Zn and transport it from roots to shoots (Brown et al. 1995; Lasat, Baker & Kochian 1996; McGrath, Shen & Zhao 1997; Shen, Zhao & McGrath 1997). Unlike many other metal-tolerant taxa, the exceptionally high tolerance of this species is associated with hyperaccumulation. Recent studies have shown that the concentrations of Zn in the shoots of T. caerulescens can reach g Zn kg 1 dry mass before any significant yield reduction is observed (Brown et al. 1995; Shen et al. 1997). The fundamental mechanisms for metal hyperaccumulation and internal detoxification are not fully understood. Krämer et al. (1996) found that enhanced production of histidine is associated with the Ni hyperaccumulation in Alyssum. It is not known if histidine is also involved in the Zn hyperaccumulation in T. caerulescens, but the affinity of histidine for Zn is about one order of magnitude less than that for Ni. The organic acid malate is abundant in T. caerulescens, and has been proposed to play a key role in detoxification by shuttling Zn from cytoplasm into vacuoles in this species (Mathys 1977). However, the affinity of malate for Zn is relatively low. Also, samples of a population of the related species Thlaspi ochroleucum Boiss. & Heldr. accumulate much less Zn in the shoots and are less tolerant to Zn than T. caerulescens, but nevertheless contain similar amounts of malate in the shoots (Shen et al. 1997). These results suggest that high concentrations of malate alone do not explain Zn hyperaccumulation and tolerance in T. caerulescens sufficiently. Compartmentalization of Zn in vacuoles has been demonstrated in the epidermal cells of leaves of T. caerulescens (Vázquez et al. 1994). Transport of Zn across tonoplast, which is possibly a vital step for both hyperaccumulation and tolerance, has not been investigated in T. caerulescens. It is well known that increasing P supply can induce or aggravate Zn deficiency in crop species, mainly by decreasing the physiological availability of Zn as a result of precipitation of sparingly soluble Zn phosphates (Cakmak & Marschner 1987). On the other hand, Zn deficiency has been shown to result in accumulation of P to toxic levels (Loneragan et al. 1982). In conventional crop species it will not be possible to test how excessive Zn Blackwell Science Ltd

2 Zinc and phosphorus interactions in the hyperaccumulator Thlaspi caerulescens 109 interferes with P nutrition, because most crop plants suffer from toxicity when Zn concentrations reach g kg 1 dry mass (Marschner 1995). Because g kg 1 dry mass of Zn is frequently observed in Zn hyperaccumulator plants, it is pertinent to ask how these plants maintain their P nutrition. In a previous paper (Shen et al. 1997), we presented results of Zn uptake and transport, and the effects on the growth of T. caerulescens and T ochroleucum. In this study, T. caerulescens material obtained from some of the previous experiments was used to determine the concentrations of total and water-soluble Zn and P, in order to examine the relationship between Zn and P over a wide range of Zn supply. In addition, we investigated how foliar supply of P affected the growth, Zn uptake and tolerance in T. caerulescens. MATERIALS AND METHODS Seeds of T. caerulescens were obtained from a population in Prayon, Belgium. Seedlings were raised hydroponically with 0 1-strength Rorison nutrient solution (Hewitt 1966), except that Zn was 1 mmol m 3. Experiments were conducted under controlled-environment conditions: 16 h day length with photon flux density of 350 µmol m 2 s 1 supplied by warm white fluorescent tubes, 20 C/16 C day/night temperature, and 60 70% humidity. Solubility of Zn and P (experiment 1) Plant material was obtained from an experiment described previously (Shen et al. 1997). T. caerulescens plants (23 d old) were grown in nutrient solutions containing 1, 10, 50, 100, 250, 500 and 1000 mmol m 3 Zn, supplied as ZnSO 4. Each treatment was replicated four times. Other nutrients included in the solutions were (in mmol