Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper

Authors


Author for correspondence: Peter B. Goldsbrough Tel: +1 765 4941334 Fax: +1 765 4940391 Email: goldsbrough@purdue.edu

Summary

  • Expression and regulation of Arabidopsis metallothionein (MT) genes were investigated to examine the functions of MTs in plants.
  • To examine the tissue-specific expression of MT genes, GUS reporter gene activity driven by promoters of MT1a, MT2a, MT2b and MT3 was analysed in transgenic plants.
  • MT1a and MT2b are expressed in the phloem of all organs and are copper (Cu)-inducible; MT2a and MT3, by contrast, are expressed predominantly in mesophyll cells and are also induced by Cu in young leaves and at root tips. Expression of MT genes is highly induced by Cu in trichomes and increases during senescence. Expression of MT4 genes is restricted to seeds.
  • We propose that plant MTs have distinct functions in heavy metal homeostasis, especially for Cu: MT1a and MT2b are involved in the distribution of Cu via the phloem, while MT2a and MT3 chaperone excess metals in mesophyll cells and root tips. These functional capabilities may allow MTs to play a role in mobilization of metal ions from senescing leaves and the sequestration of excess metal ions in trichomes.

Introduction

Metallothioneins (MTs) are a class of low molecular mass (4–8 kDa), cysteine (Cys)-rich proteins that can bind metals via the thiol groups of their Cys residues (Hamer, 1986). Since MTs were first purified from horse kidney as Cd-binding proteins in 1956, MT genes have been found throughout the animal and plant kingdoms and in some prokaryotes (Cobbett & Goldsbrough, 2002). Metallothioneins have been divided into two classes based on amino acid sequence. Class I includes primarily mammalian MTs that contain 20 highly conserved Cys residues (reviewed by Klaassen et al., 1999). Metallothioneins from plants and fungi, as well as invertebrate animals, are grouped in Class II (Robinson et al., 1993). According to the arrangement of Cys residues, plant MTs are further classified into four types (Cobbett & Goldsbrough, 2002). In Arabidopsis, for example, seven actively expressed MT genes have been identified and include representatives of these four types (Zhou & Goldsbrough, 1995; Cobbett & Goldsbrough, 2002).

Based on analysis of MT RNA expression in a number of plant species, type 1 MT genes are expressed more abundantly in roots than leaves, whereas type 2 MT genes are expressed primarily in the leaves (Zhou & Goldsbrough, 1994, 1995; Hsieh et al., 1995, 1996). Type 3 MT genes are expressed in leaves or in ripening fleshy fruits (Ledger & Gardner, 1994), while expression of type 4 MTs appears to be restricted to developing seeds (Kawashima et al., 1992; White & Rivin, 1995). Expression of Arabidopsis MT1a has been more precisely localized to the vascular tissues of roots and leaves (Garcia-Hernandez et al., 1998; Mineta et al., 2000). Moreover, in situ hybridization showed that RNA expression of type 2 MTs in Arabidopsis and Vicia faba leaves is predominant in trichomes (Foley & Singh, 1994; Garcia-Hernandez et al., 1998) while a rice MT gene was highly expressed in stem nodes (Yu et al., 1998). The diverse patterns of expression of different MT genes suggest that plant MT isoforms may differ not only in sequence but also in the functions they perform in specific tissues (Cobbett & Goldsbrough, 2002). However, there are no reports providing a comprehensive description of the expression of all MT genes in a single species. Since the Arabidopsis genome contains representatives of all four types of MT genes, Arabidopsis provides a useful model to study MT gene expression in detail.

Despite the fact that MTs have been studied for decades, the functions of MTs in plants are still unknown. In mammals, MTs maintain zinc (Zn) and copper (Cu) homeostasis and protect cells against cadmium (Cd) toxicity and oxidative stress (Palmiter, 1998; Klaassen et al., 1999; Coyle et al., 2002). In Saccharomyces cerevisiae, MTs also play a major role in Cu detoxification (Ecker et al., 1989). Because plant MTs efficiently bind metals (Kille et al., 1991; Evans et al., 1992; Murphy et al., 1997) and some plant MT genes are transcriptionally regulated by metals, MTs are also thought to play an important role in metal tolerance and homeostasis in plants (Cobbett & Goldsbrough, 2002). However, it is now well established that phytochelatins (PCs), another metal ligand in plants, are required for Cd tolerance, but these peptides have little influence on plant growth in the presence of Cu (Cobbett, 2000; Cobbett & Goldsbrough, 2002).

