Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters

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Identi¢cation of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters John R. Gittins a

a;1

î ke Strid , Mary A. Schuler b , A

a;c;

*

Biokemi och Biofysik, Institutionen fo«r Kemi, Go«teborgs Universitet, P.O. Box 462, S-405 30 Go«teborg, Sweden b Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA c º rebro Universitet, S-70182 O º rebro, Sweden Institutionen fo«r Naturwetenskap, O Received 21 August 2001 ; received in revised form 12 November 2001; accepted 20 November 2001

Abstract DNA fragments containing the 5P promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription. ß 2002 Elsevier Science B.V. All rights reserved. Keywords : Cis-elements; Electrophoretic mobility shift assay; Nuclear binding factor; Pisum sativum sad gene; Proximal promoter ; Stress-induced

1. Introduction At a relatively low £uence rate ultraviolet B radiation (UV-B, 280^320 nm) causes deleterious changes to photomorphogenesis, loss of photosynthetic activity, DNA damage, reduced protein synthesis and destruction of ATPase and auxin, which can lead to a general decline in a plant’s growth and fertility [1,2]. As global depletion of stratospheric ozone has led to a rise in the level of harmful UV-B radiation impacting the Earth [3], plant defence responses to UV-B are of growing interest. As well as genes encoding enzymes involved in the biosynthesis of protective £avonoid pigments (e.g. PAL and Abbreviations : CHS, chalcone synthase; C4H, cinnamate 4-hydroxylase ; PAL, phenylalanine ammonia lyase; PCR, polymerase chain reaction; sad, stress-induced alcohol dehydrogenase-like protein; UTR, untranslated region * Corresponding author. Present address: Institutionen fo«r Naturvetenº rebro Universitet, S-70182 O º rebro, Sweden. Fax: +46-19-303566. skap, O î . Strid). E-mail address : [email protected] (A 1 Present address: Laboratory of Plant Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin¤skiego 5A, 02-106 Warsaw, Poland.

CHS) [4^7], other defence-related genes induced by UV-B encode antioxidant enzymes [8,9], pathogenesis-related proteins [10,11] and DNA repair enzymes [12]. Conversely, genes encoding plastid-located proteins show a marked decrease in expression after UV-B exposure [1,13]. Additionally, transient decreases in transcript levels for cell cycle-related genes have been observed indicating that plant cells can delay the cell cycle to permit the repair of DNA damage caused by UV-B radiation [14,15]. The mechanism of UV-B perception by plants is at present unknown. A recent study using Arabidopsis mutants demonstrated that neither the cry1 nor the cry2 photoreceptor is involved in UV-B-induced CHS expression although phytochrome B appears to act as a negative regulator [16]. Pharmacological studies of the UV-B signal transduction pathway have shown that calcium/calmodulin, de novo protein synthesis and protein kinases all play a role in the expression of CHS in response to UV-B [17,18]. Experiments examining transcript levels of a panel of Arabidopsis genes using di¡erent chemical treatments prior to UV-B exposure and mutant lines have demonstrated that generation of reactive oxygen species (ROS) causes

