Light inducible and tissue-specific expression of a chimeric mouse metallothionein cDNA gene in tobacco

Plant Science, 76 ( 1991 ) 99--107

99

Elsevier Scientific Publishers Ireland Ltd.

Light inducible and tissue-specific expression of a chimeric mouse metallothionein c D N A gene in tobacco Indu B. Maiti, George J. Wagner and Arthur G. Hunt Plant Physiology~Biochemistry~Molecular Biology Program, Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091 (U.S.A.) (Received November 14th, 1990; revision received January 17th, 1991; accepted January 22nd, 1991)

A metallothionein cDNA was placed in sense or antisense orientation under the transcriptional control of a light-regulated promoter and the 3' flanking region from a pea ribulose bisphosphate carboxylase small subunit gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. Regenerated plants with the sense construct showed expression of metallothionein (MT) in a light-regulated, tissue-specific manner. Genetic analysis of R 1 and R2 progeny showed inheritance of the kanamycin resistance marker gene as a dominant Mendelian trait. Transgenic young seedlings with rbcS(ribulose bisphosphate carboxylase small subunit)MT construct showed slightly more tolerance to cadmium stress than did untransformed control seedlings. In addition to these studies, a comparison was made of the relative strengths of the CaMV 35S, rbcS, and mouse metallothionein promoters in plants. The MT promoter was virtually inactive in plants, even those treated with Cd.

Key words: Agrobacterium; gene expression; light-dependent metallothionein; tissue-specific tobacco

Introduction

Full exploitation of the genetic engineering approach for modifying agricultural plants requires identification of genes which confer useful traits, transfer to and expression of these genes in target plants, and the development of the capacity for organ-specific, developmentally - - or temporally -regulated, or environmentally responsive expression of foreign genes [1,2]. Genes conferring favorable traits such as herbicide insensitivity [3--7], resistance to pests [8--10], and tolerance to virus infection [11--13] have been identified and expressed in crop species using constitutively active promoters. Field crop species which have been manipulated include: tomato [14], soybean [15--16], oil seed rape [17], cotton [18--19], flax [201, alfalfa [21], sunflower [22], potato [23,24], Correspondence to: Indu B. Maiti, Plant Physiology/ Biochemistry/Molecular Biology Program, Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091 U.S.A.

tobacco [29] and others. Considerable effort is now being focused on organ-specific and developmental regulation of gene expression in plants [25--28]. As more species become amenable to manipulation by genetic transformation, it will become more important to achieve control of where, when, and by what influence introduced genes are expressed in the plant. We have expressed a mouse metallothionein (MT) gene in tobacco in an effort to assess the potential of this heavy metal chelator for partitioning the non-essential, potentially-toxic, largely pollutant metal, Cd, into unconsumed portions of crop plants [29]. Recent evidence indicates that this approach may be useful in that tobacco seedlings containing MT accumulated about 24% less Cd in leaves than did untransformed seedlings [30]. Theoretically, root-specific expression of a Cd chelator might minimize Cd accumulation in non-root tissues and be useful for minimizing Cd accumulation in leaf, grain and fruit products. Leaf-specific expression might serve to minimize

0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

100

accumulation in roots and be similarly useful for tuber and root crops. Here, we describe the introduction of MT constructs driven by a lightregulated promoter for the small subunit of the ribulose bisphosphate carboxylase gene (rbcS). We also show light dependence and organ-specificity of MT expression in transgenic tobacco. In addition, we show that the mouse MT promoter does not function in tobacco, either constitutively or in Cd-treated plants. Materials and Methods

Construction of expression vectors The construction strategy of vectors containing mouse MT cDNA gene in sense or antisense orientation is shown in Fig. 1. A 396-bp HindlII-SacI fragment from pUC MI901 [29] containing the MT coding region and a part of non-coding region was inserted in sense orientation into pKYLX5 [31] containing the light-dependent rbcS-E9 promoter. The resulting plasmid (pKYLXM508) carries (1) the 970-bp rbcS-E9 promoter, (2) 347 bp of MT sequence and (3) a 646-bp region containing the 3' non-coding sequence and polyadenylation signal of the pea rbcS-E9 gene. For the antisense construction, a 350 bp HindlII to SacI fragment from pBS MI01 [29] was cloned into the corresponding sites of the plant expression vectors pKYLX7 or pKYLX5 [31] to give rise to plasmids designated pKYLXAM720 and pKYLXAM528, respectively. In these plasmids the MT sequence, inserted in antisense orientation, was driven by either the 35S promoter from CaMV or the rbcS-E9 promoter, respectively. In order to compare the mouse MT promoter with the 35S and rbcS-E9 promoters, the EcoRI to HindlII fragment containing 35S promoter in pKYLX7 was replaced with the 1968-bp EeoRI to HindlII fragment from p8MT CAT (obtained from Dr. D. Hamer, NIH, Bethesda, MD 20892); this fragment contains nucleotides -1900 to +68 relative to the transcription start site of the mouse MT gene. The resulting plasmid was designated pKYLX9. The CAT gene was inserted into this as a HindlII to XbaI fragment from pAH10 [35]; the plasmid was termed as pKYLX9CAT. The constructions of pKYLX7CAT and pKYLX5CAT have been described previously [36].