A number of observations suggest that MT genes may be involved in Cu homeostasis and tolerance in plants. For example, MT RNA was strongly induced in Arabidopsis and rice seedlings by Cu treatment, but only slightly by Cd and Zn treatment (Zhou & Goldsbrough, 1994; Hsieh et al., 1995). Metallothionein genes from Arabidopsis complemented a MT-deficient yeast mutant and restored Cu tolerance (Zhou & Goldsbrough, 1994). In addition, the level of type 2 MT gene expression correlated closely with Cu tolerance across Arabidopsis ecotypes (Murphy & Taiz, 1995) and in Silene vulgaris (van Hoof et al., 2001). However, some observations do not support this hypothesis that MTs play a role in Cu tolerance of plants. In Brassica juncea and Vicia faba, MT gene expression was not induced by Cu treatment (Foley et al., 1997; Schafer et al., 1997). Therefore, more evidence is needed to evaluate the role of MTs in Cu homeostasis.

Senescence is characterized by the genetically programmed loss of cellular structure and metabolic function that ultimately leads to cell death (Nooden et al., 1997). Leaf senescence also involves mobilization of nutrients released after catabolism of macromolecules, including minerals such as Cu and Zn (Himelblau & Amasino, 2001), to other organs (Nooden, 1988; Mauk & Nooden, 1992; Marschner, 1995). Proteins that function as metal chelators, such as MTs, or protect against oxidative stress, such as peroxidase, may be needed to protect normal cell functions from the toxic effects of metal ions and peroxides released during senescence (Nooden et al., 1997). Dramatic increases in MT RNA levels during senescence have been shown in bean (Foley et al., 1997), Brassica (Buchanan-Wollaston, 1994), rice (Hsieh et al., 1995), and Arabidopsis (Garcia-Hernandez et al., 1998). It has been proposed that MTs may be involved in chaperoning released metal ions to protect cells from metal toxicity or metal-induced oxidative stress during the complex senescence program (Buchanan-Wollaston, 1994; Butt et al., 1998). Further analysis is necessary to examine the relationship between senescence and MT gene expression.

There is high sequence similarity within the Arabidopsis MT gene families. For example, MT1a and MT1c are over 94% identical in their coding region and intron; MT2a and MT2b are 84% identical to each other (Zhou & Goldsbrough, 1995). Therefore, it will be difficult to examine the specific expression of individual genes using nucleic acid hybridization methods. An alternative approach to study gene-specific expression of Arabidopsis MT genes is to utilize reporter gene constructs. To this end, the 5′ promoter regions of the Arabidopsis MT1a, MT2a, MT2b and MT3 genes were fused to the β-glucuronidase (GUS) reporter and transferred into Arabidopsis. Here, we show the activity of MT gene promoters in different organs and at different developmental stages in transgenic Arabidopsis. In addition, RNA blot and histochemical analysis demonstrate that these MT genes are both developmentally regulated and induced by Cu treatment, especially in the phloem and trichomes. The significance of the regulated expression of Arabidopsis MT genes will be discussed.

Materials and Methods

Plant materials and metal treatments

Arabidopsis plants (Columbia wild type) were grown in a soil mix in a growth room or greenhouse, or on half-strength Murashige and Skoog (MS) medium solidified with agar, or in hydroponic culture as indicated. For Cu treatment, plants were grown in a hydroponic system adapted from Arteca and Arteca (2000) in a growth room under 16 h–8 h light/dark cycle. The hydroponic medium was replaced weekly and maintained at pH 5.6 with 2 mm 2-(N-morpholino) ethanesulfonic acid (MES). When plants were 3–4 wk old, 25 µm or 50 µm CuSO4 was added to the medium and plants were harvested 2 d later for GUS staining and RNA extraction.

RNA isolation and RNA blot analysis

Total RNA was isolated as described by Carpenter and Simon (1998). Samples of 5 µg or 10 µg of total RNA per lane were separated on a 1.2% formaldehyde agarose gel and transferred to a nylon membrane (Hybond N+ Amersham, Piscataway, NJ, USA). Equal RNA loading was confirmed by visualization of the RNA gel under UV light. The same blot was sequentially hybridized with 32P-labeled cDNA probes using the hybridization procedure described by Zhou and Goldsbrough (1995). The blots were exposed to Kodak X-ray film at −70°C. The DNA fragments used for hybridization probes were isolated from plasmids by restriction enzyme digestion and labeled using an Ambion Decaprime II kit and 32P-dATP.

Construction of MT promoter::GUS transgenes

A 2.3-kb DNA fragment containing the Arabidopsis MT1a promoter was obtained by polymerase chain reaction (PCR) using genomic DNA as template. The PCR product contained BglII restriction enzyme sites to facilitate cloning into the BamHI site of pGEM7Z. For MT2::GUS constructs, cloned Arabidopsis genomic DNA fragments containing the MT2a and MT2b promoters were obtained from the λMT2-10 and λMT2-12 phage clones, respectively (Zhou & Goldsbrough, 1995). A 5 kb XhoI and 1.6 kb XbaI–XhoI fragment for MT2a and a 2.8 kb XhoI fragment for MT2b were cloned into pGEM7Z. A 900 bp MT3 promoter fragment was prepared by PCR using a plasmid containing the MT3 genomic DNA sequence as template. Restriction enzyme sites used to clone PCR fragments into pGEM7Z were either within the amplified DNA fragments or added in the primers used for amplification. The MT promoters were subcloned directionally from pGEM7Z into pBI101 containing a GUS reporter gene and nptII for kanamycin resistance (Jefferson et al., 1987). The plasmid constructs in pBI101 were confirmed by restriction enzyme digestion analysis.