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levels of ethylene, jasmonic acid and salicylic acid to increase. This, in turn, induces UV-B-responsive expression of some genes [10,11]. These ROS-responsive genes do not include CHS [19,20]. UV-B-induced expression of CHS has also been found to be blocked by inhibitors of nitric oxide synthase and NO scavengers [20]. The role of NO in the signalling pathway leading to CHS expression has yet to be determined. Despite the lack of knowledge concerning the putative UV-B receptor(s) and the complexity of the signal pathway network emanating from the site of perception, the ultimate impact of the UV-B stimulus is to alter gene expression. Light-regulated expression of many plant genes is controlled at the level of transcription [21]. UV-B-responsive promoters are likely to be extremely complex because many genes induced by UV-B are also stimulated by di¡erent wavelengths of light and other stresses including fungal infection and wounding [2,22^24]. Few UV-Bresponsive promoters have been studied in detail, and investigations have been con¢ned to genes induced by UV-B. The parsley CHS promoter contains two separate regulatory units which bind speci¢c transcription factors during exposure to UV-B-rich light [25]. Light regulatory unit 1 (LRU1), which is su⁄cient to mediate light-induced expression of CHS, contains at least two cis-acting elements named ACECHS (ACGT containing element) and MRECHS (Myb recognition element) that were originally called box II and box I, respectively [25]. ACECHS binds common plant regulatory factors of the bZIP type (basic region leucine zipper) [26], while MRECHS binds factors of the MYB type (cMyb homologues) [27]. These bound factors may act synergistically to cause UV-B-induced expression of this gene [28]. The promoters from UV-B-responsive CHS genes of Antirrhinum, mustard, pea and Arabidopsis are all structurally similar to the parsley promoter [29^32]. One notable exception is that the promoter for PsCHS2, the gene showing the greatest UV-B induction in pea, lacks an ACECHS element [31]. In the partially characterised UVB-responsive promoter driving the parsley gene for 4-coumarate:CoA ligase (4CL), a cis-element required for UV-B inducibility is located within the coding region [33]. Two other transcription factors have recently been implicated in UV-B-responsive gene expression. In petunia, the ZPT2-2 gene encoding a zinc ¢nger transcription factor is up-regulated by UV-B. This factor binds to the promoter of the gene encoding 5-enolpyruvyl-shikimate3-phosphate synthase, an enzyme involved in the synthesis of chorismate, a precursor of £avonoids [34]. The Arabidopsis AtMYB4 factor is a repressor for the C4H gene and to a lesser degree the genes CHS, 4CL1 and 4CL3, which encode enzymes involved in the synthesis of sinapate esters and £avonoids. This repression is lifted following UV-B irradiation or wounding which causes down-regulation of AtMYB4 transcription [35]. In a previous study using di¡erential display to identify

molecular markers for UV-B radiation in Pisum sativum we identi¢ed a novel family of genes encoding short-chain alcohol dehydrogenase homologues [36]. Compared with PAL and CHS, the sad genes are activated more rapidly and at signi¢cantly lower UV-B doses. This rapid induction indicates that the sad genes form part of an early response to this stress. As well as UV-B, ozone, wounding, salt and aluminium all induce sad transcription which indicates that the SAD proteins are involved in protection against multiple stresses [36]. This also means that the sad promoter is the location where multiple signalling pathways are integrated to produce a response to stress stimuli. Analysis of the sad promoter may permit identi¢cation of speci¢c transcription factors which will be a useful point of entry into the pathway(s) between stress perception and the molecular response. Here, in an e¡ort to expand knowledge on transcriptional regulation by UV-B and other stresses, we describe the in silico identi¢cation of putative cis-elements within promoter fragments from two members of the P. sativum sad gene family and compare our ¢ndings with the results of in vitro electrophoretic mobility shift assay (EMSA) studies.

2. Materials and methods 2.1. Plant material and stress treatments Pea seed (Pisum sativum L. cv. Greenfeast) was sown in vermiculite and grown with a 12-h photoperiod under 100 WE m32 s31 at 22‡C (day)/18‡C (night) and 70% relative humidity for 21 days. The stresses applied to separate populations of plants were low-dose UV-B and wounding. Both stresses were applied for 6 h, which has been shown to induce substantial sad expression [36], and this period was started at the same position in the daily light cycle. In each treatment, a similar number of plants were used as non-stressed controls and leaf samples were collected in an identical manner. Low-dose UV-B stress was applied by supplementing the normal white light with UV-B using a £uorescent tube (TL40W/12UV, Philips, Eindhoven, The Netherlands). To remove any UV-C radiation, a cellulose acetate screen was used as a ¢lter excluding wavelengths of 6 292 nm. The biologically e¡ective radiation (UV-BBE;300 ), normalised to 300 nm according to Caldwell [37] and Green et al. [38], was 0.093 W m32 . After a 6-h exposure, this would give a total dose of approximately 2 kJ m32 . Following irradiation, all leaves which had received the UV-B stress (i.e. non-shaded) were immediately collected, frozen in liquid N2 and stored at 380‡C. Wounding was performed by crushing one leaf in each pair with a bone clamp. Single-clamp crushing wounds were made diagonally across each leaf. Previously it has been shown that the wound stress signal induces sad ex-

