molecular cloning of a metallothionein-like gene from ... · plant physiol. (1 996) 11 2: 353-359...

Plant Physiol. (1 996) 11 2 : 353-359

Molecular Cloning of a Metallothionein-Like Gene from Nicofiana glufinosa L. and Its lnduction by Wounding and

Tobacco Mosaic Virus lnfection'

Doi1 Choi*, Hong Mo Kim, Hae Keun Yun, Jong-A Park, Woo Taek Kim, and Song Hae Bok Plant Protectants Research Unit, Korea Research lnstitute of Bioscience and Biotechnology, Korea lnstitute of

Science and Technology, P.O. Box 115, Yusung Taejeon, 305-600, South Korea (D.C., H.M.K., H.K.Y., S.H.B.); and Department of Biology, College of Science, Yonsei University, Seoul, 120-749, South Korea (J.-A.P., W.T.K.)

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance- response genes in Nicotiana gluthosa. One 530-bp cDNA clone (KC9-1 O) containing an open reading frame of 81 amino acids was characterized. Cenomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.

Metallothioneins are low-molecular-weight, Cys-rich proteins that are ubiquitous in eukaryotic organisms. Since their first description as cadmium- and zinc-binding proteins in horse kidneys, metallothionein genes and proteins have been characterized from many different organisms (reviewed by Hamer, 1986; Robinson et al., 1993). In the plant kingdom, a few metallothionein-like genes have been characterized recently, including those from peas (Evans et al., 1990), maize (De Framond, 1991), Arabidopsis (Zhou and Goldsbrough, 1994, 1995), and Sambucus nigra (Coupe et al., 1995).

Because of the nature of its metal-binding activity and induction by heavy metal ions, metallothionein is strongly believed to have a role in metal metabolism or detoxification (Hamer, 1986). However, some plant metallothionein- like genes are not stimulated by heavy metal (De Miranda et al., 1990; Kawashima et al., 1992). In recent studies with cloned Brassica and Sambucus metallothionein-like genes, the accumulation of their mRNAs in the senesced leaflet

This work was supported by a grant from the Korean govern- ment Ministry of Science and Technology to D.C. and Korea Sci- ence and Engineering Foundation Hormone Research Center Project no. 9524 to W.T.K.

* Corresponding author; e-mail doi10geri4680.geri.re.kr; fax 82- 42-861-2675.

and abscission zone, respectively, was described (Buchanan-Wollaston, 1994; Coupe et al., 1995). Conse- quently, the role and regulation of metallothionein genes in plants are probably different from those of other organisms, and the biochemical function of plant metallothioneins is not known. Other possible functions suggested for animal and funga1 metallothioneins are the control of intracellular redox potential, activated oxygen detoxification, and sulfur metabolism (Hamer, 1986), but these specula- tions lack evidence. Active oxygen species and intracellular redox potentials are very important components of plant defense, since they prevent biotic and abiotic stresses (reviewed by Baker and Orlandi, 1995). Plants produce active oxygen species during interaction with various pathogens, and these oxygen species are involved in both disease resistance and symptom development (reviewed by Tzeng and De Vay, 1993).

In this study we isolated a wound- and pathogen- inducible metallothionein-like cDNA from Nicotiana glutinosa while cloning plant disease resistance-response genes by subtractive hybridization. The cloning, characterization, and expression of this metallothionein-like gene following different treatments, including wounding and TMV inoculation, are discussed in relation to the possible role of this gene in oxidative stress in plants.

MATERIALS AND METHODS

Nicotiana glutinosa (2n), Nicotiana tabacum cv Xanthi nc (4x), and cv Samsun NN (4x) were used. The U1 strain of TMV (a gift of Dr. E.K. Park, Korea Research Institute of Ginseng and Tobacco, Taejeon, Korea) was used as an inoculum. SA and CuSO, (concentrations indicated in the figures) were dissolved in 30% acetone and sprayed onto leaf surfaces. For the wound treatment, tobacco leaves were mechanically wounded using a commercial scrubber (3M). MJ (Aldrich) was dissolved in 30% acetone. For ethylene treatment, plants were enclosed for 12 h in 4-L jars containing air, air plus ethylene (20 pL/L), or air plus NBD (7000 pL/L; Aldrich). At the end of each treatment, the leaf

Abbreviations: HR, hypersensitive response; MJ, methyl jasmonate; NBD, norbonidiene; SA, salicylic acid; TMV, tobacco mosaic virus.

