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Cadmium effects on growth and mineral nutrition of two halophytes: Sesuvium portulacastrum and Mesembryanthemum crystallinum
ARTICLE IN PRESS Journal of Plant Physiology 162 (2005) 1133—1140
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Cadmium effects on growth and mineral nutrition of two halophytes: Sesuvium portulacastrum and Mesembryanthemum crystallinum Tahar Ghnayaa, Issam Nouairib, Ine `s Slamaa, Dorsaf Messedia, c a, Claude Grignon , Chedly Abdelly , Mohamed Habib Ghorbeld a
Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, INRST, BP 95, Hammam-Lif 2050, Tunisie Laboratoire de Caracte´risation et de la Qualite´ de l’huile d’Olive, INRST, BP 95, Hammam-Lif 2050, Tunisie c Biochimie et Physiologie Mole´culaire des Plantes, Agro-M INRA, 34060 Montpellier CEDEX 1, France d Nutrition azote´e et prote ´ines de stress, Faculte´ des Sciences de Tunis, 1060 Tunis, Tunisie b
Received 23 July 2004; accepted 8 November 2004
KEYWORDS Cd accumulation; Halophytes; Mesembryanthemum crystallinum; Sesuvium portulacastrum; Nutritional disturbance; Phytoremediation
Summary Growth, cadmium accumulation and potassium and calcium status were studied in two halophytes from Aizoaceae family: Sesuvium portulacastrum and Mesembryanthemum crystallinum. After multiplication, the seedlings were cultivated on nutrient solution supplemented with NaCl (100 mM) and CdCl2 (0, 50, 100, 200 and 300 mM). After 1 month of treatment, plants were harvested and the dry weight, as well as the Cd, K and Ca concentrations in tissues were determined. Results showed that S. portulacastrum, a perennial halophyte with slow growth, is significantly more tolerant to Cd than M. crystallinum, an annual plant. Cd severely inhibited Mesembryanthemum growth even at the lowest Cd concentration in culture medium (50 mM), and did not modify significantly that of Sesuvium. For both halophytes, Cd accumulation was significantly higher in the roots than in the shoots. However, Cd concentration reached 350–700 mg g1 DM in the shoots, values characteristic of Cd hyperaccumulator plants. The addition of Cd in the culture medium led to a disturbance of Ca and especially K nutrition, suggesting the possibility to improve plant growth and Cd phytoextraction of both halophytes by increasing nutrient availability in the culture medium. & 2005 Elsevier GmbH. All rights reserved.
Corresponding author. Tel.: +216 71 430 855; fax: +216 71 430 934.
E-mail address: [email protected] (C. Abdelly). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.11.011
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Introduction Some heavy metals, such as Fe, Mn, Zn and Mo, are essential micronutrients and play important roles in plant physiology. However, high concentrations may become toxic and induce severe growth reduction, such as for Mo (Brune and Dietz, 1995) and Mn (Mukhopadhyay and Scharma, 1991). Others metals like Cd and Pb, with no identified physiological role, are non-essential and could be phytotoxic even at low concentrations (Va ´zquez et al., 1992). Over the past century, mining, manufacturing, intensive agriculture and urban activities have greatly contributed to heavy metal contamination of soils (Cunningham et al., 1995; Laughlin et al., 1996). Dietary intake of heavy metals-contaminated crop plants affect human health in the long term by damaging nervous, pulmonary and renal systems (Aoshima et al., 2003). Hu ¨ttermann et al. (1999) showed that Cd accumulation in the upper layers of forest soil, even at relatively low concentrations, impaired the natural regeneration of forest species like Pinus sylvestris, thereby contributing to forest decline. Heavy metals cannot be biodegraded, and thus should be extracted from the contaminated sites. A recent technique using plants to clean contaminated areas, or phytoremediation, is intensively studied and progressively applied to the field. One practical approach of phytoremediation is metal removal by phytoextraction (Jordan et al., 2002). Phytoextraction requires an efficient transfer of metals from roots into the shoots to maximise metal removal by cropping of plant materials. It also requires high biomass yields. For example, the Indian mustard, Brassica juncea, has been reported to moderately accumulate a variety of metals (Pb, Cu and Zn) from contaminated soils with reasonable biomass yields of 7.2 t ha1 year1 (Blaylock et al., 1997). Most plants used for metal accumulation are crop plants, including sunflower (Helianthus annuus), corn (Zea mays), pea (Pisum sativum) and mustard (B. juncea) (Huang et al., 1997; Epstein et al., 1999). All these plants are classified as glycophytes. However, the saline depressions, colonised by halophyte species, often constitute sites of accumulation of industrial effluents contaminated by heavy metals. Indeed, preliminary studies achieved in various regions of Tunisia showed that these zones are contaminated by cadmium, nickel and lead (Nouari et al., 2002). Halophytes are promising candidates for soil desalination. For example, Glenn et al. (1999) showed that Atriplex nummularia can achieve a biomass yield of 20–30 t ha1 year1 and have been shown to accumulate up to 40% NaCl in their dry
T. Ghnaya et al. matter. Recent studies suggest that halophytes may be useful for phytoremediation in contaminated salty soils (Glenn et al., 1999; Williams et al., 1994; Jordan et al., 2002). Within the framework of this approach, we analysed some physiological aspects involved in the responses to different Cd concentrations in Sesuvium portulacastrum and Mesembryanthemum crystallinum (halophytes from Aizoaceae family). We paid a particular attention to parameters implicated in plant growth (dry matter production, chlorophyll concentrations and nutrient uptake), and to Cd distribution between roots and shoots.