Plant transformation, regeneration growth and segregation analysis Transformation of Nicotiana tabacum cv. KY 14 or N. tabacum cv. Petit havana was achieved with appropriately engineered Agrobacterium tumefaciens C58C1:GV3850 containing pKYLX508, pKYLXAM528, pKYLXAM720, pKYLX7CAT, pKYLX5CAT or pKYLX9CAT as previously described [31]. Transformed plants were transplanted into soil and grown in the greenhouse to obtain seeds. Segregation analysis was done with seeds from self-pollinated transgenic plants (R1 and R2 progeny) as described earlier [30]. Analysis of DNA, RNA and protein For southern hybridization analysis, DNA was extracted from leaves of transformed and untransformed plants as described previously [29]. Total DNA (10 /zg) was digested with Sacl and HindlII, separated on a I% agarose gel, transferred to nitrocellulose, hybridized with a nick translated probe (HindIII digested pUCMI901 containing approximately 2 × 107 cpm//~g 32p), washed as described elsewhere [29], and visualized by autoradiography. Alternatively, a nonradioactive probe was prepared with HindII| digested pUCM1901 [32]. In this instance, the washed blot was visualized using mouse antidigoxigenin antibodies and goat alkaline phosphatase-conjugated anti-mouse antisera. Northern blot analysis was done as described elsewhere [29]. Total RNA was extracted from leaves of transformed plants and untransformed plants. Total RNA (30/~g) was denatured at 50"C in 2 M glyoxal, fractionated on a 1.5% agarose gel, and transferred to nitrocellulose. Northern blots were hybridized with a 32p-labelled pUCMI901 probe containing mouse MT cDNA as described earlier [29]. For the RNA dot blot analysis, total RNA was isolated from leaves and roots of homozygous KYI4M508 plants (R2 progeny) grown in presence or absence of light. Poly(A)-enriched RNA was prepared by oligo(dT) cellulose column chromatography [32]. The poly(A)-enriched RNA was denatured in 2 t~ glyoxal at 50°C and transferred to nitrocellulose paper using a Bio-dot apparatus (Bio-Rad). A uniformly 32P-labelled RNA probe (spec. act., approx. 2 × 10 9 cpm/~zg)

I01

was made by transcribing SacI-digested pBSM 101 [29] with T3 RNA polymerase [32]. Conditions for hybridization and autoradiography were as described earlier [29].

sensitive phenotype. Two other plants, nos. 2 and 6, gave a higher ratio suggesting insertion of more than one copy of T-DNA. Similar results have been obtained in earlier experiments and by others [14,30].

Determination of CA T activity in Cd-treated plants Six-week-old Petit havana seedlings with pKYLX9CAT (MT-promoter) or suspension cultures derived from these plants were exposed to concentrations of CdC12 ranging from 3 to 100 ~M. After 2 weeks, roots and leaves were harvested. Roots or calli were washed with 50 mM CaCI 2 to remove loosely-bound Cd. Tissues from these materials, and from 6-week-old Petit havana seedlings containing pKYLX7CAT and pKYLX5CAT, were extracted with 0.25 M Tris-HCI (pH 8.0), clarified by centrifugation, and aliquots containing 10 #g of soluble protein used for chloramphenicol acetyltransferase (CAT) activity determinations. CAT activity was determined according to the method of Gorman et al. [34].