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354 Choi et al. Plant Physiol. Vol. 11 2, 1996

tissues were immediately frozen in liquid nitrogen and stored at -80°C until use.

cDNA Library Construction

A11 leaf surfaces of 10-week-old N. glutinosa plants were inoculated with TMV at 32°C for 24 h for spreading and replication of virus and were then shifted to 24°C for 18 h to induce the HR. Total RNA was isolated from the inoculated leaf tissues showing the HR by a modification of the method of Parish and Kirby (1966). Poly(A)+ RNA was isolated from total RNA using oligo(dT)-cellulose (Boeh- ringer Mannheim) according to standard procedures (Aus- ubel et al., 1987). A cDNA library was constructed in a cDNA synthesis kit (AZAP, Stratagene) using 5 pg of poly(A)+ RNA.

Biotin Labeling of mRNA

mRNA (10 pg) from healthy N. glutinosa plants were labeled with photoactivatable biotin (Clontech, Palo Alto, CA) according to the method of Sive and John (1988). RNA (10 pg) was mixed with 50 pL of photoactivatable biotin and irradiated with a sunlamp (500 W) for 10 min on ice. The unlabeled biotin was removed by extracting the reaction mixture twice with 2-butanol and then extracting with chloroform. The biotin-labeled RNA was ethanol- precipitated and stored in -20°C until used for subtractive hybridization.

Subtractive Hybridization and cDNA Cloning

Single-stranded cDNA (negative strand) was obtained from the TMV-induced cDNA library according to the method of Short and Sorge (1992). Single-stranded cDNA (500 ng) was hybridized with 5 pg of biotin-labeled mRNA in 50 mM Hepes, pH 7.6, 0.2% SDS, 2 mM EDTA, 500 mM NaCl in 10 pL of reaction volume overlayed with 20 pL of mineral oil at 65°C (Sive and John, 1988). After 48 h the reaction mixture was transferred to 100 pL of the above buffer without SDS and extracted with pheno1:chloroform (l:l, v /v) to remove the cDNA/RNA hybrid. The unhy- bridized single-strand cDNA was ethanol-precipitated and subjected to one more round of subtractive hybridization. Recovered single-stranded cDNA was amplified by PCR using T7 and T3 universal primer. PCR-amplified double- stranded DNA was digested with EcoRI and XkoI, and the fragments (size range 0.3-2.0 kb) were recovered from an agarose gel using a GeneClean Kit (Bio 101, La Jolla, CA) and ligated into EcoRI/ XkoI sites of pBluescript (Strat- agene). One of the 38 clones isolated by this procedure was the partial cDNA of a metallothionein-like gene. To isolate the full-length cDNA, 90,000 plaques of a cDNA library were screened using the partial cDNA as a probe. The whole nucleotide sequence of one cDNA from 45 obtained clones was determined by the Sanger dideoxy termination method (Sanger et al., 1977) using a Sequenase kit (United States Biochemical).

Cenomic DNA lsolation and Southern Blot Hybridization

Genomic DNA from N. glutinosa (2n) and N. tabacum cv Xanthi nc (4x) leaf tissues was isolated according to previ- ously described methods (Choi et al., 1992). The genomic DNA was digested overnight with 5 times the recom- mended units of restriction enzymes. DNA (10 pg/lane) was fractionated by 0.7% agarose gel electrophoresis in Tris-acetate-EDTA buffer (40 mM Tris-acetate, pH 8.0,2 mM EDTA) and transferred to a Hybond N+ membrane (Am- ersham). The blots were hybridized with a random prime- labeled metallothionein cDNA probe using standard procedures. The hybridization was carried out in 50% formamide, 5x SSC at 42"C, and the washing conditions were the same as above, except that 2X SSC was used.

Total RNA lsolation and Northern Blot Hybridization

Total RNA was isolated from treated leaf tissues according to the method of Parish and Kirby (1966). Spectropho- tometrically measured total RNA (20 pg) was fractionated by formaldehyde-containing agarose gel electrophoresis (Ausubel et al., 1987). The loading of equal amounts of RNA was checked by duplicated gel staining with ethidium bromide. The RNA transfer and hybridization conditions were similar to genomic DNA hybridization.

