Plant Soil (2006) 288:333–341 DOI 10.1007/s11104-006-9124-1
ORIGINAL PAPER
Distinctive effects of cadmium on glucosinolate profiles in Cd hyperaccumulator Thlaspi praecox and non-hyperaccumulator Thlaspi arvense Roser Tolra` Æ Paula Pongrac Æ Charlotte Poschenrieder Æ Katarina Vogel-Mikusˇ Æ Marjana Regvar Æ Juan Barcelo´
Received: 13 July 2006 / Accepted: 1 September 2006 / Published online: 27 September 2006 Ó Springer Science+Business Media B.V. 2006
Abstract The influence of Cd on growth, Cd accumulation and glucosinolate (GS) contents was investigated in Thlaspi praecox in comparison to Thlaspi arvense. Accumulation of up to 2,700 lg Cd g-1 dry weight in shoots of T. praecox, growing in nutrient solution with 50 lM Cd without growth inhibition, confirmed this species as a Cd-hyperaccumulator. Cadmium increased the level of total GS in T. praecox without a statistically significant influence on total sulphur. This increase in GS was due to the enhancement of benzyl-GS, mainly sinalbin. In the Cd sensitive T. arvense Cd caused a shift from alkenyl-GS, mainly sinigrin, to indolyl-GS. The Cd-induced increase of total GS in T. praecox indicates that in this species Cd hyperaccumulation is not linked to trade-off of organic defences. The distinctive influence of Cd on GS profiles in Cd-sensitive T. arvense and Cd-tolerant T. praecox favouring indolyl-GS and benzyl-GS, respectively, is
R. Tolra` Æ C. Poschenrieder (&) Æ J. Barcelo´ Bioscience Faculty, Plant Physiology Laboratory, Autonomous University of Barcelona, E-08193 Bellaterra, Spain e-mail:
[email protected] P. Pongrac Æ K. Vogel-Mikusˇ Æ M. Regvar Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecˇna pot 111, SI-1000 Ljubljana, Slovenia
discussed in relation to jasmonate and salicylate as possible key molecules in Cd-stress transduction in these contrasting Thlaspi species. Keywords Cadmium hyperaccumulation Æ glucosinolate Æ sinalbin Æ Thlaspi arvense Æ Thlaspi praecox Abbreviation GS Glucosinolate INTRODUCTION Metal hyperaccumulating Thlaspi species have attracted much research efforts during the last decade (Tolra` et al. 1996; Wenzel and Jocker 1999; Baker et al. 2000; Clemens et al. 2002; Assunc¸ao et al. 2003; Peer et al. 2003; Roosens et al. 2003; Freeman et al. 2004; Papoyan and Kochian 2004; Puschenreiter et al. 2005; Hammond et al. 2006) mainly because of the potential use of these species for clean-up of heavy metal contaminated soils (Zhao et al. 2003; Chaney et al. 2005; Pilon-Smits 2005; McGrath et al. 2006). Another line of research addresses the benefits of metal hyperaccumulation for plants themselves, i.e. the advantage to be a hyperaccumulator for surviving among the much more numerous metallophytes with excluding strategy. Among different hypothesis, metal defence against biotic
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stress has deserved most attention, especially, but not exclusively, in Ni hyperaccumulators (Martens and Boyd 1994; Pollard and Baker 1997; Behmer et al. 2005, Jhee et al. 2006a, b). Based on current knowledge, we recently have proposed five different ways by which metals may contribute to enhance biotic stress resistance in plants: phytosanitary effects, elemental defence, trade-off of organic defences, metal therapy and metal-induced fortification (Poschenrieder et al. 2006a). Only few publications on plant self-protection by Cd are available (Ghoshroy et al. 1998; Mittra et al. 2004). Snails seem to be insensitive to the high-leaf Cd and/or Zn concentrations of the Zn/ Cd hyperaccumulator Thlaspi caerulescens, yet leaf consumption was rather related to low-glucosinolate (GS) concentrations than to metal concentrations in leaves of different soil-grown ecotypes (Noret et al. 2005). In contrast, feeding deterrence of thrips in T. caerulescens was attributed to Cd and not to GS because the inhibitory effect on the thrips was correlated to shoot Cd but not to shoot S or Zn concentrations (Jiang et al. 2005). However, to our best knowledge, the specific influence of Cd-hyperaccumulation on GS profiles has never been evaluated under controlled environmental conditions. In this study the influence of Cd on both total GS concentrations and GS profiles was investigated in two contrasting species; the Cd hyperaccumulator Thlaspi praecox and the non-hyperaccumulator Thlaspi arvense. This approach will contribute not only to clarify the possible role of GS in Cd-induced biotic stress defence, but also will allow to evaluate the relevance of GS production and Cd accumulation in the S balance of hyperaccumulator and a non-hyperaccumulator Thlaspi species.