Materials and methods Plant material and growth parameters S. portulacastrum was propagated by cutting. Three centimetre-long stem segments with one node and two opposite leaves were taken from mother plants growing under natural condition, in a mixture of sandy soil and organic matter and irrigated with tap water. They were disinfected for 5 min in saturated calcium hypochlorite solution, and rinsed generously with distilled water. They were then placed for 7 days in an aerated Hewitt (1966) solution diluted 10 times, supplemented with 4.5 mM Fe EDTA (Jacobson, 1951) and micronutrients (Arnon and Hoagland, 1940). Rhizogenesis took place after 1 week. Seedlings of M. crystallinum were obtained by germination. The seeds were sterilised by dipping into a 10% H2O2 solution during 20 min. They were then washed with distilled water and sown on perlite imbibed with distilled water. They remained in the dark, at room temperature for 7 days. The rooted cutting (S. portulacastrum) and the seedlings (M. crystallinum) were transferred for 15 days on 1/10 Hewitt solution supplemented with micronutrient and with 4.5 mM Fe in a glass house with mean temperatures (night–day) of 18–25 1C and relative humidity 80–70%. Thereafter, the medium was changed for full strength Hewitt solution, with micronutrients and 45 mM Fe plus 100 mM NaCl and Cd at various concentrations: 0, 50, 100, 200 and 300 mM CdCl2. The culture solution was renewed weekly. Two harvests were made, at the beginning of the treatment and 30 days of Cd treatment. At harvest, shoots were successively rinsed three times in cold water and blotted between two layers of filter paper. Roots were dipped in a cold solution of CaCl2 during 5 min to eliminate heavy metals adsorbed at the root surface (Stolt et al., 2003), then washed
ARTICLE IN PRESS Responses of Sesuvium portulacastrum and Mesembryanthemum crystallinum to cadmium
Desiccated samples were ground to a fine powder using a porcelain mortar and pestle, then digested in 4/1 (v/v) HNO3/HClO4 mixture. Cd and Ca concentrations were determined by atomic absorption spectrometry (Perkin-Elmer Analyst 300). K amounts were determined in the same homogenate by flame spectrometry (corning photometer).
Chlorophyll assay
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Figure 1. Variation of total chlorophyll concentration in young leaves of S. portulacastrum and M. crystallinum treated during 30 days by various Cd concentrations. Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
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Analyses of variance (ANOVA) with orthogonal contrasts and mean comparison procedures were used to detect differences between treatments. Mean separation procedures were carried out using the multiple range tests with Fisher’s least significant difference (LSD) (po0.05).
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Statistical analysis
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three times with cold distilled water and blotted with filter paper. The fresh weight was immediately determined, and the dry weight was measured after 48 h of desiccation in an oven at 60 1C.
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Figure 2. Changes in whole plant dry matter (g plant1) produced by M. crystallinum and S. portulacastrum treated by various Cd concentrations. Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
Plant morphology and growth After 15 days of treatment, chlorosis was visible on young leaves of plants treated by Cd, in both species. Two weeks later, chlorosis was accentuated and other toxicity symptoms were evident. In Sesuvium, petiole necrosis and leaf fall were observed at highest Cd concentrations, 200 and 300 mM. In Mesembryanthemum, young leaf acute chlorosis and necrosis occurred even at the lowest Cd concentration (50 mM). The chlorophyll concentrations (Fig. 1) confirmed the visual observation of sensitivity to cadmium more pronounced in Mesembryanthemum than in Sesuvium. At 50 mM Cd2+, the chlorophyll concentrations were diminished to 25% of the control in Mesembryanthemum leaves, and remained unchanged in Sesuvium leaves. In the absence of Cd, the biomass of Mesembryanthemum plants was five-fold larger than that of Sesuvium (Fig. 2). However, Cd severely inhibited Mesembryanthemum growth even at the
lowest concentration (50 mM) and did not modify significantly that of Sesuvium. Root growth was less sensitive to Cd than was shoot growth. Indeed, 50 mM Cd treatment resulted in decrease in shoot/root ratio of Mesembryanthemum from 12.0 to 5.4 (Table 1). For Sesuvium, the changes in shoot/root ratio were not significant.
Cadmium accumulation For both Sesuvium and Mesembryanthemum, the Cd concentration in the root tissues was higher than in shoots (Fig. 3). The Cd concentrations in roots were similar in both species, ranging from approximately 2500 (at external 50 mM Cd) to about 5000 mg g1 DM (at 300 mM Cd). In shoots, Cd concentrations differed between the two species, accumulation in Mesembryanthemum shoots being twice that of Sesuvium. But, since the shoot
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Table 1. Changes in shoot DW/root DW ratio in S. portulacastrum and M. crystallinum with Cd concentrations in the culture medium
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Figure 3. Changes in Cd concentration (mg g1 DW) in shoots and roots of S. portulacastrum and M. crystallinum. Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
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biomass of Mesembryanthemum was less than of Sesuvium in the presence of Cd, the amounts of Cd (mg plant1) accumulated in the shoots were comparable between the two species (Fig. 4). Specific differences appeared at the level of the roots: the Cd amounts found in these organs were 2–3 times higher in Sesuvium than in Mesembryanthemum.
Potassium nutrition Cd treatment resulted in a large decrease in Mesembryanthemum shoots K concentration: at 50 mM Cd, the K concentration in these organs was
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Figure 4. Distribution of the cadmium absorbed by the whole plant (mg plant1) between roots and shoots of S. portulacastrum and M. crystallinum. Means of eight replicates.
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Figure 5. Changes in potassium concentration (mmol/ g DW) in shoots of S. portulacastrum and M. crystallinum with Cd concentration in culture medium. The numbers inside the bars are the amounts of K in the shoots (mmol plant1). Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
approximately 20% of the control value (Fig. 5). A slight increase in shoot K concentration from 50 to 300 mM Cd was associated with diminishing biomass production and unpronounced changes in tissues K amount. In Sesuvium, the shoot K+ concentration was only slightly diminished (9–22% of control) in the presence of the highest Cd concentrations. The opposite behaviour was observed in roots: K concentration was diminished in Sesuvium, and was
ARTICLE IN PRESS Responses of Sesuvium portulacastrum and Mesembryanthemum crystallinum to cadmium not strongly modified in Mesembryanthemum (not shown). Changes in mean quantities of K in the shoots (mmol plant1) showed that this parameter was significantly decreased in S. portulacastrum only from 200 mM Cd in the nutrient solution. This decrease reached 45% under 300 mM Cd. In M. crystallinum, the depressive effect of Cd on K content was more pronounced, appearing from 50 mM Cd and reaching 94%, independent of Cd concentration in nutrient solution.
