Elsevier

Journal of Plant Physiology

Volume 164, Issue 11, 9 November 2007, Pages 1499-1515
Journal of Plant Physiology

Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes

https://doi.org/10.1016/j.jplph.2006.09.007Get rights and content

Summary

Sugarcane (Saccharum spp.) offers the potential to be a phytoremediator species due to its outstanding biomass production, but its prospective metal accumulation and tolerance have not been fully characterized. Sugarcane plantlets were able to tolerate up to 100 μM of copper in nutrient solution for 33 days, with no significant reduction in fresh weight, while accumulating 45 mg Cu kg−1 shoot dry weight. Higher levels of copper in solution (250 and 500 μM) were lethal. Sugarcane displayed tolerance to 500 μM Cd without symptoms of toxicity, accumulating 451 mg Cd kg−1 shoot dry weight after 33 days, indicating its potential as Cd phytoremediator. DNA gel blot analyses detected 8 fragments using a metallothionein (MT) Type I probe, while 10 were revealed for the MT Type II and 8 for MT Type III. The number of genes for each type of MT in sugarcane might be similar to the ones identified in rice considering the interspecific origin of sugarcane cultivars. MT Type I gene appeared to present the highest level of constitutive expression, mainly in roots, followed by MT Type II, corroborating the expression pattern described based on large-scale expressed sequence tags sequencing. MT Type II and III genes were more expressed in shoots, where MT I was also importantly expressed. Increasing Cu concentration had little or no effect in modulating MT genes expression, while an apparent minor modulation of some of the MT genes could be detected in Cd treatments. However, the level of response was too small to explain the tolerance and/or accumulation of Cd in sugarcane tissues. Thus, cadmium tolerance and accumulation in sugarcane might derive from other mechanisms, although MT may be involved in oxidative responses to high levels of Cd. Sugarcane can be considered a potential candidate to be tested in Cd phytoremediation.

Introduction

Phytoremediation is a plant-based approach to remove metals from contaminated soils (Salt et al., 1998), which requires a plant species efficient in metal accumulation and in high biomass production, while easily adapted to unfavorable environmental conditions (Clemens et al., 2002; Eapen and D’Souza, 2005). Sugarcane (Saccharum spp.) is a robust and vigorous tropical plant, with a high photosynthetic efficiency as a C4 plant, with superior growth over most other crop species (Alexander, 1973). Sugarcane was shown to tolerate high levels of Cd, with young plants standing up to 1 mM in nutrient solution (Fornazier et al., 2002). Metallothioneins (MTs) are related to metal sequestration in plants, and consequently candidate to be associated with metal tolerance and/or accumulation (Clemens et al., 2002).

MTs are small ubiquitous cysteine-rich metal-binding proteins (<10 kDa) directly translated from mRNAs (Cobbett and Goldsbrough, 2002). MTs are associated with metal homeostasis and reactive oxygen species scavenging in animals and fungi, but their role in plants are still unclear. Plants contain MT genes that are structurally and functionally similar to animal and fungal genes (Robinson et al., 1993; Rauser, 1999; Mir et al., 2004; Wong et al., 2004). Various MT genes have been characterized from many plant species (Cobbett and Goldsbrough, 2002). Many plant MT genes were originally identified based on analyses of differential expression affected by various types of abiotic and biotic stresses (Snowden and Gardner, 1993; Choi et al., 1996; Fordham-Skelton et al., 1998; Ma et al., 2003) and/or developmental stages (Chatthai et al., 1997; Muñoz et al., 1998; Guo et al., 2003). Often, MT genes have been detected by large scale sequencing of expressed sequence tags (ESTs) (Figueira et al., 2001) or by serial analysis of gene expression (SAGE) (Matsumura et al., 1999; Gibbings et al., 2003), frequently as one of the most abundant transcripts (Cobbett and Goldsbrough, 2002). However, most of the predicted proteins have not been isolated, except for wheat (Lane et al., 1987) and Arabidopsis MTs (Murphy et al., 1997).