Plant transformation and screening

The pBI101 constructs were transformed into Agrobacterium tumefaciens strain C58 PGV3850 by electroporation using the TransPorator Plus (BTX, San Diego, CA, USA). Plant transformation was performed by the vacuum infiltration technique (Bechtold et al., 1993). To screen for transformants, seeds were germinated on half strength MS solid medium containing 50 µg ml−1 kanamycin and 100 µg ml−1 timentin. For analysis of reporter gene expression, plants from three to five homozygous independent transgenic lines were examined for each promoter construct. Patterns of gene expression were consistent within a construct and representative plants were photographed under a stereoscopic microscope equipped with a SPOT 2 digital camera (Diagnostic Instruments, Sterling Heights, ml, USA).

Histochemical localization of GUS

Expression of MT::GUS transgenes was visualized by GUS staining. Transgenic plants or excised plant tissues were stained at 37°C for 16 h using X-Gluc solution (0.1 m NaH2PO4, 10 mm ethylenediaminetetraacetic acid (EDTA), 0.5 mm each of potassium ferricyanide and potassium ferrocyanide, 0.1% Triton X-100 and 0.5 mg ml−1 X-glucuronide cyclohexylamine salt) (Stomp, 1992). After staining, the tissues were cleared by replacing the staining solution with several changes of 70% ethanol as necessary. To prepare sections for microscopy, tissue from 4-wk-old plants was prefixed for 10 min at 4°C in freshly prepared 2% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.0. Samples were then rinsed several times with 0.1 m phosphate buffer, stained for 7 h in the X-Gluc solution described above, and fixed for a further 4 h in a 4% glutaraldehyde solution in 0.05 m phosphate buffer, pH 7.0. Chlorophyll was removed from tissues during dehydration in an ethanol series. Samples were gradually infiltrated with Technovit 8100 resin (Heraeus Kulzer, Armonk, NY, USA) before embedding in the same resin overnight at 4°C. Thin sections (4 µm) were cut using a Reichert Ultracut E ultramicrotome (Reichert-Jung, Vienna, Austria). The slides were mounted in Permount (Fisher Scientific, Chicago, IL, USA) and visualized with Nomarski (differential interference contrast) optics on a Nikon compound microscope. Images were captured with a SPOT 2 digital camera (Diagnostic Instruments).

Results

Differential expression of Arabidopsis MT genes

A total of seven active MT genes and one pseudogene have been identified in the Arabidopsis genome (Cobbett & Goldsbrough, 2002). Of the active genes, MT1a, MT1c, MT2a and MT2b have been described in some detail (Zhou & Goldsbrough, 1994, 1995). MT3 (AF013959) was identified in the Arabidopsis expressed sequences tag (EST) database (EST11988) and by amino acid sequence analysis of low molecular weight Cu-binding proteins (Murphy et al., 1997). DNA blot hybridization and analysis of the genome sequence indicate that there is only one gene (MT3) encoding a type 3 MT in the Arabidopsis genome (W. Bundithya, unpubl. data). The MT3 gene contains three exons and two introns and encodes a 69-amino-acid protein that contains two Cys-rich domains and a 34-amino-acid, Cys-free central domain. The 12 Cys residues in Arabidopsis MT3, four in the amino terminus and eight in the carboxyl terminus, are highly conserved in other plant type 3 MT proteins.

Analysis of various genomic databases revealed two additional MT genes, designated MT4a (NM129764) and MT4b (NM127888). The proteins encoded by these genes are similar to the wheat embryo MT (Kawashima et al., 1992). MT4a and MT4b are located on chromosome II but, unlike MT1a and MT1c, are not closely linked. Both genes contain a single intron whose location is conserved within the codon for the sixth Cys residue. The predicted MT4 proteins contain 85 amino acids with 17 Cys residues, which are arranged as either Cys–Xaa–Cys or single Cys residues and are distributed among three Cys-rich domains that are highly conserved in plant type 4 MT proteins (Cobbett & Goldsbrough, 2002).

Arabidopsis MT genes are expressed in a tissue-specific manner. In 7-d-old seedlings grown in MS medium, MT1a RNA was present mainly in roots, whereas MT2a and MT3 transcripts accumulated predominantly in the leaves (Fig. 1a). MT2b RNA was abundant in both roots and leaves (Fig. 1a). Similar results were observed in 4-wk-old plants (data not shown). By contrast, both MT4a and MT4b RNA was detected only in seeds (Fig. 1b). Transcripts of MT2a and MT2b were also detected in RNA from seeds, whereas MT1a and MT3 were expressed exclusively in vegetative tissues (data not shown).

