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