Other procedures Procedures for protein extraction, assay of MT by ~°9Cd binding assay, Sephadex G-75 gel filtration, and Cd-tolerance analysis of seedlings have been detailed elsewhere [30]. Results and Discussion

Transformation, regeneration, and segregation analysis of kanamycin-resistant plants The strategy for constructing the binary vectors pKYLXM508, pKYLXM528 and pKYLXM720 is shown in Fig. 1. The presence of the MT coding region in plasmids isolated from E. coli and Agrobacterium was confirmed by restriction digestion and Southern blot analysis using MT-cDNA as a probe. A,grobacterium strains carrying these plasmids were used to transform tobacco, and several primary transformed lines carrying each construction were obtained. Primary transformants obtained with pKYLXM508 were designated as KY14M508, and so on. Seed from eight inde4~,endent self-fertilized transgenic KYI4M508 lines were taken for segregation analysis of the marker gene (Table I). Six of these segregated with an expected ratio of 3:1 for the kanamycin-resistant versus kanamycin-

Integration and expression of the metallothionein gene in transgenic plants Southern analysis (Fig. 2A) of genomic DNA isolated from three different RI KY14M508 lines (nos. 1, 2 and 3) showed the presence of the MT sequence in the plant genome. The MT insert was identified as a 0.4 Kb band from HindlII-SacI digested DNA of transgenic plants (lanes 1--3) and was not present in untransformed plants (lane 5). Lane 4 represents one copy of insert per genome equivalent of pKYLXM508. By comparison with a reconstruction roughly equivalent to one copy per genome, the number of copies of the MT gene in the transgenic plant lines analyzed was roughly 2--4 copies per tobacco genome. In earlier analyses similar constructs which contained the 35S promoter were found to be present in a copy number of 2--6. The presence of the MT gene in primary transgenic plants containing the antisense construct of MT driven by either 35S or rbcS-E9 promoter were confirmed by Southern analysis (data not shown). Northern analysis (Fig. 2B) of KY 14 M508 plants (nos. 1, 2 and 3) showed the presence of MT transcripts of the expected size (compare lanes 4 6 with lane 1). The abundance of transcripts in M508-transformed plants (lanes 4 6) was about 15--30 times less than that seen in plants with the 35S promoter-MT construction (lane 1). Interestingly, the abundance of RNAs from the antisense construct driven by the 35S promoter (lane 3) was very low and the corresponding RNA was not detectable with the antisense construct driven by rbcS-E9 promoter (lane 7). Also, the antisense RNAs were much larger than expected, indicating some sort of improper processing or 3' end formation in this construction. RNA dot blot analysis of material from KYI4M508-1 (Fig. 3) showed a high abundance of MT transcript in light-grown leaves and very low levels in dark-grown leaves. In addition, no MT RNAs were seen in roots. Therefore, the previously documented tissue-specific and light-dependent

102

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Fig. 1. The structures of the micro-T-DNAs used to introduce various MT and CAT derivatives into plants in this study. The arrows show the directions of transcription of the MT or CAT genes and the kanamycin resistance gene. The positions of the left and right T-DNA borders (LB and RB, respectively), the promoters used to drive expression of the genes of interest (PROMOTER), the MT or CAT gene (hatched or solid box, respectively), the rbcS polyadenylation signal (3' REGION), and the kanamycin resistance gene are illustrated. The identity of the respective promoter (35S, rbcS, MT) is given beneath its location in the plasmid. The designation of each plasmid is given to the upper left o f each construction. The positions of Hindlll (H), Sacl (S) and Xbal (X) sites used to assemble these plasmids are shown as well.

103

Table I.

Segregation analysis of seed lots from self-pollinated KY14M508 primary transformants.

Plant no.

N u m b e r seeds tested

Percent germination a

K m R : K m sb

Ratio tested

x 2 Probability tested c

1 2 3 4 5 6 7 8

100 108 102 100 106 101 101 100

88 96 94 96 100 100 101 93

67:21 84:20 72:24 72:24 77:29 82:19 77:24 70:23

3:1 3:1 3:1 3:1 3:1 3:1 3:1 3:1

0.9-0.75 0.25-0.10 1.00 1.00 0.9-0.75 0.25-0.10 0.9-0.75 1.0-0.95

aSeeds were sterilized with ethanol (70%) for 2 min, sodium hypochlorite (0.5%) for 15 min, and sown on rooting medium containing 300 ttg/ml kanamycin. Germination efficiency was assessed after 2 weeks of growth at 22--28"C under constant light. bThe ratio of kanamycin resistant to kanamycin sensitive germinated seedlings(KmR:Km s) was evaluated after 3 weeks of growth on kanamycin-containing medium by scoring the number of green (resistant) and white (sensitive) seedlings. CThe Kmr~:Km s ratios were tested for goodness of fit to a 3:1 distribution using the X2 test. Results are presented as the range of probabilities for which that the observed ratios and the predicted ratio are the same.

