Plant Molecular Biology 10:359-366 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands 359

The 5' flanking region of a barley B hordein gene controls tissue and developmental specific CAT expression in tobacco plants

Claire Marris, Patrick Gallois, Jane Copley and Martin Kreis AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ~ UK

Received 2 October 1987; accepted in revised form 19 January 1988

Key words: barley, tissue-specific gene expression, endosperm, hordeins, tobacco

Abstract

The 549 base pairs of the 5' flanking region of a barley seed storage protein (B1 hordein) gene were linked to the reporter gene encoding chloramphenicol acetyl transferase (CAT). The chimaeric gene was transferred into tobacco plants using Agrobacterium tumefaciens. CAT enzyme activity was detected in the seeds, but not in the leaves, of the transgenic plants. Furthermore, enzyme activity was found only in the endosperm, and only from fifteen days after pollination. In contrast, the constitutive 19S promoter from cauliflower mosaic virus (CaMV) directed the expression of the CAT gene in the leaves as well as in both the endosperm and embryo and at all stages in seed development.

Introduction

B hordein is the major group of barley seed storage proteins [38]. It is encoded by a multigene family which is only expressed in the developing endosperm [8, 29, 34]. Our aim is to understand the mechanisms by which the B hordein genes are expressed in a tem- poral and cell-specific manner. It has been shown that higher plant genes that encode messenger RNA contain distinct regulatory sequences in their 5' flanking region that are required for tissue-specific gene expression (for a review, see [21]). Comparison of the 5' flanking regions of a B1 hordein gene to other cereal storage protein genes with a similar pat- tern of expression revealed three strongly conserved sequences within 563 base pairs (bp) of the ATG in- itiatorcodon, namelyasequenceat - 300 bpofabout 33 bp (the "-300 element"), a "CAAT" and a "TATN' box [9, 20]. It was suggested that the " - 300 element" might be involved in the transcriptional

control of hordein gene expression during en- dosperm development.

Plant transformation provides a good system to assay the function of regulatory elements in vivo. Although dicot genes may be better expressed than monocot genes in transgenic dicot plants [18], Lamppa et ai. [22] showed that the light-regulated and organ-specific expression of the wheat chlo- rophyll a/b binding protein gene was maintained in transgenic tobacco plants. Several other monocot genes have been transferred into dicot plants [18, 23, 36] but not all were expressed [18, 23]. In this study we have addressed the question whether the 5' flank- ing region of a barley B1 hordein gene can mediate the expression of a reporter gene in transgenic tobac- co. Our results show that a B1 hordein-CAT chimaeric gene is expressed in a tissue and develop- mental specific manner in transgenic tobacco plants, suggesting that B1 hordein cis-acting regulatory se- quences are recognized by tobacco trans-acting fac- tors.

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Materials and methods

Bacterial strains

The E. coli strains used were MC1022 and HB101 [24] and were grown at 37 °C in Luria-Bertani (LB) medium [24] with the appropriate antibiotics. The disarmed Agrobacterium tumefaciens LBA4404 strain [13] was used for tobacco transformation and was grown at 29 °C in 2x YT medium with the ap- propriate antibiotics.

Vectors used

pCATter and pl9S-CAT were both constructed and kindly donated by M. Bevan (P.B.I., Cambridge, UK). pCATter consists of Binl9 [1] with the coding sequence of the bacterial chloramphenicol acetyl transferase (CAT) gene inserted into the polylinker. Plant transcription termination and polyadenyla- tion signals are provided at the 3' end by a segment of the nos gene. Four useful restriction sites from the mpl9 polylinker (HindII, SalI, XbaI and BamHI) are left at the 5' end of the CAT coding sequence where promoter sequences can be inserted, pl9S- CAT was created by inserting the constitutive pro- moter of the 19S gene of CaMV [12, 19, 21, 40] at the 5' end of the CAT coding sequence.

Mobilization o f plasmids into Agrobacterium tumefaciens

pCATter, pl9S-CAT and pB1H-CAT were mobilized from E. coli MC1022 into A. tumefaciens LBA4404 using the helper plasmid pRK2013 [6]. A. tumefa- ciens LBA4404 cells harbouring the Binl9 deriva- tives were selected on minimal media containing 50 ttg/ml kanamycin.

