Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of ��-glucuronidase in transgenic Brassica plants

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Plant Molecular Biology 34: 549–555, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.

Short communication

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of -glucuronidase in transgenic Brassica plants Hai Ping Hong1;6 , Joanne H.E. Ross2;6 , Jean L. Gerster3 , Stamatis Rigas4 , Raju S.S. Datla1 , Polydefkis Hatzopoulos4, Graham Scoles5 , Wilf Keller1 , Denis J. Murphy2 and Laurian S. Robert3; 1

Plant Biotechnology Institute, National Research Council, 110 Gymnasium Rd, Saskatoon, Saskatchewan, S7N 0W9, Canada; 2 Department of Brassica and Oilseeds Research, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK; 3 Eastern Cereal and Oilseed Research Centre, Central Experimental Farm, Ottawa, Ontario, K1A 0C6, Canada ( author for correspondence); 4 Present address: Molecular Biology Department, Agricultural University of Athens, Greece; 5 University of Saskatchewan, Department of Crop Science and Plant Ecology, Saskatoon, Saskatchewan, S7N 5A8, Canada; 6 Hai Ping Hong and Joanne H.E. Ross have contributed equally to this paper Received 22 November 1996; accepted in revised form 4 April 1997

Key words: anther, Brassica, -glucuronidase, oleosin, promoter, tapetum Abstract To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 50 -upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene -glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 50 -upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage. The development of pollen, the male gametophyte of flowering plants, requires the expression of genes in both the gametophytic and sporophytic tissues [for reviews see 14, 16]. During microspore formation and early maturation, the tapetum is probably the most metabolically active tissue of the anther, supplying nutrients for the developing microspores, synthesizing enzymes for the dissolution of the tetrads and delivering compounds that are deposited on the exine of the pollen [for review see 28]. The production of malesterile plants by expressing genes whose products disrupt tapetum development in transgenic plants illustrates the importance of this tissue [12, 13, 31]. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers U74466 (BnOlnB;4) and Y08986 (BnOlnB;13).

*138684*

Tapetal-specific cDNAs have been isolated from several plant species, but the functions of the products encoded by most of these tapetum-specific transcripts are unknown [for review see 27]. In addition, the regulatory sequences responsible for the tapetum-specific expression of only a few of these genes have been studied [12, 19, 20, 29, 30] and the precise cis elements required for tapetal expression are still not known. Recently, we have identified a large family of B. napus genes encoding tapetal oleosin-like (OlnB) transcripts [21, 24]. Tapetal oleosin-like proteins share a 55–70 residue hydrophobic/non-polar domain with the family of seed-specific oleosins, which are proteins located on the surface of oil bodies in most lipidstoring seeds [for review see 8, 18]. During anther development, these proteins are cleaved to release the

GR: 201001947, Pips nr. 138684 BIO2KAP pla402us.tex; 5/06/1997; 8:40; v.7; p.1

550 highly variable C-terminal domain which relocates and becomes the most abundant class of proteins of the extracellular tryphine of mature pollen grains [24]. In this report, the genomic clones corresponding to the tapetal oleosin-like cDNA clones OlnB;4 (Sta 41-9; [21]) and OlnB;13 [23] were isolated. Fragments corresponding to their respective 50 upstream regions were sequenced and fused to the reporter gene -glucuronidase (GUS) to test their ability to activate transcription in transgenic B. napus plants. Sequence comparison of the 50 upstream regions of OlnB;4 and OlnB;13 To isolate the genomic clone Sta 41G(10) which corresponds to OlnB;4, a B. napus cv. Westar genomic library was constructed in  DASH II (Stratagene) using partially Sau3A-digested genomic DNA and screened as previously described [2] with the OlnB;4 (Sta 419) cDNA clone [21]. A SacI/HindIII fragment of ca. 2 kb containing the 50 -upstream region of the OlnB;4 gene was subcloned into pGEM 4Z (Promega) and sequenced using the T7 sequencing kit (Pharmacia) and by the Plant Biotechnology Institute (Saskatoon, Canada). The genomic clone corresponding to OlnB;13 was isolated from a B. napus cv. Jet Neuf genomic library constructed from partially Sau3A-digested genomic DNA cloned in EMBL3A [25]. The library was screened with the I3 oleosin-like cDNA [22]. A 3.5 kb HindIII fragment strongly hybridizing to the cDNA probe was subcloned into Bluescript II (Stratagene) and sequenced using oligonucleotide primers and the Sequenase kit (United States Biochemical Corporation). The sequences of the 1957 bp and 1906 bp fragments containing the 50 -flanking regions of genomic clones OlnB;4 and OlnB;13 respectively, were compared (Figure 1). A putative TATA box whose context compares favourably with the plant consensus sequence [10] is well conserved in both fragments. This putative TATA box occurs within a region of about 250 bp which is highly conserved (86% identity) between OlnB;4 and OlnB;13, and which also includes the untranslated leader sequence and about 150 bp of sequence upstream of the TATA box (Figure 1). This degree of similarity is considerably higher than that observed in the 50 -flanking sequence further upstream of these two genes or even within their coding regions (with the exception of the highly conserved oleosin hydrophobic domain). Such a high level of similarity

