Plant Molecular Biology 19:903-911, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

903

Localisation of cis elements in the promoter of a wheat -Amy2 gene A.K. Huttly, A.L. Phillips and J.W. Tregear

Dept of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, UK Received 27 November 1991; accepted in revised form 9 March 1992

Key words: a-amylase gene expression, aleurone protoplasts, functional analysis, cis elements, hormone regulation

Abstract

A functional analysis of the promoter from the wheat a-amylase gene e-Amy2~54 is described. Mutant e-Amy2/54 promoters containing replacements or deletions were constructed and their ability to direct expression of the reporter gene fl-glucuronidase (GUS) in gibberellin-responsive oat aleurone protoplasts analysed. Chimaeric promoters using regions of the cauliflower mosaic virus (CaMV) 35S and e-Amy2/54 promoters were also analysed. The results suggest that at least three regions within the e-Amy2~54 promoter contain cis elements that are necessary for high-level gibberellin-regulated transcription. Fusion of 1.8 kb of promoter sequence upstream from -117 bp to a minimal ( -55 CaMV 35S) promoter gave rise to hormone-independent expression implying that the region 3' to -117 bp contains an element which represses transcription in the absence of gibberellin or presence of abscisic acid.

Introduction

During germination of cereal seeds the cells of the aleurone layer are induced by gibberellins (GA) to synthesise and secrete various hydrolytic enzymes which break down the stored reserves of the endosperm [3]. The induction by GA involves an increase in transcription of the genes for at least one of these enzymes, the starch endohydrolase a-amylase, and can be inhibited by abscisic acid (ABA) [19, 8]. Our assumption is that GA and ABA directly, or indirectly, affect the proteins (trans-acting factors) involved in the initiation of transcription. Such proteins bind to short D N A sequences (cis elements) in the promoter region of a gene [ 13]. The location of such cis elements can yield not only information on the number of transacting factors involved in the transcription of a

particular gene but also on the type or class of factor. They can in addition be used as a means of identifying c D N A clones in an expression library containing trans-acting factor sequences [111. Cis elements may be identified by a number of approaches including DNase I footprinting [ 15], in vivo footprinting [ 14] and by functional analysis of the promoter. Since stable transformation of cereals is not yet a routine procedure, transient expression in transformed oat aleurone protoplasts has provided a useful method of analysing e-amylase promoter/reporter gene constructs. Huttly and Baulcombe [6] were able to show that a 289 bp sequence of the promoter from the wheat group 2 a-amylase gene e-Amy2~54 was sufficient to direct the synthesis of the reporter gene flglucuronidase (GUS) in a GA-dependent man-

904 ner, while other authors have since begun the dissection of a barley ~-amylase group 1 isozyme gene using a similar approach [9, 16]. In this paper we describe a further analysis of the wheat ~-Amy2/54 promoter and conclude that there are at least three regions that potentially contain cis elements, and which together are necessary for the high-level hormonally regulated expression observed in oat aleurone protoplasts. Furthermore, we provide evidence that one of these elements may operate through repression of transcription in the absence of GA or presence of ABA.

Materials and methods

Oat aleurone protoplast transformation

Cultivated oat, Avena sativa cv. Rhiannon, aleurone protoplasts were isolated, transformed with promoter/GUS constructs and the /~-glucuronidase produced assayed as described previously [6] with the exception that protoplasts were incubated in the presence or absence of 1 0 - T M GAl. Constructs were transformed on at least four independent occasions and the average expression level calculated. In order to compare the ability of mutant promoters to direct expression, G U S activity is expressed relative to that produced by p~2GT in the presence of GA; p0c2GT, a reference construct, contains 1.9 kb of ~-Amy2/ 54 promoter sequence fused to the G U S gene as a translational fusion [6]. An aliquot of protoplasts was always transformed with p~2GT at the same time as other constructs under investigation.

DNA constructs The 3' deletion series The plasmid p~2GT containing a 1.9 kb promoter fragment from the wheat 0c-amylase gene ~-Amy2/ 54 fused to the reporter gene G U S was linearised with Acc I and subjected to Exonuclease III digestion. Aliquots were removed from the incuba-