Calcium nutrition In Mesembryanthemum, the Ca concentration in shoots was higher in plants treated with Cd (Fig. 6). However, the corresponding total amounts of Ca were strongly diminished in these plants. Thus, the increase in Ca concentration results from growth inhibition by Cd. For Sesuvium, both the amounts of Ca and its concentrations in shoots are slightly modified by Cd. Only at 300 mM Cd, Ca contents diminished by 42%, but without any significant change in Ca concentration. The presence of Cd in the culture medium did not modify significantly the Ca root concentration in M. crystallinum, and decreased this parameter in S. portulacastrum (not shown). However, the roots of the last species remained generally more rich in Ca than those of M. crystallinum.
Discussion Addition of cadmium in the medium culture decreased growth of both halophytes and disturbed their K and Ca nutrition. From biomass data,
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S. portulacastrum appeared more tolerant than M. crystallinum. In addition, Cd was accumulated in shoots at high concentrations, reaching 350–700 mg g1 DW, respectively, in Sesuvium and Mesembryanthemum. We discuss in this section parameters which would be implied in this reduction of growth. The dry matter measured at the final harvest depends on the initial size of the plant (before the beginning of the treatments) and on its growth during the treatment. Relative growth rate (RGR), i.e. the rate of increase in total dry weight per unit of plant dry weight, is recommended to evaluate the effect of the environmental constraints on the growth activity. Analysis of the relationship between RGR and Cd concentration in shoots (Fig. 7) showed the increase of Cd concentration in photosynthetic organs is associated with a reduction in the RGR. However, the two species presented a contrasting behaviour in their reaction to Cd. In Mesembryanthemum, the RGR shifted from high (0.13 103 day1) to low values (0.03–0.04 103 day1) when Cd was present in the medium, irrespective of its concentration in tissues. In Sesuvium, the RGR was already low in the absence of Cd (ca. 0.03 103 day1), and diminished progressively when Cd accumulation was augmented. This analysis suggests that the marked sensitivity of Mesembryanthemum growth to the Cd treatment results either from a hypersensitivity of its metabolism to absorbed Cd, or is not related to the internal amount of Cd. On the contrary, the linear correlation between Cd accumulation in Sesuvium shoots and the inhibition of
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Figure 6. Changes in calcium concentration (mmol g1 DW) in shoots (A) of S. portulacastrum and M. crystallinum with Cd concentration in culture medium. The numbers inside the bars are the amounts of Ca in the shoots (mmol plant1). Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
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Figure 7. Changes in RGR with Cd concentration in the shoots tissues of S. portulacastrum and M. crystallinum. RGR measures the quantity of biomass deposited by 1 g of biomass per unit of time. It was estimated as DlnðDWÞ=Dt; where DW is the dry weight, ln stands for natural logarithm and D represents the difference between final and initial value (Hunt, 1990). Means of eight replicates.
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their growth is compatible with the hypothesis of a direct relation between the two phenomena. Analysing the effect of Cd on many plant species, Vassilev and Yordanov (1997) concluded that Cd diminishes RGR through inhibiting mainly net assimilation rate (NAR), and to a lesser extent, by restricting leaf area ratio (LAR). Inhibition of NAR is caused by disturbances of dark respiration and photosynthesis. Decreased LAR in Cd-treated plants is a consequence of the decrease of turgor potential and cell wall elasticity, resulting in smaller size of leaf cells formed with smaller intercellular space area. Some of our data may support the depressive action of Cd on cellular turgor. Indeed, in both halophytes, Cd induced shoot dehydration. However, this effect was significantly more severe in M. crystallinum than in S. portulacastrum. In plants subjected to 300 mM Cd, leaf water content decreases by 60% in the first species against 25% only in the second (not shown). In both halophytes, root growth was largely more tolerant to Cd than shoot growth. However, specific differences were observed. In M. crystallinum, these organs accumulated high concentrations of Cd, and their growth was negatively correlated to Cd accumulation (Fig. 8). On the contrary, in Sesuvium roots growth was insensitive to Cd treatment, in spite of the high values of Cd concentration in these organs (Fig. 8). The highest tolerance of the roots to Cd, relative to shoots, has been reported in several studies. For example, in P. sylvestris, growth was affected in shoots more than in roots, although Cd was mainly accumulated in the roots (Kim et al., 2003). Di Cagno et al. (1999) found a reduction in the dry weight of the leaves of H. annuus grown in soil irrigated with nutrient solution containing 2 mg Cd L1. Although Cd concentration in the roots was higher than that of the young leaves, Cd did not affect the root dry weight significantly. Several
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Figure 8. Relationship between root growth (DW, g/ plant1) and Cd concentration in these organs. Means of eight replicates.