Figure 1.

Tissue-specific RNA expression of Arabidopsis metallothionein (MT) genes. Total RNA was isolated from roots (R), leaves (L) or seeds (S) and hybridized with 32P-labeled cDNA probes for (a) MT1a, MT2a, MT2b and MT3, and (b) MT4a and MT4b.

Spatial expression of MT::GUS reporter genes in Arabidopsis

To further examine the tissue-specific expression of Arabidopsis MT genes in planta, transgenic Arabidopsis plants were produced carrying transcriptional fusions of the GUS open reading frame under the control of promoters from MT1a, MT1c, MT2a, MT2b and MT3. Homozygous T3 plants were grown under hydroponic conditions and analysed for GUS expression. Arabidopsis has two active type 1 MT genes, MT1a and MT1c, which share over 94% identity in their amino acid sequences (Zhou & Goldsbrough, 1995). In all experiments, the MT1a::GUS and MT1c::GUS reporter genes exhibited similar expression patterns in transgenic plants (data not shown). Therefore, only the results of MT1a::GUS expression are shown in this report (Fig. 2a–d). In aerial tissues, expression of MT1a::GUS was found mainly in vascular tissues of cotyledons, mature leaves, sepals and petals (Fig. 2a,b,d). Although expression of MT1a::GUS in cotyledons shown in Fig. 2a could be associated with senescence of this organ, similar vascular-specific expression was observed in cotyledons of young seedlings (data not shown). In flowers, MT1a::GUS expression was also detected in stamens where the filament attached to the anther (Fig. 2d). Strong expression of MT1a::GUS was observed in the mature region of roots, but not in the elongation zone (Fig. 2c).

Figure 2.

Histochemical analysis of expression patterns of Arabidopsis metallothionein (MT) genes. Glucuronidase (GUS) activity in transgenic Arabidopsis carrying the MT1a (a–d), MT2a (e–h), MT2b (i–l) and MT3 (m–p) promoter::GUS transgenes was analysed by staining with X-gluc. Images are shown of whole plants (a,e,i,m), mature leaves (b,f,j,n) and lateral roots (c,g,k,p) from 3-wk-old Arabidopsis plants grown in hydroponic culture. Flowers (d,h,l,p) from 6-wk-old soil-grown plants are also shown. Bar, 1 mm for whole plants and mature leaves, and 250 µm for roots and flowers.

Transgenic plants carrying MT2a::GUS transgenes with either 5 kb or 1.6 kb promoter sequence showed similar patterns of reporter gene expression (data not shown). Representative examples of histochemical staining of GUS activity driven by the 1.6 kb MT2a promoter are shown. In mature plants, MT2a::GUS expression was detected throughout the leaf with irregularly distributed spots of more intense staining (Fig. 2e,f). Expression of MT2a::GUS was low in mature root tissues (Fig. 2g). However, strong expression of MT2a::GUS was seen in the filaments and stigma (Fig. 2h), as well as at the tips of elongating lateral roots (Fig. 2g). Expression of the MT2b::GUS transgene was prominent in the vascular tissues of all organs (Fig. 2i–l). In addition, GUS staining was also observed in pollen and root hairs (Fig. 2k,l). The MT3::GUS transgene exhibited a similar pattern of expression to that of MT2a::GUS in leaves and root tips (Fig. 2m–o). The higher expression of MT2a::GUS and MT3::GUS in older leaves (Fig. 2e,m) may be associated with senescence of these organs, as discussed later. There was only a very low level of GUS activity in the pollen of plants carrying the MT3::GUS transgene (Fig. 2p). The expression patterns of the MT::GUS transgenes were not affected by stage of plant development (data not shown). Thus, the MT1a and MT2b promoters were expressed prominently in vascular tissues of most organs whereas MT2a and MT3 promoters were active in leaf mesophyll and at root tips. However, the level of expression of MT::GUS reporter genes increased as tissues aged. For example, expression of MT2a::GUS and MT3::GUS in the mature region of roots was higher in 6-wk-old plants than in 3-wk-old plants (data not shown). The expression patterns observed in transgenic plants grown in hydroponic medium were similar to those seen in plants grown in soil (data not shown).

In order to visualize expression of MT::GUS transgenes in specific tissues, sections of tissue from 4-wk-old transgenic Arabidopsis plants were stained for GUS activity and examined by light microscopy. In leaves, expression of MT1a::GUS was observed in the phloem as well as in mesophyll cells (Fig. 3a). MT2b::GUS expression was localized mainly in the phloem, and was more limited to that tissue than expression of MT1a::GUS (Fig. 3a,i). By contrast, MT2a::GUS and MT3::GUS expression was observed throughout the mesophyll (Fig. 3e,m). In the mature region of roots, expression of MT1a::GUS was high in the endodermis and lower in cortex and stele (Fig. 3c), whereas expression of MT2b::GUS was pronounced in vascular tissues, primarily the phloem (Fig. 3k). By contrast, expression of MT2a::GUS and MT3::GUS was very low in the phloem (Fig. 3g,o).