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plants were grown for 1 week in hydroponics in a greenhouse. Growth conditions were as previously described (Tolra` et al. 1996), but with minor modifications in the nutrient solution. In brief, uniform seedlings of both species were transplanted to continuously aerated nutrient solutions (pH 6, buffered with MES) of the following composition in mM: 3 KNO3, 0.5 K2SO4, 2 Ca(NO3)2, 1 NH4H2PO4, 0.5 MgSO4; and in lM 66 Fe-EDTA, 50 KCl, 46 H3BO3, 9 MnSO4, 1.5 ZnSO4, 1.5 CuSO4, 0.14 (NH4)6Mo7O24. Control plants received the basic nutrient solution, while for the Cd treatments the basic solution was supplemented with 50 lM Cd as CdCl2. Toxicity testing using root elongation as stress indicator Length of the longest root of plants was measured with a ruler before transplantation to the nutrient solution and at the end of the experiment. Given values are means ± SE from at least ten plants per species and treatment. Plant sulphur and cadmium concentrations Roots of harvested plants were rinsed with distilled water. The oven dried (70°C) plant material was acid digested in closed vessels (69% HNO3: 30% H2O2, 5:2) in a microwave digestion system (O-I Analytical, College Station, TX, USA). Concentrations of Cd were determined with an ICP-MS device (Perkin Elmer, Boston, MA, USA, model Elan-6000). Sulphur concentrations were determined using ICPOES Simultant (Perkin Elmer Optima 3200 RL). Given values are means ± SE from three samples per species, organ and treatment.
MATERIALS AND METHODS
Glucosinolate extraction and analysis
Plant material and culture conditions
Upon harvest plant material for GS analysis was weighed and immediately frozen in liquid nitrogen. Samples were stored in a deep freezer (–80°C) until analysis.
Seeds of T. praecox were collected from plants growing on a soil polluted by mining and smelting ˇ erjav (Ljubljana, Northern Sloveactivities in Z nia) (Vogel-Mikusˇ et al. 2005); T. arvense seeds were from a commercial supplier (B & T World Seeds, Aigues Vives, France). After germination and pre-culturing for 4 weeks in potting substrate,
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Total glucosinolates Total GS were extracted with 70% (v/v) aqueous methanol in a boiling water bath for 5 min. After
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cooling and centrifugation, the pellet was reextracted. The joint liquid phases were loaded onto a DEAE-Sephadex A-25 column and treated with myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1.) (Sigma, St. Louis, MO, USA). The released D-glucose was quantified using an enzyme based analytical kit (Boehringer, Mannheim, Germany). The effectiveness of the extraction method was validated using certified reference rapeseeds (CRM 367, Hampshire, UK) from the European Community Bureau of Reference (BCR, Brussels, Belgium). Given values are means ± SE from three samples per species, organ and treatment. Individual glucosinolates Extraction of individual GSs was done as previously described (Tolra` et al. 2000). Separation and quantification was performed using a liquid chromatograph equipped with a diode array detector Model HP 1090 (Hewlett Packard, San Francisco, CA, USA). Samples of 50 lL were injected onto a Lichrospher 60RP Select B column. The mobile phases used for elution were acetonitrile and water containing 0.5% acetic acid. The gradient elution was from 0 to 60% acetonitrile in 74 min at a flow rate of 1 ml/min, at 30°C and the diode array set at 230 nm. Sinigrin (allylglucosinolate) from horseradish (SigmaAldrich, Quimica, SA, Madrid, Spain) was used as an internal standard. Gluconapin (3-butenylglucosinolate) and glucotropaeolin (benzylglucosinolate) were used as external standards. Given values are means ± SE from three samples per species, organ and treatment.