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pression in both the wounded and non-wounded leaves in a pair [36], so after 6 h, both wounded and non-wounded leaves were collected from each plant, immediately frozen in liquid N2 and then stored at 380‡C. 2.2. Isolation and sequencing of sad gene promoter fragments Fragments of the promoter region from P. sativum sad genes were ampli¢ed following the GenomeWalker protocol (Clontech) using adapter-ligated pea DNA as template. High-quality genomic DNA was isolated from expanding pea leaves from 21-day-old plants following the procedure of Jofuku and Goldberg [39]. This was digested in separate reactions with a panel of ¢ve restriction endonucleases cleaving 6-bp recognition sequences to leave a blunt end (DraI, EcoRV, ScaI, SspI, StuI). A GenomeWalker adapter DNA was ligated to the ends of fragments in each digest mixture to produce ¢ve adapter-ligated libraries. Using the separate libraries as templates with adapter and gene-speci¢c primers (Table 1A), suppression/long PCR reactions were performed using Advantage polymer-

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ase mixture (Clontech) to generate large fragments of the sad promoters. Nested PCR was performed using the products of the primary reactions as template and the fragments ampli¢ed in this secondary PCR reaction were isolated by puri¢cation from an agarose gel using a Qiagen gel extraction kit. These fragments were treated with Klenow polymerase to produce blunt ends and then cloned into vector pZErO-2 (Invitrogen) cleaved with EcoRV. DNA sequencing of two clones of each of the fragments was performed using Cy5 dye-labelled primers and a Thermosequenase £uorescent sequencing kit (Amersham-Pharmacia). Reactions were analysed with a Pharmacia ALFred sequencer (Amersham-Pharmacia). Deletions were produced using ExoIII/nuclease S1 [40] to facilitate sequencing of the complete promoter fragments. Contig assembly was performed with the Seqman program in the Lasergene software package (DNASTAR). 2.3. Promoter sequence analysis The sadA and sadC promoter sequences were analysed with text searches using consensus elements having the

Table 1 Oligonucleotides used in this study Name

Positiona

Sequence

(A) GenomeWalker gene-speci¢c primers SADA-1 5P-GGTGTTGCTAATCCATTTGGCGACACAGAG-3P SADA-2 5P-CCATACGTGCAGCTTGATTGGCAAAGAGA-3P (B) sadCP 60-bp overlapping fragment oligonucleotides 1a 5P-CTACATTTTGGAAGGTTCTGAACCCGCAATGACAACA-3P 1b 5P-ATCTATTTTTAACGACAGTTTGATCTGATGTTGTCATT-3P 2a 5P-CTCAACATCAGATCAAACTGTCGTTAAAAATATTCAA-3P 2b 5P-ATCAGTTGTTTTTAGTTGAATGTTTTCGTTGAATATTT-3P 3a 5P-CTTCAACGAAAACATTCAACTAAAAACAACTTTTCA-3P 3b 5P-ATCCATATCCATGATCATGAGTTATTGAAAAGTTG-3P 4a 5P-CTTTCAATAACTCATGATCATGGATATGGATGTATT-3P 4b 5P-ATCAACAAAAGTGGGCGCCACAAAATAAATACATCCA-3P 5a 5P-CTGTATTTATTTTGTGGCGCCCACTTTTGTTGACAAT-3P 5b 5P-ATCATAGTAGCACGCCAAATTGATTTAAATTGTCAACA-3P 6a 5P-CTACAATTTAAATCAATTTGGCGTGCTACTATTTTTG-3P 6b 5P-ATCTTATTTAATTTATAAAATATGTCACCAAAAATAGT-3P 7a 5P-CTTTTGGTGACATATTTTATAAATTAAATAACAAGC-3P 7b 5P-ATCTAGTGTAATCAATGTTTCATATTTGGCTTGTTATT-3P 8a 5P-CTCAAGCCAAATATGAAACATTGATTACACTATGCTA-3P 8b 5P-ATCCTTTGAACACGGAAAGTGTATTTTAGCATAGTG-3P 9a 5P-CTGCTAAAATACACTTTCCGTGTTCAAAGGAATGTT-3P 9b 5P-ATCGTCAACCGCGGCTCTCATGAATCAAACATTCCTT-3P 10a 5P-CTATGTTTGATTCATGAGAGCCGCGGTTGACGGTCCA-3P 10b 5P-ATCGTCAGGTTGATCTTTTCAGATTGGGTGGACCGTCA-3P 11a 5P-CTGTCCACCCAATCTGAAAAGATCAACCTGACTTAGT-3P 11b 5P-ATCTACGAGACGTGATGGAATATGATACTAAGTCAG-3P (C) 4/5 overlap sub-fragment oligonucleotides sf1a 5P-AATTGTATTTATTTTGTGGCGC-3P sf1b 5P-AATTGCGCCACAAAATAAATAC-3P sf2a 5P-AATTATTTTGTGGCGCCCACTT-3P sf2b 5P-AATTAAGTGGGCGCCACAAAAT-3P sf3a 5P-AATTTGGCGCCCACTTTTGTTGA-3P sf3b 5P-AATTTCAACAAAAGTGGGCGCCA-3’ Nucleotides added to create a restriction endonuclease cleavage site or cohesive termini are in boldface letters. Position is given relative to the putative sadC transcription start site.