Figure 3.

Localization of Arabidopsis metallothionein (MT) gene expression. Plants were grown in hydroponic medium for 4 wk. For copper (Cu) treatment, plants were exposed to 25 µm CuSO4 for 2 d. Cross-sections of glucuronidase (GUS)-stained mature leaves and roots from transgenic Arabidopsis expressing MT1a (a–d), MT2a (e–h), MT2b (i–l) and MT3 (m–p) promoter::GUS transgenes are shown. Bar, 50 µm.

Copper induction of MT gene expression

Some plant MT genes have been shown to be expressed in response to Cu (Zhou & Goldsbrough, 1994, 1995; Garcia-Hernandez et al., 1998). To determine if Cu treatment altered the tissues in which MT genes are expressed, Arabidopsis plants were grown in a hydroponic system for 3 wk and then exposed to 25 µm CuSO4. After treatment for 2 d, root growth of 2-wk-old Arabidopsis plants was significantly inhibited, but plants were still green and kept growing (data not shown). This indicates that the concentration of CuSO4 used did impose stress but was not lethal to plants. RNA blots showed that the levels of MT1a, MT2a, MT2b and MT3 RNA increased after Cu treatment, but not for every gene in every tissue (Fig. 4). The Cu treatment increased RNA expression of MT1a and MT2b in roots, MT2a in leaves, and MT3 in young expanding leaves (Fig. 4). To examine the spatial distribution of Cu induction of Arabidopsis MT genes in detail, histochemical staining of GUS activity was carried out in 3- or 4-wk-old plants. Since GUS staining patterns of MT::GUS reporter genes were similar in transgenic plants exposed to 25 µm or 50 µm CuSO4, images of stained organs from plants treated with 50 µm CuSO4 are shown. As suggested by the RNA hybridization experiments, Cu did not have a dramatic effect on MT gene expression in most tissues, as visualized by reporter gene activity. However, there were a small number of tissues in which Cu had a pronounced impact on MT::GUS expression. Copper induced the expression of MT2a::GUS and MT3::GUS in young expanding leaves (Fig. 5g,h,s,t). However, expression of all MT::GUS genes was induced by Cu in trichomes and this was most prominent in expanding leaves (Fig. 5a,b,g,h,m,n,s,t). Increased expression of MT2a::GUS was localized to the base of the trichomes, whereas Cu-induced expression of the MT1a, MT2b and MT3 reporters was observed throughout the trichome. Increased expression of MT reporter genes was also observed in root tips, particularly for MT2a::GUS and MT3::GUS transgenes (Fig. 5c,e,i,k,o,q,u,w).

Figure 4.

Effect of copper (Cu) treatment on RNA expression of Arabidopsis metallothionein (MT) genes. Plants were grown in hydroponic culture for 3 wk and tissues were harvested after plants were exposed to 25 µm CuSO4 for 2 d. Total RNA was isolated from tissues as indicated and hybridized with 32P-labeled cDNA probes for MT1a, MT2a, MT2b and MT3. R, YL and ML indicate roots, young expanding leaves and mature leaves, respectively. The ethidium bromide-stained rRNA bands are shown as a loading control.

Figure 5.

Copper (Cu) induction of Arabidopsis MT::GUS reporter genes in trichomes and root tips. Plants were grown in hydroponic culture for 3–4 wk and exposed to 50 µm or 25 µm CuSO4 for 2 d as indicated. Glucuronidase (GUS) activity in transgenic Arabidopsis carrying the MT1a (a–f), MT2a (g–l), MT2b (m–r) and MT3 (s–x) promoter::GUS transgenes was analysed by staining with X-gluc. Images of trichomes on young leaves, lateral root tips (bar, 100 µm) and longitudinal sections of lateral root tips (bar, 50 µm) are shown.

To further localize the tissues where Arabidopsis MT promoters are activated in response to Cu, stained sections of roots and leaves from transgenic plants were examined under light microscopy. In mature leaves, MT1a::GUS and MT2b::GUS were highly induced in the phloem and in cells surrounding the vascular tissue (Fig. 3a,b,i,j). By contrast, Cu treatment had no obvious effect on the expression of MT2a::GUS and MT3::GUS in mature leaves (Fig. 3e,f,m,n). However, as described above, induction of MT2a::GUS and MT3::GUS expression was observed in mesophyll cells and trichomes of young leaves (Fig. 5g,h,s,t). In mature roots, expression of MT2a::GUS and MT3::GUS in the vascular tissues was slightly elevated by Cu treatment (Fig. 3g,h,o,p). By contrast, expression of MT1a::GUS and MT2b::GUS was induced in all tissues of the mature root but was highest in the phloem (Fig. 3c,d,k,l). The MT gene promoters also drive specific patterns of expression in root tips in response to Cu. For MT1a::GUS, MT2a::GUS and MT3::GUS, Cu treatment resulted in GUS expression in all cells of the root tip (Fig. 5d,f,j,l,v,x); MT2b::GUS expression increased in the phloem in response to Cu (Fig. 5p,r).