Transformation of tobacco plants

Tobacco plants were transformed using essentially the method described by Horsch et al. [14]. Four- week-old in vitro shoot cultures of Nicotiana taba-

cum (cv. Petit Havana, mutant SR1) were used as source material. Leaf discs were laid, adaxial side up, on nurse culture plates. These consisted of shoot- inducing medium containing MS salts [30] sup- plemented with 30 g/1 sucrose, 10 g/1 agar, and 1 mg/l benzyl amino purine, pH 5.6, overlaid with 2.5 ml of a 10-day-old Nicotiana rustica cell suspen- sion culture (adjusted to 0.2 g/ml) as described by Horsch and Jones [15]. The discs were incubated for 2 days, then submerged for one minute in a culture of A. tumefaciens LBA4404 harbouring the ap- propriate Binl9 derivative, blotted dry, replaced on the nurse cultures and incubated for a further two days. Then the discs were transferred to selection plates consisting of the same medium but with 200 mg/1 cefotaxime, 100 mg/1 kanamycin sulphate, only 8 g/l agar and no nurse culture. They were transferred to fresh medium regularly until green shoots developed on the edges of the leaf discs (ap- proximately three weeks after inoculation). These were excised and transferred to fresh medium. When the shoots reached 1 cm in height, they were trans- ferred to MS medium supplemented with 200 mg/1 cefotaxime, 100 mg/l kanamycin sulphate, 30 g/1 sucrose, but no hormones. After a further two weeks, the shoots which developed roots were trans- ferred to soil, hardened for two days in a micropropagator and then grown in the greenhouse. Only one kanamycin-resistant plant was regenerated from each leaf disc to ensure that each plant origi- nated from a different transformation event.

CAT assays

Transformed tobacco plants were manually self- pollinated, seeds and leaves were collected, ground in 0.25 M Tris-HC1 pH 7.5, heated to 65 °C for 10 min as described by Mercola et al. [27], and spun at 10000 g for 15 min at 4 °C. The supernatants were recovered, assayed for protein content [2], and CAT enzyme activity was measured essentially as described by Gorman et aL [10]. 100 #1 of tissue ex- tract were incubated at 37 °C for 2 h with 20 #1 of 10 mM acetyl coenzyme A and 0.5/~1 of D-threo- [dichloroacetyl-1 - 14C]-chloramphenicol (50-mCl/ mmol, Amersham International plc). The sam-

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ples were extracted twice with 100/~1 ethyl acetate, the organic layer was dried, taken up in 15 #1 of ethyl ace- tate, spotted on silica gel thin-layer plates, run with chloroform-methanol (95:5 v/v, ascending), and subjected to autoradiography.

Southern blot analysis of transgenic tobacco plants

High molecular weight DNA was extracted from tobacco plants as described by Dellaporta et al. [4] and then purified by caesium chloride gradient cen- trifugation. 10/~g DNA from each plant was digest- ed with HindIII, separated on a 0.75%o agarose gel, and transferred onto a Nytran TM membrane (0.45 /~m pore size, Schleicher and Schuell) by electro- blotting. The membrane was prehybridized with 6 × SSC (1× SSC, standard saline citrate = 0.15 M NaC1, 0.015 M trisodium citrate), 1%0 SDS, 10 × Den- hardt's (1 × Denhardt's = 0.02%o Ficoll, 0.02%o poly- vinylpyrollidone, 0.02%0 bovine serum albumin), 50 ttg/ml denatured sheared salmon sperm DNA at 42 °C for 4 h. Hybridization was performed in tax SSC, 1%0 SDS, 50% formamide, 50/~g/ml denatured sheared salmon sperm DNA at 42 °C for 15 h. Probes used were a 530 bp fragment containing the CAT coding sequence, excised from pCATter with BamHI, and a 567 bp fragment containing the 5' flanking region of the B1 hordein gene, excised from pB1H-CAT with BamHI. The DNA fragments were labelled with 32p by nick translation [35]. The mem- branes were washed in 0.1% SSC, 0.1% SDS at 65 °C.