tion at regular intervals and single-stranded D N A removed by treatment with mung bean nuclease. Promoter fragments containing deletions from 50 to 300 bp were liberated by digestion with Hind III and ligated into plasmid pUBS3 cut with Hind III and Sma I. The exact deletion endpoints were determined by D N A sequence analysis. Suitable 3' deletions were transferred to the plasmid -55CaMV::GUS. This plasmid contains the proximal 55 bp of the cauliflower mosaic virus (CaMV) 35S promoter fused to the reporter gene G U S and was generated using the Fok I site at position -60 bp in the CaMV 35S promoter followed by removal of the overhang with mung bean nuclease. The resulting series of 3' deletion constructs contained the 8 bp sequence G G G G GATC in the junction between the a-amylase promoter and CaMV 35S sequences. The plasmid p~2.58 was generated by digestion of p~2GT with Acc I and Hind III and the resultant promoter fragment was inserted into pBI201.4, a G U S expression cassette designed for transcriptional fusions [ 10]. Plasmid are named 'p~2.xxx' where xxx denotes the last base present in the 3' deletion. Internal deletions To create internal deletions of the ~-Amy2/54 promoter suitable pairs of 3' and 5' deletions, described elsewhere [6], were joined by a common Bam HI site in the polylinker sequences at the ends of the deletions. The junctions formed contain the 11 bp linker sequence G G G G G A T CCCC. Plasmids carrying these deletions are named 'p~2.Ax' where x denotes the position of the internal deletion, 1 being most distal and 4 being most proximal to the start of transcription. C a M V 35S enhancer The CaMV 35S enhancer was isolated as a 357 bp promoter fragment from the Fok I site at -60 bp to the Hinc II site at -417 bp. Constructs containing this enhancer have the additional sequence CCCC at their junction. Plasmid are named 'p~2.xxE' where xx designates the original 5' deletion (described previously [6]) to which the enhancer (E) has been fused.

905 Results

3' deletion series In a previous paper [6] it was shown, by the analysis of a series of 5' deletions, that 289 bp of the ~-Amy2/54 promoter could direct high level GA- and ABA-dependent expression of the G U S reporter gene in oat aleurone protoplasts. In order to delimit the 3' border to the critical elements of the promoter five constructs were made that contained progressively larger deletions at the 3' end. As this would in most of the constructs remove the presumed TATA box of the ~-Amy2/54 gene at position -24 to -31 bp, this was replaced by a minimal promoter from the CaMV 35S gene. A -55 bp deletion of the CaMV promoter contains the TATA box, but none of the other upstream sequences determined to be necessary for its function [2]. The -55CaMV::GUS construct gave rise to very little, or no expression and was unaffected by the presence of G A in the protoplast incubation medium (Fig. 1). Removal of part of the ~-Amy2/54 transcribed untranslated leader sequence, and its replacement by CaMV 35S sequences in plasmid p~2.58, appeared to have little effect on the level of expression of G U S relative to the full-length construct p~2GT (Fig. 1). However, when an exchange of the ~-Amy2/54 start of transcription site and TATA box with that of the CaMV 35S promoter was made in p~2.38 expression was depressed in the presence of G A to 30~o of that of p~2GT. This reduction is possibly due to the fact that in p~2.38 the CaMV 35S TATA box is positioned 26 bp further away from the end point of the ~-Amy2/54 sequence than is the native ~-Amy2/54 TATA sequence in p~2GT. However, the removal of a further 30 bp (construct p~2.68) which places the CaMV 35S TATA box to within 4 bp of the correct position relative to the remaining ~-Amy2/54 sequence, further reduced expression to 19~o of that of p~2GT. For both of these constructs, a low basal level of expression was seen in the absence of GA, similar to that found with p~2GT. In contrast, the next deletion in the series p~2.117, gave rise to a similar level of expression in the presence (12~o) or

absence (15~o) of GA (Fig. 1). Expression from this construct was also unaffected by the addition of ABA together with G A in the incubation medium (data not shown) which, in the case of the full-length ~-Amy2/54::GUS construct p~2GT, reduces expression to that seen in the absence of GA [6]. This unregulated mode of expression from p~2.117 has several implications for the organization of the ~-Amy2/54 promoter, assuming that an artificial cis element has not been accidentally created in the construction of the chimaeric promoter. Firstly, the promoter can apparently be divided into parts, consisting of a region involved in GA/ABA regulation (a hormone-regulatory element or HRE) lying in the region of -117 to -68, while upstream of -117 the promoter contains one or more enhancers which are either unaffected by GA and ABA or no longer able to respond to them, due to the loss of elements 3' to -117. Since a further 3' deletion to -175 (construct p~2.175) gave little or no expression, it seems likely that at least one such positive element is present between -175 and -117. Secondly, the unregulated expression from p~2.117 strongly suggests that the putative H R E between -117 and -68 may operate by repressing transcription in the absence of GA or the presence of ABA.

CaMV enhancer/~-Amy2/ 54 chimaeric constructs The above hypothesis cannot be tested simply by successive 5' deletions, since these would first remove the upstream enhancer(s), and the subsequent loss of hormone regulation on deletion of the H R E would be unmeasurable. Therefore, in order to attempt to identify a down-regulating HRE, a fragment of the CaMV 35S promoter from -417 to -60, containing the CaMV 35S enhancer, was positioned upstream of the previously described series of 5' deletions between -289 and -39 ([6] and Fig. 1), and expression directed by these constructs monitored in aleurone protoplasts (Fig. 1). The 35S enhancer placed upstream of the -289 deletion of ~-Amy2/54 (construct p~2.14E) gave

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