Plant Cell Reports

Plant Cell Reports (1993) 12:506-509

9 Springer-Verlag1993

The effect of different promoter-sequences on transient expression of gas reporter gene in cultured barley (Hordeum vulgate L.) cells * Ravindra N. Chibbar, Kutty K. Kartha, Raju S.S. Datla, Nick Leung, Karen Caswell, Clifford S. Mallard, and Lee Steinhauer Plant Biotechnology Institute, National Research Council Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada Received January 26, 1993/Revised version received May 4, 1993 - Communicated by F. Constabel

Abstract

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcoholdehydrogenase (Adh 1) intronl or maize shrunken locus (shl) intronl along with maize Adhl and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli I~glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoter- intron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cu Itured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells. Key words: particle bombardment; Hordeum vulgare L.; promoter screening; transient expression; gus; cereal transformation. INTRODUCTION

The ability of the 'biolistics' technique to deliver DNA into intact plant cells (Klein et al., 1987) has provided an opportunity to genetically engineer cereals. Transformed cereals such as corn, rice and wheat have been produced using this technology (Fromm et al., 1990; Gordon-Kamm et al., 1990; Christou et al., 1991; Vasil et al., 1992). However, transgenic barley plants have not been reported in the literature. Stably transformed barleycallus via PEG mediated DNA uptake by barley protoplasts has recently been reported (Lazzeri et al., 1991). Mendel et al., (1990) compared four different promoters, namely those of the cauliflower mosaic virus 35S (35S), manopine synthase (TRI' and TR2'), pea rubisco small subunit (SSU) and Vicia faba * NRCC No. 36482 Correspondence to." R. N. Chibbar

legumin (leg) for transient expression of neomycin phosphotransferase (nptll) and l~-glucuronidase (gus) marker genes in barley cells. The expression of the nptll gene was weak in barley cells as compared to the two other plant systems studied. Moreover, the activity of the nptllgene was reduced over a period of time and led to the subsequent loss of antibiotic resistance. Therefore, the identification of promoters that direct a high level of expression of introduced genes used for selection is desirable for the development of a pragmatic transformation protocol (Vasil et al., 1989). Heterologous gene expression in monocots is at variance with that in dicotyledonous plant cells. An important characteristic of heterologous gene expression in monocot cells is the enhancing effect of the presence of an intron between the promoter and the 5'end of the coding region of the gene (Callis et al., 1987). Vasil et al. (1989) reported that the maize shrunken locus (shl) intronl enhancedthe reporter gene expression severalfold as compared to maize alcohol dehydrogenase (Adhl) intronl with the same promoter in graminaceous species. The 35S (Guilley et al., 1982) promoter has been extensively used to direct high levels of gene expression in a wide variety of plants. However, the 35S promoter is 10- to 100- fold less effective in monocot cells as compared to dicot cells such as carrot and petunia (Hauptmann et al., 1988). The commonly used 35S promoter fragment is 343bp long and contains a strong transcriptional enhancer (Fang et al., 1989; Odell et al., 1985). Duplication of this transcriptional enhancer gives 10- fold higher gene expression in dicot cells such as tobacco (Kay et al., 1987) and has also been used in gene constructs to obtain stably transformed wheat callus (Vasil et al., 1991). The Adh I promoter isolated from corn was 10 -20 times more active than the 35S promoter in rice (Zhang and Wu, 1988). McEIroy et al. (1990) reported the isolation of a rice actin promoter which in combination with its first intron gave several-fold higher expression of gus gene intransgenic rice. By incorporating optimized translation context for the gus reporter gene, in combination with the 35S promoter fused to Act1 intron, a 40- fold stimulation in