Expression of MT genes during senescence

Previously, it has been demonstrated that MT1a RNA expression increases during leaf senescence in Arabidopsis (Garcia-Hernandez et al., 1998; Mira et al., 2001). To determine if other MT genes are induced during senescence, their expression was examined by RNA blot analysis (Fig. 6). Leaves at different developmental stages (based on visual characteristics as shown in Fig. 6) were collected from 7-wk-old soil-grown plants. The expression of most MT genes increased from young leaf (YL) to mature leaf (ML) and was further induced during early and late stages of leaf senescence (senescing leaf, SL; super-senescing leaf, SSL). The exception to this was MT3, whose RNA was abundant in ML and SL but decreased in SSL (Fig. 6). In this experiment, the leaves at different stages of senescence were collected from different positions on the plant and these developmental differences could also affect MT gene expression. The MT::GUS transgenic lines were used to examine the spatial distribution of MT gene expression within the leaf during senescence. The intensity of GUS staining correlated with RNA abundance (Figs 6 and 7). In addition, increased expression of MT1a::GUS and MT2b::GUS in senescing leaves was localized primarily in vascular tissue (Fig. 7b,c,n,o), whereas expression of MT2a::GUS and MT3::GUS was observed mainly in mesophyll tissues of leaves at all developmental stages (Fig. 7h,i,t,u). The induction of MT gene expression was also observed in senescing siliques. Except for MT3::GUS (Fig. 7v,w), expression of the other MT::GUS reporter genes increased throughout the carpels and false septum of senescing yellow siliques (Fig. 7d,e,j,k,p,q). Moreover, GUS activity was also observed in the stigma and the floral abscission zones between carpels and pedicel of developing siliques (Fig. 7f,l,r,x) notably after the abscission of petals and sepals (compare Figs 2d,h,l,p and 7f,l,r,x).

Figure 6.

The RNA expression of Arabidopsis metallothionein (MT) genes during leaf senescence. RNA was isolated from leaves of 7-wk-old plants at different stages of senescence. It was hybridized with 32P-labeled cDNA probes for MT1a, MT2a, MT2b and MT3. YL, ML, SL, and SSL indicate young expanding leaves (< 1 cm), mature leaves (≈ 4 cm), senescing leaves (< 50% yellow), and super-senescing leaves (> 50% yellow), respectively. The ethidium bromide-stained rRNA bands are shown as a loading control.

Figure 7.

Histochemical analysis of Arabidopsis metallothionein (MT) gene expression in senescing tissues and abscission zones. Glucuronidase (GUS) activity in 6-wk-old soil-grown transgenic Arabidopsis carrying MT1a (a–f), MT2a (g–l), MT2b (m–r) and MT3 (s–x) promoter::GUS transgenes was analysed by staining with X-gluc. Images of young leaves (a,g,m,s), mature leaves (b,h,n,t), senescing leaves (c,i,o,u), mature siliques (d,j,p,v), senescing siliques (e,k,q,w) and pistils after abscission of other floral organs (f,i,r,x) are shown. Bar, 1 mm.

Discussion

Similar to mammals, plant MTs are encoded by a multigene family. However, unlike the highly conserved sequences of mammalian MTs (Klaassen et al., 1999), the four types of plant MTs have distinct arrangements of Cys residues, suggesting they may differ not only in sequence but also in function (Cobbett & Goldsbrough, 2002). Although different types of plant MT genes have been identified in many species, only in Arabidopsis, rice and sugarcane have genes been described that encode all four types of MTs (Cobbett & Goldsbrough, 2002). The resources available with Arabidopsis make it a good model to study the functions of plant MT genes in a single species. MT1a, MT2a and MT2b have been characterized previously (Zhou & Goldsbrough, 1994, 1995). Most of the type 3 MT genes characterized in other plants are expressed primarily in fruit tissues, such as in papaya (Y08322), banana (Q40256), kiwifruit (P43389) and citrus (AB008101). Despite the high homology to type 3 MT genes of other plants expressed in fruit tissues, Arabidopsis MT3 RNA was more abundant in leaves (Fig. 1a) and cannot be detected in seeds or green siliques (Fig. 7v,w; data not shown) suggesting that MT3 is unlikely to be involved in reproductive development in Arabidopsis. Fruit anatomy may contribute to the difference in expression of type 3 MT genes between Arabidopsis and other plants.