Results

1. Construction of pB1H-CAT

A barley genomic EcoRI fragment of 2.9 kbp con- taining a B1 hordein gene was previously subcloned into pUC9 to yield the plasmid pBHR184 [9]. This genomic fragment contains 879 bp coding, 1458 bp of 3' and 563 bp of 5' flanking sequence. The latter segment contains a "TATA" box, a "CAAT"-Iike box and a putative regulatory flanking sequence at

- 300 bprelativetotheATG. A578 bpHindlII/HpaI fragment was excised from the plasmid pBHR184. This fragment contained at the 5' end 29 bp of the polylinker of pUC9 followed by 549 bp of the 5' se- quence of the B1 hordein gene beginning 15 bp up- stream of the translation initiation codon and 37 bp downstream of the transcription start site. The Hin- dIII/HpaI fragment was inserted 5' to the coding re- gion of the bacterial chloramphenicol acetyl trans- ferase (CAT) gene in the plasmid pCATter (Fig. 1), a derivative of the disarmed Ti binary vector Binl9 [I], using the following strategy (Fig. 1). The plas- mid, pCATter, contains at the 5' end of the CAT gene several unique restriction sites including HindIII and SalI. The vector was digested with SalI, blunt- ended with T4 DNA polymerase and then digested with HindIII to generate one cohesive and one blunt end. Finally, the 578 bp HindIII/HpaI restriction fragment recovered from pBHR184 was ligated into the double digested pCATter. The resulting recom- binant plasmid (called pBH1-CAT) was used to transform E. coli strain MC1022. Positive clones were selected by colony hybridization using as a probe the 578 bp HindIII/HpaI restriction flag-

~ n p t II

I j nos/npt I I ~ n ° s l i 2' end

~ CAT codirg , , I , sequence Hindll l Sai l Xbel Barn HI

• "" " . 578bp 5' l" ".j 3' Hind Ill / Hpal

t t t t T~A] t fragment Hindll l Eco RI TGTAAAQ CATC TA HI)el

- 3 0 0 element Tren$cription

start site

Fig. L Construction of pBIH-CAT used for transformation of tobacco. A 578 bp HindlII/HpaI fragment was excised from the plasmid pBHR184 [9] and inserted between the HindlI and the Sail sites immediately upstream of CAT in the vector pCATter.

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ment. The plasmid pB1H-CAT was analysed by re- striction mapping to ascertain that the B hordein 5' - flanking region was correctly fused to the CAT cod- ing region.

2. Transformation of tobacco

Tobacco leaf discs of Nicotiana tabacum ev. "Petit Havana" mutant SRI were inoculated with A. tumefaciens containing pB1H-CAT as described in Materials and Methods. Fifteen kanamycin-resistant plants were regenerated. Similarly nine kanamycin- resistant control plants were regenerated from leaf discs inoculated with LBA4404: pl9S-CAT. These transgenic plants, referred to as B1H-CAT and 19S- CAT plants respectively, were self-pollinated and the expression of the chimaeric genes was assayed by CAT enzyme activity in the leaves and seeds.

3. Seed-specific expression of the B1H-CAT chimaeric gene in transgenic tobacco

Seeds and leaves from transgenic tobacco plants were harvested, crude protein extracts were prepared and assayed for CAT activity. No enzyme activity above background was found in the leaves from the 15 transgenic B1H-CAT plants, but CAT enzyme ac- tivity was detected in the seeds from plants B1H- CAT Nos. 2, 3 and 14, indicating that the chimaeric gene functions only in the seed. Five out of the nine 19S-CAT plants showed CAT activity in the leaves, of which one (Plant No. 55) was chosen for further experiments. Figure 2 compares the CAT enzyme ac- tivities in the leaves and seeds of plants B1H-CAT No. 2 and 19S-CAT No. 55. The same seed-specific pattern was observed in plants B1H-CAT No. 3 and No. 14 but the level of CAT enzyme activity per/~g protein was lower. Plant 19S-CAT No. 55 showed several fold higher enzyme activity in leaf extracts than in seed extracts containing equal amounts of protein. The level of CAT activity under the control of a constitutive promoter will not necessarily be the same per unit soluble protein in different tissues due to their different protein compositions. Similarly Fluhr et al. [17] observed different levels of CAT ac-

Fig. 2 Seed-specific expression of the B1H-CAT chimaeric gene in a transgenic tobacco plant. CAT assays were performed as described in Materials and Methods on leaf (lanes 1, 2 and 3) and seed (lanes 4, 5 and 6) extracts containing 400 #g protein (meas- ured according to Bradford [2]). Lanes 1 and 4 show CAT enzyme activity in extracts from a non-transformed plant (NT), lanes 2 and 5 from plant B1H-CAT No. 2, and lanes 3 and 6 from plant 19S-CAT No. 55.

tivity in leaves, stems, and roots of transformed dicot plants under the control of a 35S CaMV promoter.