mechanisms were proposed to explain the capacity of the roots to accumulate large quantities of Cd without deleterious effect on their growth: (i) Cd fixation on pectocellulosic walls (Nishizono et al., 1989), even if some studies showed that these extra cellular sites may fix only small quantities of Cd (Weigel and Ja ¨ger, 1980), (ii) the immobilisation of Cd in the endocellular compartment by complexation with organic acids, like malate, citrate (Harmens et al., 1994) and malonate (Clemens, 2001), and by sequestration with phytochelatins (Clemens, 2001; Bruns et al., 2001) or metallothioneins (Goldsborough, 2000), (iii) the higher capacity to sequester Cd in the vacuoles in roots compared to shoots (Ishikawa et al., 1997). In addition to the direct effects of internal Cd on growth activity, our results show that Cd in the medium culture decreases K concentrations in different organs. Disturbances of K nutrition under Cd treatment have been reported in several studies. For example, a dramatic decrease of K shoot content was observed in white lupin (Zornoza et al., 2002) and P. sylvestris (Kim et al., 2003) under cadmium treatment. Cd seems to complex ATP and reduces the availability of energy on which the functioning of membrane transport systems depends. Consequently, this constraint can decrease the uptake of K by roots of stressed plants (Asp et al., 1994). The result is an important fall of K concentrations in different organs (Ouzounidou et al., 1994). Changes in the K and other nutrients shoot translocation could also be attributed to the alteration in the vascular system and a reduction in the number and diameter of xylem vessels (Barcelo et al., 1988). However, little is known as to whether Cd has a direct effect upon uptake and distribution of K. Our results indicate that Cd of the culture medium affected plant growth more than Ca uptake and translocation towards the shoots in the two species, resulting in an increase in Ca concentration in tissues, in spite of a decrease of Ca contents. The effect of Cd on calcium nutrition is still difficult to evaluate because they frequently show conflicting results. Some studies showed that Cd induces an increase in Ca contents in different part of stressed plants (Greger et al., 1991; Larbi et al., 2002). However, other studies showed that Cd supply reduces Ca in leaves of Fagus sylvatica (Breckle and Kahle, 1992), P. pinea, P. pinaster and F. angustifolia (Arduini et al., 1998) and in shoots of Betula pendula (Gussarsson, 1994). According to Clemens et al. (1998) Cd compete with Ca and other cations for the entry into plant cells. This result was confirmed by Kim et al. (2002), who showed the existence of a competition of Cd with
ARTICLE IN PRESS Responses of Sesuvium portulacastrum and Mesembryanthemum crystallinum to cadmium Ca for transport into root cells. Another cause of the restriction to Ca translocation to leaves might be the sequestration of this ion as oxalate crystals in the xylem vessels of Cd-treated plants (Barcelo et al., 1988). These data suggest that the induced nutritional disturbances by heavy metals are implicated in the restriction of the growth under metal constraint, and may cause a limited heavy metals phytoextraction capacity. According to recent data, S. portulacastrum, which appeared relatively tolerant to Cd, may produce 17 t ha1 year1 of dry matter (Gleen et al., 1996). On the basis of our results relating to the Cd concentration in the shoots (350 mg g1 DM), Sesuvium would enable to extract 5950 g Cd ha1 year1. This capacity of Cd extraction is comparable with that expressed by Thlaspi caerulescens, which ranged between 4160 and 8320 g Cd ha1 year1 (Robinson et al., 1998). However, the potential for Cd extraction by Sesuvium would largely exceed those of B. juncea (60–80 g ha1 year1, Blaylock et al., 1997) or Nicotinana tabacum (500 g Cd ha1 year1, Gupta et al., 2001). Since M. crystallinum accumulate a higher Cd concentration in their shoots (700 mg g1 DM), it can be also used for the phytoextraction of this metal. However, this plant suffers from a severe growth reduction, which may be due to nutritional disturbances and probably to a direct Cd toxic effect. Our results suggest that the increase in nutrient availability in the rhizosphere could improve the growth of the plants, and would increase their capacity to extract heavy metals. Indeed, it has been reported in some studies that the increase in the Ca and Mg availability increases the growth of plants subjected to Pb and Cd and reduces their concentration in tissues (Kim et al., 2002). However, the amount of extracted heavy metals that corresponds to the product of the Cd concentration by the biomass would be more important under condition of high Ca or Mg availabilities. Others studies (Epstein et al., 1999; Laurie et al., 1995; Jordan et al., 2002) showed that utilisation of chelators like EDTA and rhamnolipids facilitate plant metal uptake. In conclusion, our results showed that both halophytes accumulate large amounts of Cd in their tissues, suggesting the possibility of their use to decontaminate the saline soils polluted by Cd. Indeed, the Cd concentrations, ranging between 350 and 700 mg g1 DM, are similar to those found in hyperaccumulator plants (Brown et al., 1994). However, the high Cd accumulation is perhaps associated with a growth restriction induced (at least partially) by the disturbance of K nutrition. The implication of the nutritional disturbances in
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the growth restriction under Cd treatment will be assessed with split root systems ensuring a load of the shoots with Cd and an appropriate K nutrition.
Acknowledgments This work was performed in the Laboratory of Plant Adaptation to Abiotic stresses. It was supported by the PRC project ‘‘Utilisation des halophytes pour la re´habilitation et la valorisation des sols salins’’ of the Tunisian Secretariat of State of Scientific Research and Technology.