RESULTS

lsolation and Sequence Analysis of Metallothionein-Like cDNA from N. glutinosa

The cloning and characterization of genes expressed during the plant response to pathogens could bring us one step closer to understanding the signal transduction mechanism of host-pathogen interaction. We used subtractive hybridization for cloning pathogen-response genes. A cDNA library constructed with mRNA from N. glutinosa plant tissues showing a systemic HR to TMV was subjected to subtractive hybridization with excess mRNA from healthy plant tissues. A total of 38 clones were selected after two rounds of subtraction. A11 of the clones were partially sequenced and the sequence data were analyzed by the BLAST program (Altschul et al., 1990). Northern blot hybridization of those clones with TMV-induced and unin- duced RNA was carried out to determine whether expression of the gene is induced by TMV. The deduced amino acid sequence of one clone (KC-9) had a high sequence similarity to metallothionein proteins, and the mRNA was strongly induced in TMV-infected tissues. This cDNA clone was 0.4 kb in size and contained only a partial sequence for the metallothionein-like protein cDNA.

To obtain the full-length clone, we screened 90,000 plaques of the original library and obtained 45 positive clones. One clone, KC9-10, had a cDNA insert and appeared to contain the full-length sequence of metallohonein-like protein. It was 530 bp in length and had one open reading frame consisting of 81 amino acids (Fig. 1). The deduced amino acid sequence had a high sequence similarity with metallothioneins from other organisms, including plants (Fig. 1). It also had eight and six Cys residues in its N- and



Wound- and Tobacco Mosaic Virus-lnducible Metallothionein-Like Gene 355

ACTTTCTAGTTTTAGAAAATOTCTTGCTGTGGAGGAAACTGTGGTTGTGGATCTGGCTGC

61 AAGTGCGGCAATGGCTGTGGCGGATGCAAGATGTACCCAGATTTGAGCTACAACGAAAGCK C G N G C G G C K M Y P D L S Y N E S

121 ACCACAACCGAGACTTTGGTGCTTGGGGTGGGCCATGAGAAGACAAGCTTTGGCACAATGT T T E T L V L G V G H E K T S F G T M

181 GAAATGGGTGAATCACCTGCTGCTGAAAATGGCTCCAAATGTGGATCTGAGTGCAAGTGTE M G E S P A A E N G C K C G S E C K C

241 AACCCCTGCGCTTGTTCTAAGTGAACAAATTAAATCTTAAAACGGAGCAGAGATGGATGTN P C A C S K

301 TAGCCTTTATTGGCTAAGACCAAAAAATAACTATAGTTTATAGTTCTTGTGTTTGTTGAA361 AACAAGGGGGAAATTATGTGTTTTTTTCCCTCTGAATAAGAAATAGTAATGGAATCTGAA421 C AATGCATGGGTTCTTGTTATTGGATACAAGTAGTTGTGTGGTTTGTAACATGAGATCTC4 81 TGCTTCTTGGTATTTTAACCTATGTTTTGGAAGTAAAAAAAAAAAAAAA

KMYPDLSYNES-TTTETLVLGVGHEKTSFGTMEMKMYPDMSYTESSTTTETLVLGVGPEKTSFGAMEMKMYPDMSFSEK-TTTETLVLGVGAEKAHFEGGEMKMYPDLGFSGESTTTETFVFGVAPAMKNQYEASG

NgLeReBe

NgLeReBe

MSMSMSMS

CCGGNCGCGSGCKCGNGCGGCCCGGNCGCGSSCKCDNGCGGCCCGGNCGCGSGCKCGNGCGGCCCGGNCGCGSGCKCGNGCGGC

GESPAAENG-GESPVAENG-GWGAEEGG-EG--VAENDR

CKCGSECKCNPCACCKCGSDCKCNPCTCCKCGDNCTCNPCTCCKCGSDCKCDPCTC

KMYKMYKMYKMY

SKSK

--

Figure 1. Nucleotide and deduced amino acid sequences and align-ment of some plant metallothionein-like proteins. A, Nucleotide anddeduced amino acid sequences of N. glutinosa metallothionein-likecDNA (GenBank accession no. U46543). Cys-rich N- and C-terminaldomains are underlined. B, Alignment of some plant metallothion-ein-like proteins. Ng, N. glutinosa (GenBank accession no. U46543);Le, Lycopersicon esculentum (GenBank accession no. Z68138); Re,Ricinus communis (GenBank accession no. L02306), Be; Brassicacampestris (GenBank accession no. L31940). Cys-rich N- and C-terminal domains are boxed and shaded.

C-terminal domains, respectively, and the pattern of Cysrepeats in this domain was the same as in other deducedplant metallothioneins (Fig. 1).