activity of the rbcS-E9 promoter is retained in our constructions. To demonstrate the occurrence of metallothionein capable of binding Cd in leaves of KY14M508, gel filtration and 1°9Cd binding was used. Sephadex G-50 gel filtration analysis of leaf extracts showed the presence of a 1°9Cd binding activity in KY14MS08-3 that is characteristic of rodent MT (Fig. 2C). Untransformed tobacco and transformed tobacco with the antisense construct did not show the presence of this macromolecule (data not shown). The amount of MT in transgenic plants driven by rbcS-E9 promoter was about 0.003% of soluble leaf protein as compared to 0.06-~0.1% reported previously for transformants containing the 35S promoter. Thus, expression of MT was about 20--30 times more in 35S-MT transgenic plants compared to these transgenic plants with the light-dependent rbcS-E9 promoter.

Comparative Cd tolerance of transformed KY14M508 and untransformed KY14 seedlings We have previously reported that seedlings of tobacco lines that express MT under control of the 35S promoter were more resistant to Cd than were controls. In order to extend this observation to the rbcS-MT lines, seeds from transformed homozygous KY14M508 and untransformed lines

were germinated in presence of different concentrations of CdC12 (0--1000 #M). At 1000 #M Cd, transformed 4-leaf stage seedlings accumulated 16% of the fresh weight of untreated seedlings over the same growth period (Fig. 4). Untransformed controls, on the other hand, accumulated less than. 2% of the fresh weight of untreated controls at the same CdCI2 concentration. Chlorophyll content was severely reduced in both untransformed and transformed seedlings. Using accumulation of fresh weight as a parameter, the rbcS-MT transformant tested here was more resistant to Cd in this system than untransformed controls. We would note, however, that the degree of tolerance seen here is less than what we have seen with seedlings carrying a 35S-MT gene [30]. Seedlings carrying a 35S promoter-MT construction retained 63% of the fresh weight and 29% of the chlorophyll (with respect to untreated controls [30]), whereas the rbcS promoter-MT containing plants analyzed here retained just 20% of the fresh weight and none of the chlorophyll under similar conditions (Fig. 4). In tobacco seedlings in which the MT gene is driven by the 35S promoter, about 24% less Cd accumulates in leaves than in leaves of untransformed controls [30]. We see no such differences between KYI4M508 plants and KYI4 controls (data not shown). This is probably due to the

104

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Fig. 2. Analysis of the expression of the MT genes in transgenic plants. (A) Southern blot analysis oftransgenic RI plants containing the MT DNA sequence. Genomic DNA ( l0 p,g) was digested with Sacl HindIll and separated on a 1% agarose gel. Shown are samples isolated from leaves of KY14MS08 plants l, 2 and 3 (lane 1--3), KYI4 non-transformed control plant (lane 5) and a reconstruction representing one copy per genome equivalent of pKYLXMS08 (lane 4). Immunological detection was done using a non-radioactive probe as described in Materials and Methods. (B) Northern blot analysis of total RNA isolated from leaves of KYI4M508 plants. Shown is an autoradiograph of a blot containing: RNA (30 #g) from leaves of KY 14M707 plants (these carry a 35S-MT construction; lane 1): liver RNA (10/~g) from a Cd-injected mouse (lane 2); leaf RNA (30 ~g) from a KYI4AM720 plant (these carry a 35S-MT antisense construction; lane 3); leaves of individual KY 14M508 plants 1 3 (these plants carry the rbcS-MT construction: lanes 4~6); and leaf RNA (30 t~g) from antisense construct KY 14 AM528 (these plants carry the rbcS-MT antisense construction; lane 7). Filters were hybridized with nick-translated 32p-labelled pUC MI901 (this plasmid contains a mouse MT cDNA). Markers at the left of the autoradiograph indicate position and size in kbp ofHindlil-digested h DNA. The size difference between the plant and rat-derived MT RNAs reflects the presence of an additional 250 or so nucleotides in the plant-derived RNAs that are due to the presence of rbcS 3'-non-coding sequences in the plant gene expression vector. (C) Sephadex G-50 gel filtration analysis of MT in transgenic plants. Protein was extracted from mature fully expanded leaves. One hundred milligrams was loaded onto a Sephadex G-50 column (90 x 15 cm) and analyzed as described earlier [30]. The profile shows the presence of metallothionein (MTI in a leaf-extract from KYI4M508 plant no. 1 (RI progenyl. A 4.3-kd Cd-binding component was found in KY 14M508 plants which was like that found earlier in KYI4M707 plants [30] but not in untransformed plants. The nature of this component is not clear.