m 3 ) 400 Ca(NO 3 ) 2, 200 MgSO 4, 200 K 2 HPO 4, 9 2 H 3 BO 3, 1 8 MnSO 4, 0 2 Na 2 MoO 4, 0 32 CuSO 4, 10 8 Fe(III)-EDTA (ethylenediaminetetra-acetic acid ferric monosodium salt), and the ph was 5 7. Nutrient solutions were aerated continuously and renewed every 3 d. Plants were harvested on day 35 after treatments were started. Shoots and roots were separated, washed with tap water and then with deionized water, and blotted dry with tissue paper. Samples were dried at 80 C for 16 h, and ground in an agate ball mill. Plant materials were digested with a mixture of HNO 3 /HClO 4 and the total concentrations of Zn and P were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES; Fisons ARL Maxim III) (Zhao, McGrath & Crosland 1994). Water-soluble Zn and P were extracted with 1 mol m 3 MES (2-morpholinoethanesulphonic acid) buffer at ph 6 0 (Cakmak & Marschner 1987), and the concentrations determined by ICP-AES. MES does not complex with either Zn or P, and serves as a ph buffer only. Insoluble Zn and P were calculated as the difference between the total and water-soluble concentrations. One-way analysis of variance was performed on all data sets. Where appropriate, linear or quadratic equations were fitted to the relationships between the concentrations of total, water-soluble or insoluble Zn and P. Effects of foliar P supply (experiment 2) Seedlings of T. caerulescens were raised in 0 1 strength Rorison nutrient solution containing 1 mmol m 3 Zn for 36 d before being used for the experiment. Six plants were transferred to a 1 dm 3 polycarbonate vessel. There were six treatments, consisting of factorial combinations of three Zn concentrations (10, 1000 or 3000 mmol m 3, supplied as ZnSO 4 ) and two foliar sprays of deionized water or 15 mol m 3 NaH 2 PO 4 (both containing 100 cm 3 m 3 Triton X-100, ph 5 2). Foliar sprays were applied every 5 d on four occasions, with a volume of 3 cm 3 per pot on each occasion. Each treatment was replicated in four vessels. The concentrations of other nutrients in the nutrient solutions were (in mmol m 3 ): 400 Ca(NO 3 ) 2, 200 MgSO 4, 50 K 2 HPO 4, 300 KCl, 9 2 H 3 BO 3, 1 8 MnSO 4, 0 21 Na 2 MoO 4, 0 31 CuSO 4 and 10 8 Fe(III)-EDDHA (ethylenediamine-di(o-hydroxyphenylacetic acid). MES (2 mol m 3 ) was used to buffer solution ph at 6 0. Nutrient solutions were aerated continuously and renewed every 3 d. Plants were harvested 24 d after Zn treatments started. Shoots and roots were separated, washed with tap water and then with deionized water, and blotted dry with tissue paper. Fresh weights were determined. Samples were dried at 80 C for 16 h, and dry weights determined. Total and water-soluble Zn and P were determined as described above. Two-way analysis of variance was performed on all data sets. RESULTS The activities of free Zn 2+ ions in different nutrient solutions (Table 1) were calculated using GEOCHEM-PC (Parker, Norvell & Chaney 1995). In all treatments, free Zn 2+ accounted for 50 95% of the total Zn in solution. The program predicted formation of the Zn 3 (PO 4 ) 2 solid in the nutrient solutions containing 250 mmol m 3 Zn, with increasing percentages of the P added in the solution present as Zn 3 (PO 4 ) 2 (Table 1). Solubility of Zn in T. caerulescens shoots and roots (experiment 1) Total Zn concentration in shoots increased from 0 2 g kg 1 dry mass in the 1 mmol m 3 Zn treatment to 27 g kg 1 dry mass in the 1000 mmol m 3 Zn treatment (Fig. 1a). A large percentage of the total Zn in shoots was water soluble, and this percentage increased from 60 with 1 mmol m 3 Zn in solution to > 80 in the treatments with 50 mmol m 3 Zn in solution (Fig. 1c; P < 0 001). For ions that are present in soluble forms in plant cells, it would be physiologically more relevant to express their concentrations on the volumetric basis of tissue water. The concentration of watersoluble Zn in the tissue water of T. caerulescens shoots increased from 400 to mmol m 3 when solution Zn concentrations increased from 1 to 1000 mmol m 3.