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Statistics Data were analysed by factorial ANOVA using species and treatment as independent factors (Statistica version 6.0, StatSoft Inc., St. Tulsa, OK, USA). Significance of differences was determined at p < 0.05 using Fisher LSD test. RESULTS Toxicity symptoms, root elongation and Cd accumulation Thlaspi arvense plants exposed to Cd exhibited toxicity symptoms in leaves in the form of chlorosis. No such symptoms were observed in T. praecox. Under control conditions T. praecox exhibited considerably higher root elongation rates than T. arvense. Cadmium exposure inhibited root elongation in T. arvense by 80%, while in T. praecox the high-elongation rate remained unaffected (Fig. 1). Low Cd concentrations were detected in control plants (Fig. 2). Under Cd treatment, T. arvense accumulated higher root Cd concentrations than T. praecox. In contrast, shoot Cd concentrations were substantially higher in T. praecox, where more than 2,500 lg Cd g–1 dry weight was found (Fig. 2). Total sulphur and total glucosinolate concentrations The concentrations of total GS tended to increase under Cd exposure. However, only in T. praecox
Mass spectrometric analysis For identification of individual GSs extracts were analysed by LC-APCI-MS using a liquid chromatograph system Agilent 1100 (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a mass spectrometer Esquire 3000 (Bruker Optik, Ettlingen, Germany) and an APCI interface spray chamber. Individual GS with a peak area less than 1% of the total area of peaks in a chromatogram were reported as ‘‘traces only’’.
Fig. 1 Root elongation (mm week–1) in Thlaspi arvense and Thlaspi praecox grown in nutrient solutions without (control) or with 50 lM Cd. Values are means ± SE for the longest root of ten plants per species and treatment. Asterisks denote significant differences (p < 0.05%)
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a statistically significant difference was observed (Fig. 3). No statistically significant differences in total S concentrations in roots and shoots among species and treatments were found (Fig. 4). Figure 5 shows the per cent fraction of GS-S in relation to the total S content. GS-sulphur usually represented between 15 and 25% of the total plant S, with exception of control roots of T. arvense (34%) and roots of T. praecox treated with Cd where GS-S accounted for 58% of total S. Individual glucosinolates HPLC-MS allowed identifying different GS in Thlaspi species under control or Cd supply conditions. GSs with different alkenyl-, benzyl- and indolyl-side chains were identified in T. praecox, while benzyl-GS were not detectable in T. arvense (Table 1). As an example Fig. 6 shows the chromatograms of roots of T. praecox (Fig. 6a) and T. arvense (Fig. 6b) exposed to Cd. Aromatic GS like sinalbin were only found in T. praecox
Fig. 2 Cadmium concentrations in Thlaspi arvense and Thlaspi praecox shoots (a) and roots (b) grown in control or 50 lM Cd-containing nutrient solutions. Numerical values are given for control plants because bars were smaller than lines used for drawing them. Values are means ± SE of three samples per species, organ and treatment. Asterisks denote significant differences (p < 0.05%)
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(Fig. 6a). The absence of a peak with the specific retention time of 16.1 min in the chromatogram of T. arvense (Fig. 6b) made sinalbin undetectable in this species. GSs with alkenyl side chains were the most abundant fraction in T. arvense, accounting for more than 50% of the total GS content. Cadmium supply did not alter the relative proportion of indolyl- and alkenyl-GS in roots of T. arvense, while in shoots the proportion of indolyl-GS increased by almost a 20% (Fig. 7). The Cd-induced increase of total GS found in roots and shoots of T. praecox (Fig. 4) was mainly due to an increase of sinalbin, the main benzyl-GS in this species (Fig. 7).