Tobacco Metallothionein Genes Are Encoded by a SmallGene Family

The genomic DNA of N. glutinosa (diploid) digested withEcoRl, Hindlll, and BamHl was probed with KC-9 cDNA.The Southern blot hybridization of N. glutinosa genomicDNA detected one major band of 0.5, 4.0, and 5.0 kb bythose enzyme digestions, respectively (Fig. 2A). On theother hand, the amphidiploid N. tabacum cv Xanthi nc(hybrid between N. tomentosiformis and N. silvestris, withthe TMV-resistant locus N introgressed from N. glutinosa)has two distinct hybridizing fragments when probed withthis cDNA (Fig. 2B). None of the hybridized restrictionfragments in N. tabacum was of the same size as thefragment of N. glutinosa, and this may suggest that themetallothionein-like gene(s) in N. tabacum was not intro-gressed from N. glutinosa. These results also suggest thatdiploid plants of Nicotiana spp. such as N. glutinosa probablyhave only one copy of this gene (Fig. 2). The weak hybridiza-tions present in the lanes suggest that there may be homolo-gous genes present in both species of plants (Fig. 2).

Expression of Tobacco Metallothionein-Like Gene

Tobacco cultivars containing the TMV resistance gene Nshow HR against TMV in a temperature-sensitive manner.Above the permissive temperature (28°C), N-containing

plants are susceptible to TMV, and the virus replicates andspreads systemically. However, below 28°C, all of the in-fected cells show HR to TMV. Consequently, systemic HRcould be obtained by the temperature shifts in the TMV-infected tobacco plants. Because the cDNA library wasconstructed from mRNA isolated from tissues showingsystemic HR, we tested the possibility of heat-shock-mediated induction of this gene expression. The KC-9cDNA probe was hybridized with total RNA from systemicHR (by temperature shifting), TMV-infected (HR withouttemperature shifting), and healthy plant tissues. The up-regulated level of 0.5-kb mRNA was detected in both sys-temic HR and HR but not in healthy tissues (Fig. 3). Thisresult clearly suggests that expression of the metallothionein-like gene is not heat-shock-mediated but is induced by theTMV-mediated disease resistance response. To confirm thisresult, we tested different chemicals and stresses for theinduction of metallothionein-like gene expression. North-ern blot hybridization revealed that the expression of thetobacco metallothionein-like gene is induced by wounding

BH B

23.0

0.53

Figure 2. Southern blot hybridization of tobacco DNA with N. glu-tinosa metallothionein cDNA probe. DNA (10 jug/lane) from N.glutinosa (diploid plant, A) and N. tabacum cv Xanthi nc (amphidip-loid, B) was digested with FcoRI (E), H/ndlll (H), and BamHl (B) andthen blotted onto a nylon membrane and hybridized with a 32P-labeled cDNA probe.



356 Choi et al. Plant Physiol. Vol. 112, 1996

- 25S

— 18S

Figure 3. Induction of N. g/ut/nosa metallothionein mRNA by TMVinfection. Total RNA (20 jug/lane) was isolated from infected tissuesshowing systemic HR (48 h at 32°C and then 24 h at 24°C after TMVinfection), local legion (72 h of TMV infection at 24°C), and healthyplant tissues. Blots were hybridized with a 32P-labeled metallothio-nein cDNA probe. Migrations of 25S and 18S rRNA are indicated.

or TMV infection, but not by SA or copper treatment (Fig.4). As a positive control of TMV, SA, and copper treat-ments, the same RNA blot was hybridized with thepathogenesis-related protein-1 cDNA probe (H.K. Yunand D. Choi, unpublished data), which is known to beinduced by TMV infection or SA treatment (Fig. 4).

Metallothionein

Pathogenesis-relatedprotein-l

^jjjjMjU

Figure 4. Northern blot hybridization of metallothionein mRNA afterdifferent stress treatments. Total RNA (20 /ig/lane) was isolated fromtreated tissues after 48 h of wounding, 5 mM SA treatment, 10 ITIMCuSO4 treatment, and TMV inoculation. Duplicated RNA blots werehybridized with metallothionein and pathogenesis-related protein-1cDNA probes. Migrations of 25S and 18S rRNA are indicated.