a

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+644 to +615 +166 to +138 3400 to 3366 3341 to 3375 3370 to 3336 3311 to 3345 3340 to 3306 3281 to 3315 3310 to 3276 3251 to 3285 3280 to 3246 3221 to 3255 3250 to 3216 3191 to 3225 3220 to 3186 3161 to 3195 3190 to 3156 3131 to 3165 3160 to 3126 3101 to 3135 3130 to 396 371 to 3105 3100 to 366 341 to 375 3280 3263 3274 3257 3268 3250

to to to to to to

3263 3280 3257 3274 3250 3268

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following tolerances : TCA motif (TCATCTTCTT with two mismatches) [41], G-box (CACGTG with no mismatches) [42], H-box (CCTACCN7 CT with two mismatches) [43], P-box (CCAa/cCa/tAACc/tCC with two mismatches) [44], A-box (CCGTCC with one mismatch) [44], L-box (t/aCTc/aACCTAc/aCc/a with two mismatches) [44], box IV (TAATTAAT with one mismatch) [45], GT-1 box (GGTTAA with no mismatches) [46], SBF1 [(g/t)(a/t)(a/g)TNGTTAA(a/t)3 with one mismatch] [47], TGACG box (with no mismatches) [21], ACE box (CACGT with no mismatches) [32], MRE (t/aCNa/cACCTAc/aC with two mismatches) [32], MYB.Ph3 [(a/ g)2 AGTTAGTTAc/g with one mismatch] [48], WRE1 (ATAAAAATTTC with two mismatches) [49], WRE2 (TTAGTATAA with one mismatch) [49] and WRE3 (CCACCT with one mismatch) [49]. Additional searches for other plant-speci¢c regulatory elements were done us-

ing the TRANSFAC search program (http://molsun1.cbrc.aist.go.jp/htbin/) and with tolerance thresholds set at 87.0. 2.4. Nucleus isolation and extract preparation Nuclei were isolated from frozen pea leaf material (30^ 50 g) and protein extracts were prepared using the procedure of Manzara and Gruissem [50]. Proteins in the extracts were quanti¢ed using the ¢lter paper dye binding assay of Minamide and Bamburg [51]. 2.5. Production of sadC promoter sub-fragments to act as EMSA probes and competitors Eleven overlapping 60-bp DNA fragments covering a 360-bp region of the proximal sadC promoter were gener-

Fig. 1. The promoter, 5P UTR and 5P coding sequences of the P. sativum sadA and sadC genes. The 1625-bp sadA (GenBank accession AF242183) and the 749-bp sadC (GenBank accession AF242182) promoter fragment sequences including cis-elements were drawn using mismatch tolerances outlined in Section 2. The putative transcription initiation sites de¢ned by alignment with the full-length sadC cDNA [36] are designated with an arrow at +1 and the sequences are numbered relative to this position. Mismatches from each consensus cis-element are designated with a slash (/) in the line/box representing a potential regulatory element. Single nucleotide variations between the two sequenced clones for each promoter fragment are written using IUPAC ambiguous nucleotide codes (i.e. M = A or C, R = A or G, S = C or G, Y = C or T). In the sadA 5P UTR, the location of the three-nucleotide deletion relative to the sadA cDNA sequence reported in Brosche¤ and Strid [36] is marked by a triangle. Regions conserved in the sadA and sadC promoters are shaded.