3 110 F. J. Zhao et al. Experiment 1 Experiment 2 Treatment (Zn mmol m 3 ) log[zn 2+ ] % of P as Zn 3 (PO 4 ) 2 log[zn 2+ ] % of P as Zn 3 (PO 4 ) Table 1. Concentrations of Zn in nutrient solutions, calculated activities of free Zn 2+ ions ( log[zn 2+ ]) and percentages of the added P precipitated as Zn 3 (PO 4 ) 2 in the two experiments with Thlaspi caerulescens In the roots, total Zn concentrations increased from 0 08 g kg 1 dry mass in the 1 mmol m 3 Zn treatment to 44 g kg 1 dry mass in the 1000 mmol m 3 Zn treatment (Fig. 1b). The percentages of water-soluble Zn in the total were considerably lower than those in the shoots, varying between 27 and 57 (Fig. 1c). In contrast to the values obtained in the shoots, the percentage of water-soluble Zn decreased as the concentration of Zn in solution increased, particularly from 1 to 250 mmol m 3. The Zn treatments had little effect on the concentration of water-soluble P in the roots (Fig. 3b). Insoluble P was relatively small in the low Zn treatments, but increased markedly as the Zn concentration in solution increased. There was a close positive correlation between insoluble P and insoluble Zn in the roots (Fig. 3c). Relationships between Zn and P concentrations in shoots (experiment 1) Concentration of total P in the shoots of T. caerulescens was maintained at 3g kg 1 dry mass in the treatments containing mmol m 3 Zn in solution. Total P began to decrease slightly in the 500 mmol m 3 Zn treatment, and was about 2 3 g kg 1 dry mass with 1000 mmol m 3 Zn in solution (Fig. 2a). A quadratic equation described the relationship between total P and total Zn in the T. caerulescens shoots satisfactorily, but the negative quadratic component was only important at high Zn concentrations (Fig. 2a). Water-soluble P accounted for 75% of the total P in the shoots in the treatments containing mmol m 3 Zn, but the percentage began to decrease in the 500 mmol m 3 Zn treatment and was only 57% in the treatment with highest Zn. The relationship between water-soluble P and water-soluble Zn (Fig. 2b) was similar to that between total P and Zn, except that the decline in water-soluble P was larger. In contrast to the water-soluble fractions, insoluble P in the shoots increased slightly as insoluble Zn increased (Fig. 2c). Relationships between Zn and P concentrations in roots (experiment 1) Roots of T. caerulescens had much higher concentrations of total P (Fig. 3a) than the shoots. Furthermore, increasing the concentration of Zn in solution from 1 to 1000 mmol m 3 doubled the concentration of P in the roots. A close positive relationship between total P and total Zn in the roots was evident (Fig. 3a). Figure 1. Concentrations of total and water-soluble Zn in (a) the shoots, (b) the roots, and (c) the percentage of the total Zn that was water-soluble in T. caerulescens. Bars represent SEs. Where no error bar is shown, the standard error was smaller than the size of the symbol.

4 Zinc and phosphorus interactions in the hyperaccumulator Thlaspi caerulescens 111 The effects of foliar P spray were generally consistent across the three different concentrations of Zn in the nutrient solution. Thus, there were no significant interactions between the Zn and P treatments. Increasing solution Zn increased the concentration of total P in the roots, but decreased the concentration of P in the shoots (Table 3). Foliar P spray increased total P in both shoots and roots, with the shoots showing greater increases than the roots. In the roots, the increase of total P attributable to foliar spray was greater in the high-zn treatments than the low-zn treatments. The Zn treatment had large effects on the concentrations of total and water-soluble Zn in the shoots and roots (Table 3), but the largest concentrations of total Zn in the shoots Figure 2. Relationships between the concentrations of (a) total P and total Zn, (b) water-soluble P and water-soluble Zn and (c) insoluble P and insoluble Zn in the shoots of T. caerulescens. The concentrations (mmol m 3 ) of Zn in the nutrient solutions were: 1 l; 10 s; 50 n; 100 ; 250 t; 500 ; The slope of the linear regression was 0 3, a value close to the P:Zn ratio of 0 31 in Zn 3 (PO 4 ) 2. Effects of foliar P spray (experiment 2) T. caerulescens plants grown with 1000 mmol m 3 Zn were smaller than those with 10 mmol m 3 Zn, although no toxicity symptoms occurred in the former treatment. The plants grown with 3000 mmol m 3 Zn showed severe toxicity symptoms, and the shoot and root dry mass were decreased by 63% and 99%, respectively, compared to the 10 mmol m 3 Zn treatment (Table 2). Foliar P spray had no significant effect on the shoot dry mass, but decreased the root dry mass and main root length significantly (Table 2). Figure 3. Relationships between the concentrations of (a) total P and total Zn, (b) water-soluble P and water-soluble Zn and (c) insoluble P and insoluble Zn in the roots of T. caerulescens. The concentrations (mmol m 3 ) of Zn in the nutrient solutions were: 1 l; 10 s; 50 n; 100 ; 250 t; 500 ; 1000.