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the gus expression was observed in transformed rice cells (McEIroy et al., 1991). In the above reports these four promoters were not compared under identical conditions. In this communication we compare the relative efficiency of these promoters in combination with introns in the transient expression of gus (Jefferson, 1987) in cultured barley (Hordeum vulgare L) cells. These cultured cells provide an efficient recipient system for biolistically delivered DNA to assess the transient expression of marker genes (Kartha et al., 1989). MATERIAL AND METHODS Cell culture Cell suspension cultures of barley (Hordeum vulgare L. cv. Heartland) used in this study were maintained by weekly sub-cultures and prepared for particle bombardment as described earlier (Kartha et al., 1989). Plasmid construction The gene constructs used and their sources are listed in Table 1. The steps involved in the generation of gene constructs specifically assembled for this work are briefly described~ All the promoter-intron combinations constructed in this study are transcriptional fusions to the gusgene. The maize shl intronl, as a 1 kb EcoRV - Smal fragment, was cloned into the Smal site of pBI221 (Clontech) or pBI-364 (Bekkaoui et al., 1990) to give 35S:shl intronl :gus:nos and E35S:shl intrqnl :gus:nos, respectively. The maize Adh I intron 1, as a 450bp Bcll - BamH 1 fragment, was cloned into the BamH1 site of pBI-364 (Bekkaoui et al., 1990) to give the E35S: Adhl intronl : gus:nos construct. Particle bombardment The cells were bombarded with the gene constructs described in Table 1, using a helium-driven particle bombardment system (DuPont PDS 1000). Ten pg of the respective plasmid DNA was coated on 2 pg of tungsten M10 particles (mean diameter 0.71Jm) with calcium chloride and spermidine as described in the owners manual, and resuspended in 65 I.JLof ethanol. Table 1.

For each bombardment a 10 IJL aliquot was loaded on a macro-carrier and allowed to air dry. There was a gap of 6 mm between the macro-carrier and the 1100 psi rupturedisc. The macro-carrier and micro-carrier travel distances were 6 and 60 mm, respectively. The sample chamber was evacuated to 75 cm of mercury during the bombardment. GUS assays Forty-eight hours after bombardment the GUS expressing foci were histochemically localized by incubating the cells with 500 mL X-gluc (0.2%) stain for 24h (Chibbar et al., 1991). The number of blue GUS expressing foci were counted under a dissecting microscope. A fluorogenic assay was done 48h after bombardment. At least three independent bombardments were separately assayed in each case. The preparation and purification of the cell extracts for fluorogenic assay, as well as the procedure used, was essentially as described earlier (Chibbar et al., 1991). Protein determination for normalization of G US specific activity (pmole 4-methyl umbelliferone (MU) produced / min / mg protein) was done by the dye-binding assay (Bradford 1976). Results and Discussion The GUS expressing foci represented by the cells showing blue color (Fig 1) were present only in the cells bombarded with gus containing gene constructs (Table 1). In the 30 independent control bombardments using either no plasmid DNA or plasmid DNA lacking the gus, no blue-colored GUS expressing foci were detected. This was confirmed by the fluorogenic assay in which the level of fluorescence was the same as that with boiled extracts (Fig 2). This lack of intrinsic GUS-like activity (Hu et al., 1990), and a previously determined direct correlation between the amount of DNA introduced and transient expression (Kartha et al., 1989), make this barley cultured-cell line an ideal system for quantitative assessment of various promoter-intron combinations to direct foreign gene expression using gus as a reporter gene in monocot cells.

The description and the source of constructs with gusused in this study and the histochemical assay of GUS expression effected by them. 1Gone construction Control (puc9 or pKSBlu) 35S:gus:nos (pBI 221) 35S :Adhl intron 1:gus-nos 35S:Shl intronl :gus:nos E35S:gus:nos E35S:Adh1intronl :gus:nos E35S:sh1 intron 1:gus:nos Adhl :Adhl intronl :gus:nos Act1 :Act1 intronl : gus:nos

Source Stratagene Clontech Chibbar et al. 1991 This work Bekkaoui et al. 1990 This work This work Klein et al. 1988 McEIroy et a1.1990

2GUS exoressine foci 0 (3r/= 30) 113 + 27 ( n = 6) 460 + 61 ( n = 9) 335 + 21 ( n = 3) 340 + 20 (n = 9) 189 + 125 ( n = 9) 505 + 381 ( n = 3) 247 + 60 (n = 18) 656 + 105 (n = 18)

1 The order of sequences assembled is 9promoter: intron (+ or - ): gus reporter gene:nos terminator. 2 The number of GUS- expressing foci ( + S.E. ) from the blue spots shown in Fig1. 3 n -- number of independent bombardments analyzed.