suggests an important role for this part of the upstream region of these OlnB genes. The 150 bp conserved sequence immediately upstream of the putative TATA box shows 83% identity and the size and position of this region is somewhat consistent with previous studies suggesting that the cis elements required for correct tapetal expression were located within a region upstream of the TATA box consisting of at most 176 bp in the tobacco gene TA29 [12] and 295 bp in the Arabidopsis thaliana gene A9 [20]. Comparison of the 150 bp region just upstream of the putative TATA box from the two Brassica OlnB genes with the equivalent region from other tapetalspecific promoters [12, 19, 20] showed that the highest level of similarity was to the promoter of the A9 gene from the closely related species A. thaliana [20]. This alignment revealed some conserved motifs between the Arabidopsis and Brassica sequences (Motifs I–IV, Figure 1). Motif II is also similar to motif V found in the Arabidopsis anther-expressed atgrp genes which encode oleosin-like proteins [5] and shows similarity to part of sequence (II) found in the promoter of the Brassica pollen pectin esterase gene Bp19 and other pollen-expressed genes [1]. Motif IV resembles repeats found in the promoter of the Arabidopsis pollen expressed -tubulin gene TUA1 [3]. These motifs (I– IV) are not as well conserved and/or occur in different relative positions in the promoters of the tapetal genes from the more distant species, TA29 from tobacco [12] and tap2 from Antirrhinum majus [19]. Whether these motifs represent functional cis elements important for the determination of tapetal expression will require further analysis. Searches for additional consensus sequences believed to be involved in the regulation of antherexpressed genes in the region further upstream of the conserved sequences of OlnB;4 and OlnB;13 yielded equivocal results. A search for repeated sequences revealed a region of the OlnB;4 upstream fragment from approximately positions ,1125 to ,1957 which is highly repetitive (Figure 1). This region is also associated with long open reading frames (ORFs) in both orientations which show no similarity to known proteins. Interestingly, an ORF was also found within the upstream region of a seed-specific B. napus oleosin promoter [11]. This ORF was recently shown to encode a functional peptide methionine sulphoxide reductase and the intergenic region did possess divergent promoter activity [26].

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Figure 1. Nucleotide sequence of the 50 sequences flanking the translational start site ( 1) of genomic clones OlnB;4 and OlnB;13. The putative TATA box is shown in bold. Motifs I–IV conserved among the Brassica napus OlnB;4, OlnB;13 and Arabidopsis A9 tapetal promoters are double underlined. Motif IV is found within a palindromic sequence outlined in bold. The CAAAAAAAAA repeats conserved between OlnB;4 and OlnB;13 are underlined. The highly repetitive region found between 1125 and 1957 on OlnB;4 is underlined, with some examples of direct repeats (A) 50 -ATAAACCAAAAGATAAA and (B) 50 -GGACCAACA/GGATAAA/GCCA being double-underlined.