4. Expression o f the BIH-CAT chimaeric gene during the development of the transgenic tobacco plant

During normal barley endosperm development, B hordeins are first detected around 15 days after anthesis and the level increases until seed maturity at 40 days [34]. To determine whether this develop-

mental control of expression is retained in the trans- genic tobacco plants, seeds from self-fertilized trans- genic B1H-CAT No. 2 and 19S-CAT No. 55 plants were collected at different stages after pollination and tested for CAT activity. Fig. 3 shows that CAT activity under the control of the B1 hordein 5' flank- ing region was first measured 15 days after pollina- tion, reached a maximum level at 19 days and re- mained constant until maturity at 30 days. In contrast, the seeds from plant 19S-CAT No. 55

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Fig. 3. Developmental specific expression of the B1H-CAT chimaeric gene in a transgenic tobacco plant. Protein was extracted from tobac- co seeds collected at different stages after pollination and assayed for CAT enzyme activity as described in Materials and Methods. Each sample contained 50 seeds. Lanes I and 2 show CAT enzyme activity in seeds from plant 19S-CAT No. 55, lane 3 from a non-transformed (NT) plant and lanes 4 to 10 from plant BIH-CAT No. 2.

showed the same levels of CAT activity at all stages of development. Thus the regulatory sequences of the barley B1 hordein gene confer developmental specific expression of CAT in the transgenic tobacco plant containing the B1H-CAT chimaeric gene.

5. Endosperm-specific expression of the B1H- CAT chimaeric gene in transgenic tobacco plants

In barley the B hordeins are only synthesized in the

endosperm, where they are deposited in protein bod- ies [28, 34]. The major tobacco storage proteins, however, are synthesized and accumulated both in the endosperm and embryo [11, 37]. To determine in which part of the tobacco seed the B1H-CAT gene was expressed, mature seeds from self-fertilized transgenic plants were dissected into endosperms, embryos and testas, which were assayed for CAT ac- tivity separately. Enzyme activity was present only in the endosperms of plants B1H-CAT No. 2 and 3 (No. 14 was not assayed), whereas it was also present

Fig. 4. Endosperm-specific expression of the BIH-CAT chimaeric genein a transgenic tobacco plant. Mature tobacco seeds were manually dissected under a microscope and CAT assays were performed on protein extracts from testas (a), endosperms (b), embryos (c), or whole seeds (d) as described in Materials and Methods. Lanes 1 and 6 show CAT enzyme activity in whole seeds from a non-transformed (NT) plant, lanes 2 to 5 in tissues from plant BIH-CAT No. 2, and lanes 7 to 10 in tissues from plant 19S-CAT No. 55. In Fig. 4A, each sample contained 50 seeds or parts of seeds, in Fig, 4B, each sample contained 100.

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in the embryo of plant 19S-CAT No. 55 (Fig. 4). The testa, which is a dead tissue consisting only of ligni- fled cell walls and no cytoplasm [26], showed no CAT activity either in the B1H-CAT or in the 19S- CAT plants. When driven by the constitutive 19S promoter, CAT activity in 100 embryos is lower than in 100 endosperms. This is probably due to the fact that the embryo accounts for only 30% of the tobac- co seed tissue [37].

6. Molecular analysis o f the transgenic tobacco plants containing the B1H-CAT and 19S-CAT genes

A Southern blot analysis of independently trans- formed tobacco plants which were used in the ex- pression experiments described above is shown in Fig. 5. Plant DNA digested with HindlII was frac-

tionated and transferred to a nylon membrane. The membrane was first hybridized at moderately low stringency to the 32p-labeUed 530 bp BamHI restric- tion fragment containing only the CAT coding re- gion. The probe was stripped off from the mem- brane which was then hybridized to the labelled 5' flanking region (567 bp) of the B1 hordein gene. Genomic DNA from transgenic plants B1H-CAT