l o w e r levels o f M T in K Y I 4 M 5 0 8

with respect to

KY14M707 plants (20~30X less; see a b o v e ) . Because cadmium binds to other plant comp o n e n t s s u c h as cell w a l l m a t e r i a l , a c e r t a i n t h r e s h h o l d o f M T m a y be n e e d e d to a t t a i n the dif-

f e r e n c e s in d i s t r i b u t i o n t h a t w e r e p o r t e d e a r l i e r ; i f t h e M T levels a r e n o t o f t h e s a m e m a g n i t u d e o f t h e s e o t h e r C d - b i n d i n g c o m p o n e n t s , a m a r k e d diff e r e n c e in

overall

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Fig. 3. Tissue specific and light-regulated expression of the rbcS-MT gene in transgenic tobacco. Poly(A)-enriched RNA ( 1 ~ 4 p,g) isolated from leaves of KYI4M508 homozygous plants (R2 progeny) grown in light or dark and from roots was immobilized on nitrocellulose filters, hybridized with a 32p_ labelled antisense RNA probe transcribed from Sacl digested pUC MI901, washed, and the results visualized by autoradiography.

0

I00

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$00

750

1000

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120 lOO

Comparison of the MT, 35S, and rbcS E-9 promoters In addition to using different plant promoters to express MT in tobacco, we were interested in determining whether or not the mouse MT promoter might be useful as a tool for obtaining Cdinduced expression in plants, as has been demonstrated in mouse cells and other organisms [37]. Consequently, we assembled a promoter MTCAT gene and compared its activity in transgenic plants with the activities of the 35S and rbcS-E9 promoters (Fig. 5, pKYLX9-, pKYLX7-, and pKYLX5-CAT, respectively). In plant cells not treated with Cd, we found that the MT promoter was less than 10% as active as the rbcS promoter, and less than 1% as active as the 35S promoter, as judged by relative levels o f CAT gene expression. In addition, expression of the MT-CAT gene was not increased by treatment of the transgenic seedlings carrying this gene with Cd (data not shown). Pautot et al. reported that the mouse MTpromoter was not active in Ri plasmid mediated transformed tobacco root cells [38]. Thus, it appears that the mouse M T promoter is not suitable for driving gene expression in tobacco.

.<

0

100

250

500

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I.tM CdCi 2 Fig. 4. Comparative cadmium tolerance analysis of KYI4M508 and untransformed KY14 very young seedlings, Cd-tolerance analysis of transgenic and control seedlings were done as described earlier [30]. The top panel shows a comparison of the relative growth (assessed as mg fresh weight per 100 seedlings and normalized against untreated controls as described earlier [30]) of transformed and control seedlings in the presence of increasing concentrations of CdCI 2, The bottom panel shows a comparison of the relative chlorophyll contents (assessed as mg chlorophyll per 100 seedlings and normalized against untreated controls as described earlier [30]) of transformed and control seedlings in the presence of increasing concentrations of CdCI.~. Error bars represent 10% of the mean o f each column.

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Fig. 5. Functioning of the MT promoter in transgenic tobacco calli. CAT activity in transformed plants carrying the pKYLX7-CAT, pKYLX5-CAT and pKYLX9-CAT constructions was assessed as described earlier. Shown is an autoradiograph of a chromatographic separation of acetylated (Ac) from non-acetylated 14C-labelled chloramphenicol (CM). Chloramphenicol acetyltransferase activity was determined in extracts containing 10/~g of total protein isolated from plant callus material carrying pKYLX5-CAT (rbcS-E9 promoter; lane 1), pKYLX7-CAT(35S promoter; lane 2) and pKYLX9 (MT promoter: lane 3). Lane 4 is an assay of an equivalent sample prepared from untransformed tobacco cells.

Acknowledgements We thank Dr. Brad Mogen for assistance with graphics and Iris Deaton for assistance with preparing the manuscript.

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