5 112 F. J. Zhao et al. Treatment Shoot DM Root DM Root length Root length Zn (mmol m 3 ) P (spray) (g pot 1 ) (g pot 1 ) at 10 d (cm) at 22 d (cm) 10 P P P P P P SED a ANOVA F ratio Zn 46 8 *** 81 8 *** 81 6 *** *** P 1 5 NS 4 5 * 11 9 ** 4 5 * Ζn P 0 8 NS 3 2 NS 1 9 NS 2 8 NS Table 2. Effects of foliar P spray and solution Zn concentration on shoot and root dry matter and the length of main root of T. caerulescens a Standard error of difference for the foliar P treatment (error d.f. = 18). * P < 0 05; ** P < 0 01; *** P < 0 001; NS, not significant; DM, dry mass. Table 3. Effects of foliar P spray on the concentrations of total and water-soluble P and Zn in the shoots and roots of T. caerulescens P concentration (g kg 1 dry mass) Zn concentration (g kg 1 dry mass) Shoot Root Shoot Root Treatment Zn(mmol m 3 ) P spray Total Soluble Total Soluble Total Soluble Total Soluble 10 P P P P P nd nd + P nd nd SED a ANOVA F ratio Zn 20 0*** 28 2*** 203 9*** 3 2 NS 350 2*** 444 2*** 479 4*** 628 1*** P 18 2*** 30 1*** 82 9*** 6 52** 0 3 NS 0 5 NS 25 5*** 1 2 NS Zn P 3 6* 6 0** 20 6*** 1 3 NS 0 1 NS 0 3 NS 15 1*** 1 3 NS a Standard error of difference for the foliar P treatment (error d.f. = 18); nd, samples not enough for the determination; * P < 0 05; ** P < 0 01; *** P < 0 001; NS, not significant. was obtained with 1000 mmol m 3 Zn instead of the 3000 mmol m 3 Zn. Foliar P spray had little effect on the concentrations of total and soluble Zn in the shoots (Table 3). In contrast, the total Zn concentration in the roots was increased significantly by the foliar P spray, particularly in the highest Zn treatment, which showed a 40% increase. Water-soluble Zn in the roots appeared to be little affected by the foliar P spray. DISCUSSION Previous studies using hydroponic culture have shown that the Zn hyperaccumulator T. caerulescens can accumulate Zn in the shoots to g kg 1 dry mass without showing toxicity symptoms or significant growth depression (Brown et al. 1995; Shen et al. 1997). The present study shows that a large proportion of the Zn accumulated in the shoots of this species was present in water-soluble forms. The proportion of water-soluble Zn in shoots exceeded 80% when total Zn was > 5 g kg 1 dry mass. This is much higher than the range of 30 60% found with cotton and citrus leaves, which contained < g kg 1 total Zn (Cakmak & Marschner 1987). The high proportion of water-soluble Zn in the shoots of T. caerulescens rules out apoplast precipitation as an important detoxification mechanism, but suggests that the excess Zn is complexed with soluble organic compounds and stored in the vacuoles. Evidence from X-ray microanalysis also shows that Zn was accumulated predominantly in the vacuoles of epidermal cells of T. caerulescens leaves (Vázquez et al. 1994). It is not clear which soluble organic compounds are responsible for chelating Zn in T. caerulescens. Shoots of T. caerulescens contain abundant amounts of malate (Mathys 1977; Shen et al. 1997), but this is a constitutive property of the species (Tolrà et al. 1996; Shen et al. 1997). Furthermore, malate has a low affinity to chelate Zn, and there is little evidence

6 Zinc and phosphorus interactions in the hyperaccumulator Thlaspi caerulescens 113 that the Zn-malate complex exists. Malate may be involved in Zn chelation in vacuoles because of its high concentration, but it does not explain why T. caerulescens can hyperaccumulate Zn in the shoots (Shen et al. 1997). Binding of Zn with myoinositol hexakisphosphate (phytate) in root cortical cells has been suggested as a detoxification mechanism in a Zn-tolerant ecotype of Deschampsia caespitosa (L.) Beauv. (Van Steveninck et al. 1987). If Zn is precipitated with P-containing compounds or with inorganic phosphate in T. caerulescens, there should be a positive relationship between insoluble P and Zn with a slope