Plant MT-predicted proteins can be categorized into four types according to the distribution of Cys over the two domains (Cobbett and Goldsbrough, 2002). The various plant MT types are encoded by small gene families. There are nine genes in the Arabidopsis (Zimeri et al., 2005) and rice genomes (Wong et al., 2004). Each type of MT gene displays a distinct spatial and temporal expression pattern. In general, MT Type I tends to be more abundant in roots than in shoots of Arabidopsis, while Type II and III are more expressed in shoots than in roots (Zhou and Goldsbrough, 1995; Murphy et al., 1997), with Type III also being more commonly detected in fleshy fruits (Garcia-Hernandez et al., 1998; Cobbett and Goldsbrough, 2002). The diversity in types and expression patterns suggest that MT might play distinct roles in plant tissues (Cobbett and Goldsbrough, 2002).

Plant MT genes can efficiently complement yeast mutant deficient for the MT gene (cup1Δ), re-establishing Cu tolerance (Zhou and Goldsbrough, 1994), while improving, in some cases, tolerance to other metals, such as Cd, Zn and Pb (Zhang et al., 2004). Similarly, over-expression of plant MT genes in transgenic Arabidopsis was shown to cause an increase in copper accumulation in relation to controls (Fordham-Skelton et al., 1997), and to improve tolerance to Cu and Cd (Zhang et al., 2004). Additionally, some MT genes are transcriptionally regulated by metals, particularly Cu in Arabidopsis (Murphy and Taiz, 1995; Cobbett and Goldsbrough, 2002). All these evidences support the hypothesis that MTs may present an important role on metal tolerance and homeostasis in plants.

Genes encoding all four types of plant MTs have been identified from a large EST database of sugarcane, with Type I being the most abundantly expressed, followed by Type II, with a similar pattern of expression described for MT in Arabidopsis (Figueira et al., 2001). The level of expression of MT transcripts in sugarcane under no particular induction indicates that these genes are constitutively highly expressed, particularly MT Type I.

Based on previous work (Fornazier et al., 2002), we hypothesized that sugarcane should accumulate and tolerate elevated levels of Cd and Cu, with increasing expression of MT genes as part of the tolerance mechanism. Studying the differential gene expression by metal treatment in sugarcane would help to elucidate the role of MTs in sugarcane metal tolerance to allow future plant manipulations to improve its ability to stand higher metal concentration in the soil. The objectives of this work were to investigate the metal tolerance and accumulation of sugarcane plantlets grown in nutrient solution with increasing dosages of Cu or Cd and the regulation of transcription of three types of MT genes (MT I, II and III).

Section snippets

Plant material

Rooted plantlets derived from in vitro meristem cultures of the commercial sugarcane (Saccharum spp. L.) cultivar ‘SP80-3280’ were obtained from ‘Centro de Tecnologia Canavieira’ (Piracicaba, SP, Brazil).

Plant cultivation in nutrient solution

The plantlets were acclimatized in the greenhouse by cultivation in 40 L plastic tray containing 120th strength of nutrient solution (Hoagland and Arnon, 1950), [1×=1180 mg L−1 Ca(NO3)2·4H2O; 505 mg L−1 KNO3; 136 mg L−1 KH2PO4; 462 mg L−1 MgSO4·7 H2O; 7.15 mg L−1 H3BO3; 4.52 mg L−1 MnCl2·4H2O; 0.55 mg L−1 Zn

Analyses of sugarcane plantlet growth in increasing doses of Cu

Plant fresh weight and tissue concentration of Cu in roots and shoots were estimated at 11 and 33 days after exposition to Cu, sampling two plants per pot, from each of the three pots per metal treatment. Plant fresh weight was significantly reduced at the highest doses of Cu (100, 250 and 500 μM) in comparison to controls 11 days after imposition of treatments based on Tukey's test (Table 1). Conversely, the 50 μM level of Cu did not cause a significant decrease in plantlet fresh weight at 11

Metal tolerance and accumulation in sugarcane

An ideal plant species for phytoremediation should tolerate the presence of high concentration of metals; accumulate metals in the above-ground tissues; be fast growing and high biomass yielder; easily harvestable; and have a dense root system (Clemens et al., 2002; Eapen and D’Souza, 2005). Sugarcane can be considered a potential candidate for phytoremediation because of its outstanding biomass production (commercially average >100 ton ha−1), but its prospective metal tolerance had not been

Acknowledgments

We would like to acknowledge the financial support by FAPESP (02/02004-9) and fellowships received (MLS, AF) from CNPq, and the technical assistance by Cristiane C Borges and Anileda PRM Vicente. The help from Dr. Virgilio Nascimento Filho on metal analyses was greatly appreciated.

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