No. 2, 3 and 14 show several HindIII restriction frag- ments hybridizing to the CAT coding region (Fig. 5, lanes 1, 2 and 3). A reconstruction experiment (Fig. 6, lanes 7, 8, 9 and 10) suggests that the copy number of the transferred gene ranges from 3 to about 20 copies per haploid genome. The same frag- ments hybridized when the filters were probed with the 5' flanking region of the BI hordein gene (not shown). Genomic DNA from plant 19S-CAT No. 55

contained one HindIII fragment which hybridized to the 530 bp CAT coding region (Fig. 5, lane 4) but not to the 5' flanking region of the B1 hordein gene. No hybridization was observed with DNA isolated from non-transformed plants (Fig. 5, lanes 5 and 6). The above results clearly show that the number of genes transferred differs in each of the transformed plants. No correlation was observed between gene copy number and the level of CAT enzyme activity. This agrees with previous reports that have shown variation in gene expression independent of copy

Fig. 5. Southern blot analysis of transgenic tobacco plants con- taining B1H-CAT and 19S-CAT. 10 #g of DNA from tobacco plants were digested with HindlII, separated on an 0.75070 agarose gel and transferred to a nylon membrane. Hybridization and washing was as described in Materials and Methods. Lanes 1, 2, and 3 represent plants B1H-CAT No. 2, 3 and 14 respectively. Lane 4, transgenic plant 19S-CAT No. 55. Lanes 5 and 6, non- transformed (NT) plants. In lanes 7 to 10 the 530 bp BamHI re- striction fragment containing the CAT coding region was used to estimate the gene copy number (2.18 pg fragment is equivalent to 1 gene copy/haploid genome).

number in individual transformed organisms by us- ing A. tumefaciens or other gene transfer methods [3, 16, 17, 31, 33]. However, a recent report shows dosage-dependent expression of a potato gene trans- ferred into potato and tobacco plants [39]. The phenomenon of varying expression of artificially in- troduced genes could be due to a position effect resulting from a random integration of the trans- ferred DNA [16].

Discussion

This is the first report that shows regulated expres-

sion of a barley seed protein-CAT chimaeric gene in

transgenic plants. Our observations indicate that the

nuclear transcription apparatus present in the dicot

tobacco plant recognizes the relevant regulatory se-

quences of the monocot barley hordein gene, and

that the necessary information for regulated gene ac-

tivation is contained within a barley genomic frag- ment which begins 511 bp 5 ' to the transcription start

site. It is interesting to observe that the B1 hordein

gene regulatory sequences direct the synthesis of

CAT only in the endosperm of transgenic tobacco

plants. This contrasts with endogenous tobacco

storage proteins which are synthesized in embryo

and endosperm. In addition the barley regulatory se-

quences confer temporal specific expression to the

CAT gene. More experiments are required to define

the sequences which confer the tissue and temporal

specificity and also to identify the factors which

regulate the gene.

Although the regulatory sequences of the barley

B1 hordein and the wheat chlorophyll a /b binding

protein [32] genes drive expression of a CAT reporter

gene in transgenic tobacco, similar sequences from

genes for the wheat small subunit of ribulose-l,5-

bisphosphate carboxylase [18] and a maize alcohol

dehydrogenase gene (Adhl) [23] were not expressed.

Subsequent work with the Adhl gene showed high

expression in tobacco when enhancer-like sequences

from the 35S gene of CaMV which function in dicot

plants were placed in front of the maize promoter

[51. The cis-acting sequences that control B hordein

gene expression had not previously been character- ized. We have shown that the regulatory elements of

the B hordein gene function with high fidelity in

tobacco. This renders their identification feasible by

analysis of deletion and substitution mutants in

transgenic plants. Two main conclusions can be

deduced from the experiments reported here: (1) the

nucleotide sequences necessary for tissue and de- velopmental specific expression are present in the 549 bp 5' flanking region of the B1 hordein gene; (2) cellular components are present in tobacco en-

dosperms that recognize the regulating sequences 1o-

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cated in the 5' flanking region of the B1 hordein gene.

Acknowledgement

This research was carried out in the framework of

contract No. BAP-0099-UK and training contract No. BT-015 of the Biotechnology Action Programme

of the European Community. We are grateful to Dr

M. Bevan (PBI) for providing the plasmids pl9S-

CAT and pCATter, and to Drs B. G. Forde, B. J.

Miflin and P. R. Shewry for suggestions and discus-

sions during the course of this work.

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