close to the P:Zn ratio in the particular compound formed. The relationship between insoluble P and insoluble Zn in the shoots of T. caerulescens was weak, and linear regression between the two produced a slope of 0 07, which is much smaller than the P:Zn ratios of 0 31, 0 95 and 1 43 for Zn 3 (PO 4 ) 2, Zn 3 - phytate and Zn 2 -phytate, respectively. Therefore, precipitation of Zn with inorganic phosphate or phytate is unlikely to play a significant role in the Zn tolerance in the shoots of T. caerulescens. This is in agreement with the observations of Vázquez et al. (1994), who found small P:Zn ratios in the globular crystals in many vacuoles of epidermal cells of T. caerulescens leaves. In the shoots of T. caerulescens, the concentrations of total P remained relatively constant up to total Zn concentrations of 20 g kg 1 dry mass. Significant decreases in the concentrations of total and water-soluble P in the shoots occurred when total Zn exceeded 20 g kg 1 dry mass. This suggests that the transport of P from roots to shoots was suppressed, and possibly a small amount of coprecipitation of P with Zn in shoots. The relationship between Zn and P in the roots of T. caerulescens was very different to that found in the shoots. The concentration of total P in the roots increased sharply with total Zn, solely because of the increases of insoluble P. The slope of the linear regression between insoluble P and Zn in the roots was close to the P:Zn ratio of Zn 3 (PO 4 ) 2, indicating precipitation of Zn 3 (PO 4 ) 2 in the root tissues. This is not surprising, because computations using GEOCHEM-PC indicated that Zn 3 (PO 4 ) 2 would form in the nutrient solutions containing 200 mmol m 3 P and 250 mmol m 3 Zn. Precipitation of Zn 3 (PO 4 ) 2 is likely to occur on the root surface or in the apoplast. Availability of P is likely to be low in soils that are heavily contaminated with heavy metals such as Zn and Pb. Huang, Chen & Cunningham (1997) found that the weed species goldenrod (Solidago bicolor L.) grown in Pb-contaminated soils showed severe P deficiency symptoms, and foliar sprays of P increased shoot and root dry mass by more than fourfold. With a high concentration of Zn in solution and hyperaccumulation of Zn in the shoots of T. caerulescens, it may be expected that plants could become deficient in P, as shown by significant decreases of both total and water-soluble P in the shoots. However, the present study showed no positive effects of foliar P supply on the growth of T. caerulescens with Zn concentrations in solution ranging from 10 to 3000 mmol m 3. In fact, root length and root dry mass were decreased significantly by foliar P. The reason for the decreased root growth resulting from foliar P is not clear. However, it can be concluded that extra P supply through foliar sprays does not increase the tolerance to Zn in T. caerulescens. It is also interesting to note that the foliar P treatment increased the concentration of P in the roots more than that in the shoots. Foliar P sprays also increased the concentration of Zn in the roots significantly, especially as the concentration of Zn in the nutrient solution increased. In the 10 and 1000 mmol m 3 Zn treatments where root samples were sufficient for analysis of water-soluble Zn, the increase in total Zn caused by the foliar P treatment did not result in an increase of watersoluble Zn in the roots, indicating that increased co-precipitation of P and Zn took place in the roots. CONCLUSION The present study shows that T. caerulescens is able to take up and transport a large quantity of Zn to the shoots, and to maintain 80% of the total Zn there in water-soluble forms, without inducing P deficiency. Detoxification of Zn in the shoot cannot be explained by co-precipitation with inorganic phosphate or phytate. 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