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T. Ghnaya et al. Jacobson L. Maintenance of iron supply in nutrient solutions by a single addition of ferric-potassiumethylene-diamine-tetraacetate. Plant Physiol 1951; 26:411–3. Jordan FL, Robin-Abbott M, Maier RM, Glenn EP. A comparison of chelator-facilated uptake by a halophyte and a glycophyte. Environ Toxic Chem 2002; 21:2698–704. Kim C-G, Bell JNB, Power SA. Effects of soil cadmium on Pinus sylvestris L. seedling. Plant Soil 2003;257:443–9. Kim YY, Yang YY, Lee Y. Pb and Cd uptake in rice roots. Physiol Plant 2002;116:368–72. Larbi A, Morales F, Abadı´a A, Gogorcena R, Lucena J, Abadı´a J. Effects of Cd and Pb in sugar beet plants grown in nutrient solution: induced Fe deficiency and growth inhibition. Funct Plant Biol 2002;29:1453–64. Laughlin MJ, Tiller KG, Naidu R, Stevens DG. Review: the behaviour and environmental impact of contaminants in fertilizers. Aust J Soil Res 1996;34:1–54. Laurie SH, Tancock N, McGrath SP, Sanders JR. Influence of EDTA complexation on plant uptake of manganese (II). Plant Sci 1995;109:231–5. Mukhopadhyay MJ, Scharma A. Manganese in cell metabolism of higher plants. Bot Rev 1991;57:118–49. Nishizono H, Kubota K, Suzuki S, Ishii F. Accumulation of heavy metals in cell walls of Polygonum cuspidatum roots from metalliferous habitats. Plant Cell Physiol 1989;30:595–8. Nouari I, Ghnaya T, Messeddi D, Zarrouk M, Abdelly C, Ghorbel MH. Etude de la croissance et de la capacite d’accumulation des me´taux lourds chez deux halophytes: Mesembryanthemum crystallinum et Sesuvium portulacastrum. XIIIe `mes J Biol l’Assoc Tunis Sci Biol 2002:112. Ouzounidou G, Elefthriou EP, Karataglis S. Ecophysiological and ultrastructural effects of copper in Thlaspi ochroleucum (Cruciferae). Can J Bot 1994;70:947–57. Robinson BH, Meblanc L, Petit D, Brooks RR, Kirkman JH, Greg PEH. The potential of Thlaspi caerulescens for phytoremediation of contaminated soils. Plant Soil 1998;203:47–56. Stolt JP, Sneller FEC, Brynelsson T, Lundborg T, Schat H. Phytochelatin and cadmium accumulation in wheat. Environ Exp Bot 2003;49:21–8. Vassilev A, Yordanov I. Reductive analysis of factors limiting growth of cadmium-treated plants: a Rev Bulg. J Plant Physiol 1997;23(3–4):114–33. Va ´zquez MD, Poschenrieder C, Barcelo ´ J. Ultrastructural effects and localization of low cadmium concentrations in bean roots. New Phytol 1992;120:215–26. Weigel HJ, Ja ¨ger HJ. Subcellular distribution and chemical from of cadmium in bean plants. J Plant Physiol 1980;65:480–2. Williams TP, Bubb JB, Lester JN. The occurrence and distribution of trace metals in halophytes. Chemosphere 1994;28:1189–99. Zornoza P, Va ´zquez S, Esteban E, Ferna ´ndez-Pascual M, Carpena R. Cadmium-stress in nodulated white lupin: strategies to avoid toxicity. Plant Physiol Biochem 2002;40:1003–9.
www.elsevier.de/jplph
Cadmium effects on growth and mineral nutrition of two halophytes: Sesuvium portulacastrum and Mesembryanthemum crystallinum Tahar Ghnayaa, Issam Nouairib, Ine `s Slamaa, Dorsaf Messedia, c a, Claude Grignon , Chedly Abdelly , Mohamed Habib Ghorbeld a
Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, INRST, BP 95, Hammam-Lif 2050, Tunisie Laboratoire de Caracte´risation et de la Qualite´ de l’huile d’Olive, INRST, BP 95, Hammam-Lif 2050, Tunisie c Biochimie et Physiologie Mole´culaire des Plantes, Agro-M INRA, 34060 Montpellier CEDEX 1, France d Nutrition azote´e et prote ´ines de stress, Faculte´ des Sciences de Tunis, 1060 Tunis, Tunisie b
Received 23 July 2004; accepted 8 November 2004
KEYWORDS Cd accumulation; Halophytes; Mesembryanthemum crystallinum; Sesuvium portulacastrum; Nutritional disturbance; Phytoremediation
Summary Growth, cadmium accumulation and potassium and calcium status were studied in two halophytes from Aizoaceae family: Sesuvium portulacastrum and Mesembryanthemum crystallinum. After multiplication, the seedlings were cultivated on nutrient solution supplemented with NaCl (100 mM) and CdCl2 (0, 50, 100, 200 and 300 mM). After 1 month of treatment, plants were harvested and the dry weight, as well as the Cd, K and Ca concentrations in tissues were determined. Results showed that S. portulacastrum, a perennial halophyte with slow growth, is significantly more tolerant to Cd than M. crystallinum, an annual plant. Cd severely inhibited Mesembryanthemum growth even at the lowest Cd concentration in culture medium (50 mM), and did not modify significantly that of Sesuvium. For both halophytes, Cd accumulation was significantly higher in the roots than in the shoots. However, Cd concentration reached 350–700 mg g1 DM in the shoots, values characteristic of Cd hyperaccumulator plants. The addition of Cd in the culture medium led to a disturbance of Ca and especially K nutrition, suggesting the possibility to improve plant growth and Cd phytoextraction of both halophytes by increasing nutrient availability in the culture medium. & 2005 Elsevier GmbH. All rights reserved.
Corresponding author. Tel.: +216 71 430 855; fax: +216 71 430 934.
E-mail address: [email protected] (C. Abdelly). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.11.011
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Introduction Some heavy metals, such as Fe, Mn, Zn and Mo, are essential micronutrients and play important roles in plant physiology. However, high concentrations may become toxic and induce severe growth reduction, such as for Mo (Brune and Dietz, 1995) and Mn (Mukhopadhyay and Scharma, 1991). Others metals like Cd and Pb, with no identified physiological role, are non-essential and could be phytotoxic even at low concentrations (Va ´zquez et al., 1992). Over the past century, mining, manufacturing, intensive agriculture and urban activities have greatly contributed to heavy metal contamination of soils (Cunningham et al., 1995; Laughlin et al., 1996). Dietary intake of heavy metals-contaminated crop plants affect human health in the long term by damaging nervous, pulmonary and renal systems (Aoshima et al., 2003). Hu ¨ttermann et al. (1999) showed that Cd accumulation in the upper layers of forest soil, even at relatively low concentrations, impaired the natural regeneration of forest species like Pinus sylvestris, thereby contributing to forest decline. Heavy metals cannot be biodegraded, and thus should be extracted from the contaminated sites. A recent technique using plants to clean contaminated areas, or phytoremediation, is intensively studied and progressively applied to the field. One practical approach of phytoremediation is metal removal by phytoextraction (Jordan et al., 2002). Phytoextraction requires an efficient transfer of metals from roots into the shoots to maximise metal removal by cropping of plant materials. It also requires high biomass yields. For example, the Indian mustard, Brassica juncea, has been reported to moderately accumulate a variety of metals (Pb, Cu and Zn) from contaminated soils with reasonable biomass yields of 7.2 t ha1 year1 (Blaylock et al., 1997). Most plants used for metal accumulation are crop plants, including sunflower (Helianthus annuus), corn (Zea mays), pea (Pisum sativum) and mustard (B. juncea) (Huang et al., 1997; Epstein et al., 1999). All these plants are classified as glycophytes. However, the saline depressions, colonised by halophyte species, often constitute sites of accumulation of industrial effluents contaminated by heavy metals. Indeed, preliminary studies achieved in various regions of Tunisia showed that these zones are contaminated by cadmium, nickel and lead (Nouari et al., 2002). Halophytes are promising candidates for soil desalination. For example, Glenn et al. (1999) showed that Atriplex nummularia can achieve a biomass yield of 20–30 t ha1 year1 and have been shown to accumulate up to 40% NaCl in their dry
T. Ghnaya et al. matter. Recent studies suggest that halophytes may be useful for phytoremediation in contaminated salty soils (Glenn et al., 1999; Williams et al., 1994; Jordan et al., 2002). Within the framework of this approach, we analysed some physiological aspects involved in the responses to different Cd concentrations in Sesuvium portulacastrum and Mesembryanthemum crystallinum (halophytes from Aizoaceae family). We paid a particular attention to parameters implicated in plant growth (dry matter production, chlorophyll concentrations and nutrient uptake), and to Cd distribution between roots and shoots.