Two additional MT genes, designated MT4a and MT4b, were identified in the Arabidopsis EST database as homologues of the wheat Ec protein. Compared with Ec homologues from wheat (P30569) and maize (U10696), the positions of Cys residues are conserved in all type 4 MT proteins. Similar to wheat and maize Ec homologues (Kawashima et al., 1992; White & Rivin, 1995), expression of the Arabidopsis MT4 genes is abundant in seeds and not detected in vegetative tissues (Fig. 1b) suggesting that type 4 MTs are involved in seed development both in monocot and dicot plants. Arabidopsis type 4 MT genes contain promoter sequences with homology to abscisic acid (ABA)-response elements. The MT4a promoter includes one putative ABA-response element at −322, CTAACCA, which is identical to a transcription factor binding site in the promoter of the dehydration-responsive gene rd22 in Arabidopsis (Abe et al., 1997; Busk et al., 1997). Three elements in the MT4b promoter, at −115 and −122 (both CACATG), and −1354 (CATGCA) upstream from the ATG, are similar to functional elements in the promoters of rd22 (Abe et al., 1997; Busk et al., 1997) and a napin seed storage protein gene (Ezcurra et al., 1999), respectively. Together with the localized expression of the wheat Ec homologue in microspores, which is induced by ABA and osmotic stress, these results suggest that MT4 proteins may function in pollen embryogenesis and preparing seed tissues for desiccation (Kawashima et al., 1992; White & Rivin, 1995). Seed-specific type 4 MTs may also provide a mechanism for storing Zn and other metals that are required for growth after germination (Kawashima et al., 1992; White & Rivin, 1995). Manipulating the expression of type 4 MTs in seeds of transgenic plants may provide a mechanism to alter metal concentrations in seeds (Lucca et al., 2001) and produce nutritionally enhanced grains.

To examine the specific expression of individual genes, chimeric GUS reporter genes were used. Expression of the GUS reporter under the control of various MT promoters in transgenic Arabidopsis demonstrated spatial and temporal patterns of expression that corresponded well with RNA expression data. This indicates that the MT gene promoter sequences used contained elements sufficient for appropriately regulated expression. Expression of MT::GUS transgenes under control conditions or after Cu treatment agree with RNA blot analysis (Figs 1–5; Zhou & Goldsbrough, 1994, 1995) and in situ hybridization data (Garcia-Hernandez et al., 1998), where Cu induced the expression of MT1a, but not MT2a and MT2b, in phloem tissue of Arabidopsis leaves. High expression of MT1a::GUS and low expression of MT2a::GUS in mature regions of roots was shown. Accumulation of MT2a protein (Murphy et al., 1997) and Cu induction of MT2a::GUS expression (Mineta et al., 2000) in Arabidopsis root tips have been reported previously. However, the expression of MT2a in root tips and Cu-inducible expression of MT1a and MT2a in roots (Figs 3 and 5) were not observed by in situ hybridization (Garcia-Hernandez et al., 1998). These discrepancies may reflect the sensitivity of the GUS reporter system compared with in situ hybridization, as well as possible differences in the growth of plants and how they were exposed to Cu. It is interesting to note that the promoter of a type 1 MT gene from pea directed similar GUS expression in transgenic Arabidopsis as MT1a::GUS (Fig. 2a–d) (Fordham-Skelton et al., 1997). This indicates conservation of MT promoter activity across plant species.

Based on patterns of gene expression, we can divide the Arabidopsis MT genes into two groups: MT1a and MT2b, whose expression is mainly localized in the phloem in both leaves and roots and is inducible by Cu; and MT2a and MT3, which are expressed mainly in leaf mesophyll cells and highly induced by Cu in young leaves and root tips (Figs 3 and 5).

Both MT1a and MT2b may play a major role in Cu homeostasis in the phloem, a tissue that is important for long-distance transport of excess Cu between tissues (Marschner, 1995). Moreover, the high basal expression of MT2b and responsiveness of MT1a expression to Cu (Fig. 3a,d,i–l) further suggest that MT2b is a housekeeping MT whereas MT1a may be responsible for dealing with rapid changes in Cu concentration in the phloem. It is also possible that constitutively expressed MT2b protein acts as a Cu storage mechanism in the vascular tissue to provide Cu for lignification of xylem vessels and cell walls (Marschner, 1995). In addition, the Cu-induced expression of GUS activity driven by the MT1a and MT2b promoters in the root cortex correlates with the tissues where Cu accumulates in roots (Fig. 3; Mineta et al., 2000), suggesting that MT1a and MT2b are also involved in Cu tolerance in the basal root zones. Cosegregation of elevated SvMT2b transcript levels with Cu tolerance in S. vulgaris provides further evidence that MT2b contributes to mechanisms for ameliorating Cu toxicity in roots (van Hoof et al., 2001).