Differential Expression of Metallothionein-Like mRNAfollowing Wounding and TMV Infection

From the results shown in Figure 4, we hypothesizedthat the induction of the metallothionein-like mRNA levelby TMV infection could be mediated by wounding, whichis essential for TMV inoculation. To test this hypothesis,time-course RNA blot hybridization experiments followingwounding and TMV infection were carried out. Woundingalone can induce the expression of the metallothionein-likemRNA. The induced level of mRNA was detected within6 h after wounding, and the maximum level of mRNA wasdetected 12 h after wounding (Fig. 5A). TMV-infectedplants (local lesion) showed very similar patterns of mRNAexpression within 12 h of inoculation. But, in contrast to

A Time(hr) 0 6 12 24 48 72

Wounding

TMV

Ethidium BromideStaining of RNA

B

12 24 36 48 60 72

Time(hr)

Figure 5. Differential expression of tobacco (N. tabacum cv Xanthinc) metallothionein gene following wounding and TMV infection. A,Northern blot results of RNA samples isolated from treated tissues atdifferent times after wounding and TMV infection at 24°C. B, Quan-tification of the results of A using a densitometer (Pharmacia LKB).One result from two independent experiments with similar trends ispresented.



Wound- and Tobacco Mosaic Virus-lnducible Metallothionein-Like Gene 357

wounded tissues, TMV-infected tissues showed that theinduced level of mRNA was maintained even 48 and 72 hafter inoculation (Fig. 5B). This result may suggest thatTMV infection may prolong the wound response or pro-vide an additional signal transduction mechanism to pro-long the expression of the metallothionein-like gene.

Expression of Metallothionein-Like mRNA afterTreatment with CuSO4 and MJ

RNA samples were isolated from plant tissues treatedwith different concentrations of MJ (4, 40, and 400 ng/mL),which is an inducer of several wound-inducible plantgenes (Creelman et al., 1992; Farmer and Ryan, 1992; Choiet al., 1994). None of the three different concentrations ofMJ affected the metallothionein mRNA level at these par-ticular times (Fig. 6A). As much as 10 /xg/mL MJ also didnot affect the mRNA level. However, when 100 /xg/mL MJwas sprayed onto the tobacco leaves the mRNA level wasdramatically down-regulated (data not shown). Metal ionsare known to be important inducers of metallothionein

A Methyl jasmonate

B CuS04

Figure 6. Expression of metallothionein mRNA after MJ and CuSO4treatment. A, MJ was dissolved in 10% acetone and concentrations of0, 4, 40, and 400 ng/mL were sprayed directly onto tobacco leaves.Total RNA was isolated 48 h after treatment. B, CuSO4 was dissolvedin 10% acetone and concentrations of 0, 1, 10, and 50 mM weresprayed onto tobacco leaves. Total RNA was isolated 48 h aftertreatment.

Metallothionein

ACC Oxidase

Figure 7. Effects of ethylene and NBD on metallothionein and ACCoxidase mRNA expression. Total RNA (20 jtg) isolated from intactleaves that had been incubated for 12 h with air or air plus ethylene(20 p.L/L) and from wounded leaves incubated with air or air plusNBD (7000 jiL/L) were analyzed by northern blotting using 32P-labeled cDNA probes of metallothionein or ACC oxidase, respec-tively. The same trends were observed in three independentexperiments.

gene expression in mammals and yeast (Hamer, 1986; Furstet al., 1988; Mett et al., 1993). CuSO4 was tested as aninducer of tobacco metallothionein gene expression bytreating RNA tissue samples with different concentrationsof CuSO4 and subjecting them to northern blot hybridization.An approximately 2-fold increase of the metallothionein-likemRNA was detected when 1 or 10 mM CuSO4 was sprayedon tobacco leaves (Fig. 6B). A higher concentration (50 mM)of CuSO4 reduced the level of message, which may be theresult of phytotoxicity of copper. Thus, the induced level ofthe metallothionein-like mRNA by copper treatment wasrelatively mild when compared with wounding or TMVinfection (Figs. 3 and 4).

Effect of Ethylene and Ethylene Action Inhibitor onWound Induction of Metallothionein-Like mRNA

Ethylene is considered as a possible inducer of metallo-thionein gene expression because some plant metallothio-nein genes are induced during senescence (Buchanan-Wollaston, 1994; Coupe et al., 1995) and by ethylene(Coupe et al., 1995). To test the possibility that wound-induced ethylene is the causal agent of metallothioneingene expression, ethylene-action inhibitor NBD was ap-plied during wounding. The result was that NBD could notinhibit the wound-induced expression of the metallothionein-like gene (Fig. 7). As a positive control for the ethylenetreatment, a duplicate RNA blot was hybridized with anACC oxidase cDNA probe that is known to be induced byethylene (Knoester et al., 1995). ACC oxidase gene expres-sion was slightly induced by wounding (Fig. 7) and stronglyinduced by ethylene treatment (Fig. 7). These results clearlysuggest that the wound-induced accumulation of this metal-lothionein-like mRNA is not ethylene-mediated.