Materials and methods Plant material and growth parameters S. portulacastrum was propagated by cutting. Three centimetre-long stem segments with one node and two opposite leaves were taken from mother plants growing under natural condition, in a mixture of sandy soil and organic matter and irrigated with tap water. They were disinfected for 5 min in saturated calcium hypochlorite solution, and rinsed generously with distilled water. They were then placed for 7 days in an aerated Hewitt (1966) solution diluted 10 times, supplemented with 4.5 mM Fe EDTA (Jacobson, 1951) and micronutrients (Arnon and Hoagland, 1940). Rhizogenesis took place after 1 week. Seedlings of M. crystallinum were obtained by germination. The seeds were sterilised by dipping into a 10% H2O2 solution during 20 min. They were then washed with distilled water and sown on perlite imbibed with distilled water. They remained in the dark, at room temperature for 7 days. The rooted cutting (S. portulacastrum) and the seedlings (M. crystallinum) were transferred for 15 days on 1/10 Hewitt solution supplemented with micronutrient and with 4.5 mM Fe in a glass house with mean temperatures (night–day) of 18–25 1C and relative humidity 80–70%. Thereafter, the medium was changed for full strength Hewitt solution, with micronutrients and 45 mM Fe plus 100 mM NaCl and Cd at various concentrations: 0, 50, 100, 200 and 300 mM CdCl2. The culture solution was renewed weekly. Two harvests were made, at the beginning of the treatment and 30 days of Cd treatment. At harvest, shoots were successively rinsed three times in cold water and blotted between two layers of filter paper. Roots were dipped in a cold solution of CaCl2 during 5 min to eliminate heavy metals adsorbed at the root surface (Stolt et al., 2003), then washed
ARTICLE IN PRESS Responses of Sesuvium portulacastrum and Mesembryanthemum crystallinum to cadmium
Desiccated samples were ground to a fine powder using a porcelain mortar and pestle, then digested in 4/1 (v/v) HNO3/HClO4 mixture. Cd and Ca concentrations were determined by atomic absorption spectrometry (Perkin-Elmer Analyst 300). K amounts were determined in the same homogenate by flame spectrometry (corning photometer).
Chlorophyll assay
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Figure 1. Variation of total chlorophyll concentration in young leaves of S. portulacastrum and M. crystallinum treated during 30 days by various Cd concentrations. Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
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Analyses of variance (ANOVA) with orthogonal contrasts and mean comparison procedures were used to detect differences between treatments. Mean separation procedures were carried out using the multiple range tests with Fisher’s least significant difference (LSD) (po0.05).
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Statistical analysis
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three times with cold distilled water and blotted with filter paper. The fresh weight was immediately determined, and the dry weight was measured after 48 h of desiccation in an oven at 60 1C.
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Figure 2. Changes in whole plant dry matter (g plant1) produced by M. crystallinum and S. portulacastrum treated by various Cd concentrations. Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
Plant morphology and growth After 15 days of treatment, chlorosis was visible on young leaves of plants treated by Cd, in both species. Two weeks later, chlorosis was accentuated and other toxicity symptoms were evident. In Sesuvium, petiole necrosis and leaf fall were observed at highest Cd concentrations, 200 and 300 mM. In Mesembryanthemum, young leaf acute chlorosis and necrosis occurred even at the lowest Cd concentration (50 mM). The chlorophyll concentrations (Fig. 1) confirmed the visual observation of sensitivity to cadmium more pronounced in Mesembryanthemum than in Sesuvium. At 50 mM Cd2+, the chlorophyll concentrations were diminished to 25% of the control in Mesembryanthemum leaves, and remained unchanged in Sesuvium leaves. In the absence of Cd, the biomass of Mesembryanthemum plants was five-fold larger than that of Sesuvium (Fig. 2). However, Cd severely inhibited Mesembryanthemum growth even at the
lowest concentration (50 mM) and did not modify significantly that of Sesuvium. Root growth was less sensitive to Cd than was shoot growth. Indeed, 50 mM Cd treatment resulted in decrease in shoot/root ratio of Mesembryanthemum from 12.0 to 5.4 (Table 1). For Sesuvium, the changes in shoot/root ratio were not significant.
Cadmium accumulation For both Sesuvium and Mesembryanthemum, the Cd concentration in the root tissues was higher than in shoots (Fig. 3). The Cd concentrations in roots were similar in both species, ranging from approximately 2500 (at external 50 mM Cd) to about 5000 mg g1 DM (at 300 mM Cd). In shoots, Cd concentrations differed between the two species, accumulation in Mesembryanthemum shoots being twice that of Sesuvium. But, since the shoot
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Table 1. Changes in shoot DW/root DW ratio in S. portulacastrum and M. crystallinum with Cd concentrations in the culture medium
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Figure 3. Changes in Cd concentration (mg g1 DW) in shoots and roots of S. portulacastrum and M. crystallinum. Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
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biomass of Mesembryanthemum was less than of Sesuvium in the presence of Cd, the amounts of Cd (mg plant1) accumulated in the shoots were comparable between the two species (Fig. 4). Specific differences appeared at the level of the roots: the Cd amounts found in these organs were 2–3 times higher in Sesuvium than in Mesembryanthemum.