By contrast, expression of the MT2a and MT3 promoters was highly induced by Cu only in root tips and young leaves (Fig. 5). This suggests that MT2a and MT3 function in metal homeostasis in the mesophyll cells of leaves, especially young leaves, and in protecting the root apex, the first tissue to absorb excess Cu from the soil, from Cu toxicity. A correlation between MT2a RNA expression and Cu tolerance has been observed in different ecotypes of Arabidopsis (Murphy & Taiz, 1995). Moreover, MT2 RNA levels were also correlated with recovery of root growth from Cu inhibition (Murphy & Taiz, 1995). These results provide further evidence to support a role for MT2a and MT3 in Cu tolerance in root tips. Our studies have focused on MT gene expression in response to copper. However, MTs in plants may also be involved in mechanisms of homeostasis for other metals, such as Zn, although the expression of Arabidopsis MT genes is less affected by Cd and Zn than Cu (Zhou & Goldsbrough, 1994).

The large volume of trichome cells, compared with most leaf cells, allows them to serve as substantial reservoirs for toxic metal ions. Accumulation of heavy metals (Zn, Cd, Cu) in trichomes has been observed in Arabidopsis halleri (Kupper et al., 2000; Sarret et al., 2002). In Brassica juncea, Cd accumulates preferentially in trichomes (Salt et al., 1995). These observations indicate that trichomes may play an important role in metal detoxification in leaves. The dramatic induction of Arabidopsis MT genes in trichomes by Cu, especially in young leaves (Fig. 5), further supports this hypothesis. Metallothioneins may function either in chaperoning excess metal ions into trichomes, or as a chelator of some metal ions in these specialized cells.

The concentrations of several metal ions, such as those for Cu, Fe, Mo and Zn, drop dramatically in senescing Arabidopsis leaves (Himelblau & Amasino, 2001), suggesting that these metal ions are recycled from senescing leaves to growing organs (Mauk & Nooden, 1992; Marschner, 1995). However the sudden release of metal ions during senescence could be highly toxic. All of the Arabidopsis MT genes expressed in vegetative tissues were upregulated in senescing leaves (Fig. 6). The high affinity of plant MTs for metal ions (Evans et al., 1992; Murphy & Taiz, 1996) allows MTs to function as chelators that protect cells from metal ion toxicity during senescence. The specific expression of MT1a and MT2b in vascular tissues during leaf senescence suggests that MT1a and MT2b could also be involved in transport and redistribution of released metal ions, from senescing leaves to developing organs, which takes place via the phloem (Marschner, 1995). Because Arabidopsis MT genes are not significantly induced by Cd and Zn (Zhou & Goldsbrough, 1994) and plant MTs have the highest affinity for Cu (Tommey et al., 1991), MTs may function mainly in Cu homeostasis during senescence in plants.

Arabidopsis contains homologues of many key components of the Cu homeostasis system described in yeast and mammals, including the Cu chaperone AtCCH and Cu transporter AtRAN1 (Himelblau & Amasino, 2000). Expression of AtCCH and AtRAN1 are also upregulated in senescing leaves (Himelblau et al., 1998; Himelblau & Amasino, 2000, 2001). However, the expression of AtCCH is reduced by Cu treatment (Mira et al., 2001) and an AtCCH-deficient mutant is normal in terms of nutrient mobilization from senescing leaves (Himelblau & Amasino, 2001). These results suggest that MT expression in senescing leaves may be independent of other genes in the Cu homeostasis system, including AtCCH and AtRAN1.

Senescing leaves have been shown to contain increased concentrations of H2O2 (Stohs & Bagchi, 1995), and the production of such reactive oxygen species can be stimulated by ions of Cu and Fe (Orendi et al., 2001). Several studies in animals have demonstrated that MTs act as antioxidants against reactive oxygen species (Palmiter, 1998). Moreover, some antioxidant enzymes, such as catalase, do not appear to be induced by oxidative stress in senescing Arabidopsis leaves (Orendi et al., 2001). Therefore, plant MTs may also provide protection against oxidative stress during senescence.

It has been suggested that specific expression of some genes in leaf abscission zones may be mediated by reactive oxygen species (Michaeli et al., 2001). Accumulation of MT RNA has been observed in the abscission zone of Sambucus nigra leaflets (Coupe et al., 1995). Together with high levels of GUS activity driven by Arabidopsis MT promoters detected in the abscission of floral organs (Figs 2 and 7), these results indicate that plant MTs may also act as antioxidants in abscission zones to attenuate free radicals produced during the abscission of leaves and floral organs.

In conclusion, in this report we have described the expression of the complete MT gene family in Arabidopsis; this is the first time this has been done for any plant species. The tissue-specific expression of Arabidopsis MT genes and their responses to Cu treatment indicate they function independently but may have a complementary relationship in Arabidopsis. Additional studies of both MT proteins and MT-deficient mutants will be necessary in order to clarify the importance of MTs in plants.

Acknowledgements

This research was supported by a grant from the USDA-NRI. Weenun Bundithya was supported by the Royal Thai Government. We thank Mary Alice Webb and Debby Sherman for their advice on microscopy.

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