DISCUSSION Cadmium hyperaccumulation is a rare phenomenon that has mainly been investigated in T. caerulescens and, to a lesser extent, in Arabidopsis halleri
Fig. 3 Concentrations of total glucosinolates in shoots (a) and roots (b) of Thlaspi arvense and Thlaspi praecox grown in control or 50 lM Cd-containing nutrient solutions. Values are means ± SE of three samples per species, organ and treatment. SE value for shoots of Thlaspi arvense exposed to Cd was smaller than the line used to draw the error bars. Asterisks denote significant differences (p < 0.05%)
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Fig. 4 Sulphur concentrations in shoots (a) and roots (b) of Thlaspi arvense and Thlaspi praecox grown in control or 50 lM Cd-containing nutrient solutions. Values are means ± SE of three samples per species, organ and treatment. Means were not statistically significant at p < 0.05%
(McGrath et al. 2001). Only recently a third species, T. praecox, growing on Cd-contaminated soil in Slovenia, has also been found to hyperaccumulate Cd (Vogel-Mikusˇ et al. 2005, 2006). Results from hydroponic experiments presented here clearly confirm this Cd hyperaccumulator character of T. praecox, as well as its extraordinary Cd tolerance (Figs. 1, 2). A shoot Cd concentration of nearly 2,700 lg g–1 d.w. without growth reduction situates
Fig. 5 Proportion (%) of glucosinolate sulphur in total sulphur content of roots and shoots of Thlaspi arvense and Thlaspi praecox grown in control or 50 lM Cd-containing nutrient solutions
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this species close to the T. caerulescens ecotypes with the highest Cd shoot concentrations (Lombi et al. 2000). In contrast to the Cd sensitive T. arvense, T. praecox did not show any toxic effects or growth inhibition when exposed to 50 lM Cd in solution. Nonetheless, Cd influenced the plant’s secondary metabolism. The concentrations of total GS in roots and shoots were substantially enhanced in Cd hyperaccumulating T. praecox. This contrasts with the recent finding in Ni hyperaccumulator Streptanthus polygaloides where no difference in total GS between low- and high-Ni plants was observed (Jhee et al. 2006b). The Cd-induced increase of GS levels found in T. praecox is not in line with the trade-off hypothesis according to which metal hyperaccumulation may help to save resource allocation to energy-demanding organic defences (Boyd and Martens 1998). The Cd-induced enhancement of total GS levels in T. praecox was not matched by a statistically significant increase in root or shoot S concentrations (Fig. 4). In consequence, the proportion of S used in GS synthesis increased under Cd exposure, especially in roots of T. praecox, while the opposite trend was observed in roots of Cd sensitive T. arvense (Fig. 5). Enhanced herbivore resistance in Cd-hyperaccumulating T. caerulescens has been attributed to Cd-based elemental defence due to a lack of Cd effects on total root or shoot S (Jiang et al. 2005). In the view of our results such a conclusion should be drawn with caution, yet GS levels can increase without a substantial change in total S concentrations. Taken together results from this study and current data available in the literature indicate that interactions between Cd accumulation and biotic stress can be much more complex than envisaged by the trade-off or elemental defence hypothesis. The sulphur metabolism provides multiple points of interactions between ion toxicity and biotic stress in plants (Poschenrieder et al. 2006a, b). Sulphur compounds are known to play a crucial role in defence of plants against Cd. Phytochelatin-mediated Cd sequestration is implied in the low-level Cd tolerance of non-metallicolous species, like T. arvense, but not in the hyper-tolerance of T. caerulescens (Schat et al. 2003). Maintenance of high levels of reduced
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338 Table 1 Glucosinolates with different side chains detected in shoots and/or roots of Cd hyperaccumulator Thlaspi praecox and nonaccumulator Thlaspi arvense (n.d., not detected)
a
Traces only
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Type of side chain Alkenyl2-propenyl3-butenyl4-pentenyl2-hydroxy–4-pentenylAromatic p-hydroxybenzyl2-phenylethylIndolyl3-indolylmethyl4-hydroxyindolylmethyl 4-metoxyindolylmethyl 1-metoxyindolylmethyl
Trivial name
Thlaspi praecox
Thlaspi arvense
Sinigrin Gluconapin Glucobrassicanapin Napoleiferin
Root and Shoot Root Root and Shoota Shoota
Root and Shoot Root n.d. n.d.
Sinalbin Gluconasturtiin
Root and Shoot Roota
n.d. n.d.
Glucobrassicin 4-OH-glucobrassicin 4-metoxy-glucobrassicin Neoglucobrassicin
n.d. Root Root n.d.