Potassium nutrition Cd treatment resulted in a large decrease in Mesembryanthemum shoots K concentration: at 50 mM Cd, the K concentration in these organs was
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Figure 4. Distribution of the cadmium absorbed by the whole plant (mg plant1) between roots and shoots of S. portulacastrum and M. crystallinum. Means of eight replicates.
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Figure 5. Changes in potassium concentration (mmol/ g DW) in shoots of S. portulacastrum and M. crystallinum with Cd concentration in culture medium. The numbers inside the bars are the amounts of K in the shoots (mmol plant1). Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
approximately 20% of the control value (Fig. 5). A slight increase in shoot K concentration from 50 to 300 mM Cd was associated with diminishing biomass production and unpronounced changes in tissues K amount. In Sesuvium, the shoot K+ concentration was only slightly diminished (9–22% of control) in the presence of the highest Cd concentrations. The opposite behaviour was observed in roots: K concentration was diminished in Sesuvium, and was
ARTICLE IN PRESS Responses of Sesuvium portulacastrum and Mesembryanthemum crystallinum to cadmium not strongly modified in Mesembryanthemum (not shown). Changes in mean quantities of K in the shoots (mmol plant1) showed that this parameter was significantly decreased in S. portulacastrum only from 200 mM Cd in the nutrient solution. This decrease reached 45% under 300 mM Cd. In M. crystallinum, the depressive effect of Cd on K content was more pronounced, appearing from 50 mM Cd and reaching 94%, independent of Cd concentration in nutrient solution.
Calcium nutrition In Mesembryanthemum, the Ca concentration in shoots was higher in plants treated with Cd (Fig. 6). However, the corresponding total amounts of Ca were strongly diminished in these plants. Thus, the increase in Ca concentration results from growth inhibition by Cd. For Sesuvium, both the amounts of Ca and its concentrations in shoots are slightly modified by Cd. Only at 300 mM Cd, Ca contents diminished by 42%, but without any significant change in Ca concentration. The presence of Cd in the culture medium did not modify significantly the Ca root concentration in M. crystallinum, and decreased this parameter in S. portulacastrum (not shown). However, the roots of the last species remained generally more rich in Ca than those of M. crystallinum.
Discussion Addition of cadmium in the medium culture decreased growth of both halophytes and disturbed their K and Ca nutrition. From biomass data,
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S. portulacastrum appeared more tolerant than M. crystallinum. In addition, Cd was accumulated in shoots at high concentrations, reaching 350–700 mg g1 DW, respectively, in Sesuvium and Mesembryanthemum. We discuss in this section parameters which would be implied in this reduction of growth. The dry matter measured at the final harvest depends on the initial size of the plant (before the beginning of the treatments) and on its growth during the treatment. Relative growth rate (RGR), i.e. the rate of increase in total dry weight per unit of plant dry weight, is recommended to evaluate the effect of the environmental constraints on the growth activity. Analysis of the relationship between RGR and Cd concentration in shoots (Fig. 7) showed the increase of Cd concentration in photosynthetic organs is associated with a reduction in the RGR. However, the two species presented a contrasting behaviour in their reaction to Cd. In Mesembryanthemum, the RGR shifted from high (0.13 103 day1) to low values (0.03–0.04 103 day1) when Cd was present in the medium, irrespective of its concentration in tissues. In Sesuvium, the RGR was already low in the absence of Cd (ca. 0.03 103 day1), and diminished progressively when Cd accumulation was augmented. This analysis suggests that the marked sensitivity of Mesembryanthemum growth to the Cd treatment results either from a hypersensitivity of its metabolism to absorbed Cd, or is not related to the internal amount of Cd. On the contrary, the linear correlation between Cd accumulation in Sesuvium shoots and the inhibition of
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Figure 6. Changes in calcium concentration (mmol g1 DW) in shoots (A) of S. portulacastrum and M. crystallinum with Cd concentration in culture medium. The numbers inside the bars are the amounts of Ca in the shoots (mmol plant1). Means of eight replicates. Bars marked with same letter are not significantly different at p ¼ 0:05:
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Figure 7. Changes in RGR with Cd concentration in the shoots tissues of S. portulacastrum and M. crystallinum. RGR measures the quantity of biomass deposited by 1 g of biomass per unit of time. It was estimated as DlnðDWÞ=Dt; where DW is the dry weight, ln stands for natural logarithm and D represents the difference between final and initial value (Hunt, 1990). Means of eight replicates.
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their growth is compatible with the hypothesis of a direct relation between the two phenomena. Analysing the effect of Cd on many plant species, Vassilev and Yordanov (1997) concluded that Cd diminishes RGR through inhibiting mainly net assimilation rate (NAR), and to a lesser extent, by restricting leaf area ratio (LAR). Inhibition of NAR is caused by disturbances of dark respiration and photosynthesis. Decreased LAR in Cd-treated plants is a consequence of the decrease of turgor potential and cell wall elasticity, resulting in smaller size of leaf cells formed with smaller intercellular space area. Some of our data may support the depressive action of Cd on cellular turgor. Indeed, in both halophytes, Cd induced shoot dehydration. However, this effect was significantly more severe in M. crystallinum than in S. portulacastrum. In plants subjected to 300 mM Cd, leaf water content decreases by 60% in the first species against 25% only in the second (not shown). In both halophytes, root growth was largely more tolerant to Cd than shoot growth. However, specific differences were observed. In M. crystallinum, these organs accumulated high concentrations of Cd, and their growth was negatively correlated to Cd accumulation (Fig. 8). On the contrary, in Sesuvium roots growth was insensitive to Cd treatment, in spite of the high values of Cd concentration in these organs (Fig. 8). The highest tolerance of the roots to Cd, relative to shoots, has been reported in several studies. For example, in P. sylvestris, growth was affected in shoots more than in roots, although Cd was mainly accumulated in the roots (Kim et al., 2003). Di Cagno et al. (1999) found a reduction in the dry weight of the leaves of H. annuus grown in soil irrigated with nutrient solution containing 2 mg Cd L1. Although Cd concentration in the roots was higher than that of the young leaves, Cd did not affect the root dry weight significantly. Several
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µg Cd /g DW roots
Figure 8. Relationship between root growth (DW, g/ plant1) and Cd concentration in these organs. Means of eight replicates.