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Fig 1. GUS expressing foci in cultured barley cells. Four day- old suspension cultures of barley cells were bombarded with (a) control, lacking DNA; (b) 35S:Adh1 :gus:nos (c) E35S: Adhl :gus:nos and (d) Act1 :Act1 :gus:nos and after 48 h were stained with X-Gluc. Observations were made 24 h after staining (magnification 180X)

The blue GUS expressing foci were observed in all the constructions with gusgene (Table 1). The number of GUS expressing foci was greater than 600 with rice actin promoter-intron construction and as low as 100 with 35S promoter alone. Another interesting observation was that the apparent size of the foci was much larger with rice actin promoter intron fusions as compared to other constructions (Fig 1). Therefore, the counting of foci may not be a precise representation of G US activity. As a result fluorogenic assays were employed in addition to the counting of foci to determine the relative strengths of the different promoter intron combinations. The number of GUS expressing foci, as well as the amount of MU produced, was lowest in constructs with 35S promoter alone. Cells bombarded with the E35S constructs showed higher GUS activity than 35S promoter constructs (Table 1; Figures 2 and 3). The fusion of introns with both these promoters enhanced the levels of gus expression. At least 2.5- and 7- fold higher GUS activity was observed when Adh I intron and sh I intronl, respectively, were fused to:the 35S promoter. However, E35S promoter fusions with these two introns showed 3.5- fold higher GUS activity in both the cases (Fig 3). There is a difference in the absolute values of pmoles MU produced in the two independent experiments because of the nature of the gene delivery method used. However, the comparison of the two common treatments 35S and 35S fused to Adhl intron reveal similar trends in both experiments (Fig 2 and 3). Cells bombarded with the Adhl promoter with its first intron showed the same level of GUS activity as with 35S promoter fused to Adhl intronl (Fig 2). The highest number of GUS expressing foci was observed in cells bombarded with a construct carrying the rice actin promoter and its first intron (Table 1 ; Fig 1). These cells also exhibited the highest G US activity in the fluorogenic assay (Fig 2). The results obtained with the 35S promoter and the enhancement of transient foreign gene expression observed by fusion of introns with promoters is in agreement with our earlier results using chloramphenicol

acetyl transferase as a marker gene in cultured barley cells or immature zygotic embryos of barley and wheat (Kartha et al., 1989; Chibbar et al., 1991). That the highest level of G US activity was observed with the rice actin promoter and intron combination isvery interesting. This is in agreement with the results obtained in rice (McEIroy et al., 1990; McEIroy et al., 1991). However, in a heterologous system Picea mariana and Larix x eurolepis the rice actin promoter has been shown to give the same level of expression as the 35S promoter (Duchesne and Charest, 1992). The shl intronl isolated from corn had the most stimulatory effect on foreign gene expression in combination with various promoters studied in corn as compared to two other grass species studied (Vasil et al., 1990). Similarly, the enhancing effect of Adh I intronl was most pronounced in combination with Adhl promoter as compared to other promoters in corn (Callis et al., 1987). These results indicate that maximal expression of a foreign gene in a particular species occurs when the gene is driven by a promoter-intron combination isolated from the same species i.e. in a homologous situation.

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Fromm M E, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Bio/Technology 8:833 - 839

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Fang R-X, Nagy F, Sivasubramaniam S, Chua N-H (1989) Plant Cell 1:141 - 150

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Fig3. Effect ofthe maize alcoholdehydrogenasel (Adhl) intronl and maize shrunken locus1 (sh 1) intronl in combination with 35S (A) or E35S (B) promoter on the GUS activity in cultured barley cells.

Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start W J, O'Brien JV, Chambers SA, Adams WR Jr, Wilets NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Plant Cell 2:603 - 618. Guilley H, Dudley RK, Jonard G, Balazas E, Richards KE (1982) Cell 30:763 - 773 Hauptmann RM, Ashraf M, Vasil V, Hannah LC, Vasil IK, Ferl R (1988) Plant Physiol. 88:1063 - 1066 Hu C-y, Chee PP, Chesney RH, Zhou JH, Miller PD, O'Brien WT (1990) Plant Cell Reports 9:1 - 5 Jefferson RA (1987) Plant Mol. Biol. Rep. 5:387 - 405

In conclusion, the rice actin promoter with its first intron wasthe most effective promoter in cultured barley cells. Similar results were obtained with cultured cells and immature zygotic embryos of wheat (unpublished results). The rice promoter-intron combination has been used to successfully express a gus:npt fusion gene (Datla et al., 1991) in transgenic wheat cell lines (Chibbar et al. ms under preparation). Acknowledge me nts: Drs. M.Fromm (Monsanto St Louis, USA), D.McEIroy and Ray Wu (Cornell University, Ithaca, NY, USA) are kindly acknowledged for the constructs Adhl :Adhl intron:gus: nos and ActlD:gus:nos used in this work. The shl locus1 intron was kindly provided by Dr L.C.Hannah, University of Florida, Gainesville, USA. Dr Monica B~ga is acknowledged for readingthe manuscript. References

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