Root n.d. n.d. Root and Shoot
glutathione under high-metal ion stress is essential to both metallicolous and non-metallicolous species (Howden et al. 1995; Boominathan and Doran 2003, Freeman et al. 2004). In our hydroponic system sulphur supply was clearly not a limiting factor. If there was a Cd-induced requirement for a surplus of reduced sulphur, this had no negative influence on the level of total GSs either in the Cd sensitive T. arvense or in the Cd tolerant and hyperaccumulating T. praecox. Cadmium, however, had a strong and species specific influence on the GS profiles. In Cd
sensitive T. arvense a Cd-induced shift from alkenyl-GS to indolyl-GS was found, especially in shoots. Each type of GS derives from different amino acids: alkenyl-GS from methionine and indolyl-GS from tryptophan. Cadmium can induce elicitor-like responses in plants and, in fact, jasmonate has been found to be involved in the signal transduction pathway of Cd (Xiang and Oliver 1998). Jasmonate is a strong elicitor of indolyl-GS (Jost et al. 2005; Mewis et al. 2005). Thus the shift from alkenyl to indolyl-GS observed in our Cd-stressed T. arvense plants
Fig. 6 (a) Base peak chromatogram of the molecular ion of sinalbin (retention time 16.1 min) in Thlaspi praecox treated with 50 lM Cd. Insert shows the mass spectrum of sinalbin. (b) Base peak chromatogram of the molecular ion of sinalbin in Thlaspi arvense exposed to 50 lM Cd. No
sinalbin was observed (absence of peak with retention time 16.1 min). This chromatogram is shown on a different scale (104) than that of Thlaspi praecox (106) because on a 106 scale the base peak chromatogram for peak 184 of Thlaspi arvense is completely empty
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Fig. 7 Cadmium-induced changes in profiles of glucosinolates in roots and shoots of Thlaspi arvense and Thlaspi praecox grown in control (inner ring) or 50 lM Cd-containing nutrient solutions (outer ring). The per cent value of each type of GS is represented as the arc length of a circle; the sum of total GS fractions (100%) is the circle’s perimeter. Numbers inside and outside the rings are per cent values of each fraction for controls and Cd-treated plants, respectively
could have been jasmonate-mediated. In Cd tolerant T. praecox the Cd-induced increase of total GS concentrations was mainly a consequence of enhanced p-hydroxybenzyl-GS (sinalbin) concentrations. Sinalbin can be synthesized from tyrosine or as a secondary GS, from phenylalanine. In transgenic Arabidopsis that in contrast to the wild-type was able to synthesize sinalbin, aromatic GSs stimulate salicylic acid-mediated defences, while suppressing jasmonate-dependent defences (Brader et al. 2006). Enhanced susceptibility to the fungus Alternaria brassicicola, but increased resistance towards the bacterial pathogen Pseudomonas syringae was observed as a consequence of this change in signalling pathway caused by sinalbin production. Salycilate has also been implied in the enhanced Ni tolerance of Thlaspi goesingense (Freeman et al. 2004). The distinctive features of Cd-induced changes in GS profiles in the Cd-tolerant hyperaccumulator T. praecox and the Cd-sensitive non-accumulator T. arvense found in this study, suggest clear differences between these contrasting species in Cd-induced signalling pathways for GS production and biotic stress defences. The observed
correlation between Cd hyperacumulation and the production of the aromatic GS sinalbin in T. praecox indicates a Cd-induced enhacement of the shikimate pathway. Further investigations are required to see if a Cd-induced stimulation of this pathway also favours salycilate production and salycilate mediated defense responses. In conclusion, this is the first investigation that compares the influence of Cd on GS profiles in Cdhyperaccumulating and non-hyperaccumulating Thlaspi species. A Cd-induced increase of total GS in T. praecox does not support the trade-off hypothesis in the case of Cd-hyperaccumulation. Specific Cd-induced enhancement of benzyl-GS in Cd tolerant hyperaccumulator T. praecox and the Cd-induced shift from alkenyl-GS to indolyl-GS in Cd-sensitive T. arvense indicates complex consequences of Cd pollution on plant-biotic stress interactions. Acknowledgements Supported by the Spanish and Catalonian Governments (DGICYT, BFU 2004-02237CO2-01 and Grup de Recerca 2005GR 0078). The grant by COST Action 859 to P. Pongrac for her research stay at the Autonomous University of Barcelona is gratefully acknowledged.
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