DISCUSSION

The cloning and characterization of genes expressed dur-ing the plant disease-resistance response could be an initial



358 Choi et al. Plant Physiol. Vol. 11 2, 1996

step toward elucidating the complex mechanisms of disease resistance. In this study we have isolated a wound- and TMV-inducible metallothionein-like cDNA from a N. glutinosa cDNA library by subtractive hybridization. Me- tallothionein genes are ubiquitous in many eukaryotic organisms, but the exact role of this protein is largely un- known (Hamer, 1986). In the case of animal and yeast metallothionein genes, it has been suggested that they play a role in metal metabolism and detoxification (Hamer, 1986; Furst et al., 1988). Expression of animal metallothionein genes in plants confer metal tolerance of the transgenic plants that could be indirect evidence for the role of those genes in metal detoxification (Misra and Gedamu, 1989; Yeargan et al., 1992; Elmayan and Tepfar, 1994; Hattori et al., 1994; Pan et al., 1994). Severa1 plant metallothionein-like protein genes have been cloned (Evans et al., 1990; De Framond et al., 1991; Zhou and Goldsbrough, 1994, 1995; Coupe et al., 1995), and some results showed that they are not induced by heavy metals (De Miranda et al., 1990; Kawashima et al., 1992); other recent results suggested that the genes are induced in senesced plant tissues (Buchanan-Wollaston, 1994) or induced by ethylene (Coupe et al., 1995). From these results, it can be assumed that the regulation and roles of the metallothionein-like gene in plants could be different from the gene in animals. The metallothionein-like gene from N. glutinosa is strongly induced by the vira1 pathogen TMV (Fig. 3), but subse- quent experiments revealed that it is also induced by me- chanical wounding (Fig. 4). To explain the signal molecules that induce tobacco metallothionein-like gene expression, we tested severa1 signal molecules as possible inducers. MJ, an inducer of some wound-inducible gene expression, did not affect the steady-state mRNA leve1 of this metallothionein-like gene (Fig. 6A). SA, an inducer of pathogenesis- related protein gene expressions in tobacco and other plants (Ward et al., 1991), also did not affect the accumulation of mRNA in this gene (Fig. 4). Copper, an inducer of metallothionein gene expression in animals and yeasts, only induced the accumulation of the mRNA by 2-fold (Figs. 3 and 5). Because some plant metallothionein genes were reported to be expressed in senesced tissues, and because their expressions were reported to be induced by ethylene (Coupe et al., 1995), we tested ethylene as a possible inducer but failed to find ethylene-induced expression of this gene (Fig. 7). This result rules out ethylene produced by wounding or pathogen infection as a signal recognized by this pathway. To date, the research on the function of metallothionein has focused on its role in metal metabolism and detoxification in biological organisms. Re- cent results from studies of cloned plant metallothionein genes, including the present study, suggest that metallothionein genes in plants may have different roles from those described for animals or yeasts.

Metals are involved in complex biological processes, which include a role as a cofactor of enzymes involved in biochemical oxidation and reduction. Some suggested roles of metallothionein in living organisms are the control of intracellular redox potential and activated oxygen detoxification (Hamer, 1986). Plants undergo oxidative stresses when exposed to wounding or pathogen infection (Baker

and Orlandi, 1995). Active oxygen species produced during those stresses affect disease resistance as well as disease symptom development in plants (Tzeng and DeVay, 1993). One possible role of the wound- and pathogen-inducible metallothionein gene is involvement in regulating avail- able metal ions, which subsequently affects the intracellular active oxygen species produced in stressed plants. Overexpression or inhibition of gene expression using an- tisense RNA techniques could provide insight into the role of this gene in plants.

ACKNOWLEDCMENTS

The authors thank to Dr. E.K. Park for the TMV strain and Mr. Mark A. Baayen for correcting the English during preparation of the manuscript.

Received April 18, 1996; accepted June 14, 1996. Copyright Clearance Center: 0032-0889/96/ 112/0353/ 07.

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molecular cloning of a metallothionein-like gene from ... · plant physiol. (1 996) 11 2: 353-359...

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