mechanisms were proposed to explain the capacity of the roots to accumulate large quantities of Cd without deleterious effect on their growth: (i) Cd fixation on pectocellulosic walls (Nishizono et al., 1989), even if some studies showed that these extra cellular sites may fix only small quantities of Cd (Weigel and Ja ¨ger, 1980), (ii) the immobilisation of Cd in the endocellular compartment by complexation with organic acids, like malate, citrate (Harmens et al., 1994) and malonate (Clemens, 2001), and by sequestration with phytochelatins (Clemens, 2001; Bruns et al., 2001) or metallothioneins (Goldsborough, 2000), (iii) the higher capacity to sequester Cd in the vacuoles in roots compared to shoots (Ishikawa et al., 1997). In addition to the direct effects of internal Cd on growth activity, our results show that Cd in the medium culture decreases K concentrations in different organs. Disturbances of K nutrition under Cd treatment have been reported in several studies. For example, a dramatic decrease of K shoot content was observed in white lupin (Zornoza et al., 2002) and P. sylvestris (Kim et al., 2003) under cadmium treatment. Cd seems to complex ATP and reduces the availability of energy on which the functioning of membrane transport systems depends. Consequently, this constraint can decrease the uptake of K by roots of stressed plants (Asp et al., 1994). The result is an important fall of K concentrations in different organs (Ouzounidou et al., 1994). Changes in the K and other nutrients shoot translocation could also be attributed to the alteration in the vascular system and a reduction in the number and diameter of xylem vessels (Barcelo et al., 1988). However, little is known as to whether Cd has a direct effect upon uptake and distribution of K. Our results indicate that Cd of the culture medium affected plant growth more than Ca uptake and translocation towards the shoots in the two species, resulting in an increase in Ca concentration in tissues, in spite of a decrease of Ca contents. The effect of Cd on calcium nutrition is still difficult to evaluate because they frequently show conflicting results. Some studies showed that Cd induces an increase in Ca contents in different part of stressed plants (Greger et al., 1991; Larbi et al., 2002). However, other studies showed that Cd supply reduces Ca in leaves of Fagus sylvatica (Breckle and Kahle, 1992), P. pinea, P. pinaster and F. angustifolia (Arduini et al., 1998) and in shoots of Betula pendula (Gussarsson, 1994). According to Clemens et al. (1998) Cd compete with Ca and other cations for the entry into plant cells. This result was confirmed by Kim et al. (2002), who showed the existence of a competition of Cd with
ARTICLE IN PRESS Responses of Sesuvium portulacastrum and Mesembryanthemum crystallinum to cadmium Ca for transport into root cells. Another cause of the restriction to Ca translocation to leaves might be the sequestration of this ion as oxalate crystals in the xylem vessels of Cd-treated plants (Barcelo et al., 1988). These data suggest that the induced nutritional disturbances by heavy metals are implicated in the restriction of the growth under metal constraint, and may cause a limited heavy metals phytoextraction capacity. According to recent data, S. portulacastrum, which appeared relatively tolerant to Cd, may produce 17 t ha1 year1 of dry matter (Gleen et al., 1996). On the basis of our results relating to the Cd concentration in the shoots (350 mg g1 DM), Sesuvium would enable to extract 5950 g Cd ha1 year1. This capacity of Cd extraction is comparable with that expressed by Thlaspi caerulescens, which ranged between 4160 and 8320 g Cd ha1 year1 (Robinson et al., 1998). However, the potential for Cd extraction by Sesuvium would largely exceed those of B. juncea (60–80 g ha1 year1, Blaylock et al., 1997) or Nicotinana tabacum (500 g Cd ha1 year1, Gupta et al., 2001). Since M. crystallinum accumulate a higher Cd concentration in their shoots (700 mg g1 DM), it can be also used for the phytoextraction of this metal. However, this plant suffers from a severe growth reduction, which may be due to nutritional disturbances and probably to a direct Cd toxic effect. Our results suggest that the increase in nutrient availability in the rhizosphere could improve the growth of the plants, and would increase their capacity to extract heavy metals. Indeed, it has been reported in some studies that the increase in the Ca and Mg availability increases the growth of plants subjected to Pb and Cd and reduces their concentration in tissues (Kim et al., 2002). However, the amount of extracted heavy metals that corresponds to the product of the Cd concentration by the biomass would be more important under condition of high Ca or Mg availabilities. Others studies (Epstein et al., 1999; Laurie et al., 1995; Jordan et al., 2002) showed that utilisation of chelators like EDTA and rhamnolipids facilitate plant metal uptake. In conclusion, our results showed that both halophytes accumulate large amounts of Cd in their tissues, suggesting the possibility of their use to decontaminate the saline soils polluted by Cd. Indeed, the Cd concentrations, ranging between 350 and 700 mg g1 DM, are similar to those found in hyperaccumulator plants (Brown et al., 1994). However, the high Cd accumulation is perhaps associated with a growth restriction induced (at least partially) by the disturbance of K nutrition. The implication of the nutritional disturbances in
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the growth restriction under Cd treatment will be assessed with split root systems ensuring a load of the shoots with Cd and an appropriate K nutrition.
Acknowledgments This work was performed in the Laboratory of Plant Adaptation to Abiotic stresses. It was supported by the PRC project ‘‘Utilisation des halophytes pour la re´habilitation et la valorisation des sols salins’’ of the Tunisian Secretariat of State of Scientific Research and Technology.
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