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552 Analysis of OlnB;4 and OlnB;13 promoter activity in transgenic plants The 50 -flanking regions of OlnB;4 (1957 bp) and OlnB;13 (1906 bp) were fused to the GUS gene to form binary plant transformation vectors pOB4G (OlnB;4/GUS) and pOB13G (OlnB;13/GUS) respectively. The OlnB;4 promoter fragment was obtained from the subclone described above by PCR using the primer 50 (,) Sta41 III (50 -ATAGGATCCTGATGGTTTGGTGATGGG30 ), which is complementary to bases at positions ,6 to ,24 upstream from the translational start ATG (Figure 1), in combination with the SP6 promoter primer of pGEM 4Z. PCR amplifications were done as previously reported [21]. The OlnB;4 promoter fragment was subcloned as a HindIII/BamHI fragment upstream of the GUS gene of the binary vector pRD 420 [4] resulting in vector pOB4G. The OlnB;13 promoter fragment was obtained from the subclone described above by PCR using the primer JR101 (50 -GCGCGGATCCATGGATTTTCTTATGGTTTGG-30), which is complementary to bases at positions ,1 to ,17 upstream of the translational start ATG in OlnB;13 (Figure 1), in combination with the T3 promoter primer of Bluescript II. Amplifications were performed as previously described [23]. The 1.9 kb OlnB;13 promoter fragment was subcloned upstream of the GUS gene of pGNOS [32] and the promoter-GUS fusion ligated into the binary vector pCGN1559 [15] resulting in vector pOB13G. The B. napus cv. Westar was transformed with the pOBG4 or pOBG13 vectors via Agrobacteriummediated transformation [17] with some modifications for vector pOBG4 [7]. Analyses were carried out on 27 and 15 independent transformants containing pOB4G or pOB13G respectively. To verify the spatial expression of these constructs, GUS assays were performed on different plant parts as described previously [7, 23]. Figure 2 shows typical results from fluorometric assays performed on transgenic B. napus plants containing pOB4G or pOB13G. With both promoter fragments, GUS activity was detected specifically within the anthers and the activity in stem, leaf, sepal, petal or pistil tissue was similar to levels found in untransformed plants (results not shown). GUS activity was localized within the developing anthers of plants transformed with pOB4G by histochemical staining following the procedure of [9] with some modifications [7]. As shown in Figure 3 (A–E), the OlnB;4 promoter fragment directs GUS expres-

Figure 2. Fluorometric analysis of GUS activity in the different organs of transgenic B. napus plants. Transformants 6 and 35 contain the pOB4G construct and transformants G32f and G56 contain the pOB13G construct.

sion specifically to the tapetum beginning in 2–3 mm buds (Figure 3B) at which time the microspore are at the early uninucleate stage. No staining was observed in other transformed plant tissues or in anthers of untransformed plants (results not shown). Histochemical GUS assays were also performed on transgenic B. napus plants containing pOB13G as described previously [23]. The OlnB;13 promoter fragment directed GUS expression to the tapetum in T0 (Figure 3F) and T1 plants (results not shown). Some staining can be observed on microspores in the sections corresponding to the later stages of anther development (Figure 3D, E, F). This is probably due to the GUS enzyme being released into the locule upon tapetal degeneration since isolated microspores show no GUS activity (results not shown). To verify if the OlnB;13 promoter was able to control the correct temporal expression of the GUS gene, fluorometric assays were performed throughout anther development. GUS expression became detectable in 3 mm buds and peaked in 4 mm buds (Figure 4). Therefore the patterns of expression observed for pOB4G and pOB13G are in close agreement with previous northern blot and in situ hybridization data [21, 24] and indicate that both promoters possess the necessary cis regulatory elements for correct tapetal expression.

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Figure 4. Fluorometric analysis of GUS activity during anther development in B. napus transformants G32f and G56 containing pOB13G.

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GUS activity in root, leaf, petiole, anther, petal, sepal and carpel tissue. This indicates firstly, that important differences occur in these two promoter fragments possibly outside the conserved upstream region and secondly, that promoter analyses in heterologous host plants must be interpreted with care. Other examples of unfaithful promoter activity of anther genes in heterologous hosts have been reported [6, 33]. Hence, tobacco will not be appropriate for the further characterization of the promoters regulating the expression of these B. napus tapetal oleosin-like genes and may not always be a useful model system for other heterologous anther-specific gene promoters. It will be useful to compare the promoter sequences of other oleosin-like genes and to investigate whether the motifs and homologous regions identified in this study are involved in temporal and spatial gene regulation during male gametophyte development.

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We wish to thank Joe Hammerlindl (PBI, Saskatoon) for advice with Brassica transformation and Dr A. Ryan, University of Cambridge, U.K., for the generous gift of the B. napus cv. Jet Neuf genomic library. H.P.H. was supported by a graduate studies stipend from Agrevo Canada. J.R. and D.J.M. were funded by a BBSRC competitive strategic grant to the John Innes Centre. S.R. was funded by a British Council/Greek Ministry of Science and Technology grant and P.H. by a NATO Science Council Grant.

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