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ated using 11 pairs of complementary oligonucleotides (Table 1B, Fig. 2A). These oligonucleotides were between 35 and 38 nucleotides in length, the variation being due to the inclusion of additional residues at their 5P termini to generate £anking restriction sites when the fragments were cloned in a plasmid vector. Individual oligonucleotide pairs were complementary over 10 residues at their 3P ends so that they could be annealed and extended to generate double-stranded DNAs of between 61 and 65 bp. This primer extension was performed using Pwo DNA polymerase (Roche Molecular Biochemicals) in a thermocycling reaction. The conditions used were like those of a normal PCR but without added template. The reaction mixture was composed of 0.6 WM of both phosphorylated oligonucleotides, 200 WM of each of the four dNTPs, 1Ureaction bu¡er and 1.25 U Pwo DNA polymerase. The incubation was composed of an initial denaturation period of 30 s at 94‡C during which the Pwo polymerase was added to produce a ‘hot start’, followed by a thermocycle of 94‡C for 30 s, 55‡C for 30 s and 72‡C for 1 s, which was repeated 30 times. Reactions were separated on a non-denaturing 8% TBE-polyacrylamide gel and fragments eluted in a bu¡er containing 0.5 M NH4 -acetate, 1 mM EDTA and puri¢ed by ethanol precipitation [52]. Each fragment was cloned into vector pLITMUS38 (NEB) cleaved with EcoRV and clones were selected by digestion with restriction endonucleases EcoRV and BglII which con¢rmed that the fragments were in the required orientation. Selected plasmid clones were sequenced to con¢rm that they were error-free and then stocks were generated for use in the preparation of EMSA probes. As competition in the ¢rst EMSA reactions, the same 11 fragments of the proximal sadC promoter were used. These were generated by separate annealing and extension of the 11 complementary oligonucleotide pairs (Table 1B, Fig. 2A) as described above. All fragments were puri¢ed from a non-denaturing 8% TBE-polyacrylamide gel. As unlabelled competitors in the second EMSA, to localise factor binding sites within the proximal sadC promoter, half-fragments covering the region of interest were created by annealing oligonucleotide pairs 2b/3a, 3b/4a, 4b/5a and 5b/6a (Table 1B, Fig. 3A). Annealing mixtures of 100 Wl contained approximately 10 Wg of each oligonucleotide in 0.1 M NaCl. These were heated to 90‡C and then cooled slowly to room temperature. The annealed fragments were gel-puri¢ed on a non-denaturing 8% TBE-polyacrylamide gel as above. For ¢nal localisation of the factor binding sites, three additional sadC promoter sub-fragments were produced using complementary oligonucleotide pairs sf1a/sf1b, sf2a/sf2b and sf3a/sf3b (Table 1C). Once annealed these oligonucleotides produced sub-fragments of 18 bp (sf1, sf2) or 19 bp (sf3) overlapping one another by 12 bp (Fig. 4A). Annealing was performed as above and the fragments were gel-puri¢ed from a non-denaturing 12% TBE-polyacrylamide gel. The composition of the elution

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bu¡er used was 0.25 M NaCl, 10 mM MgCl2 , 1 mM EDTA and 10 mM Tris^HCl (pH 7.5), which was found to be more e¡ective than the standard NH4 -acetate bu¡er for the isolation of small fragments. 2.6. Probe preparation for EMSA experiments Radiolabelled probe preparation and EMSAs were performed according to the method of Schindler and Cashmore [52]. As the probe in the initial experiment an equimolar mixture of all 11 constructs containing the separate sub-fragments of the sadC promoter (1 Wg of each) was digested with restriction endonucleases EcoRV and then EcoRI. The excised fragments of between 78 and 82 bp were radiolabelled with [K-32 P]dATP using Klenow polymerase and then gel-puri¢ed from non-denaturing 6% TBE-polyacrylamide gels to produce a mixed probe. For subsequent assays, individual constructs containing fragments 4 and 5 (10 Wg) were treated as above to generate radiolabelled single-fragment probes. For the ¢nal binding site localisation EMSA, 100 ng of the gel-puri¢ed sub-fragment sf2 was radiolabelled with [K-32 P]dATP using Klenow polymerase to ¢ll the 5P extension (5P-AATT-3P) and isolated using a Qiagen nucleotide removal kit. The activity of all radiolabelled probe fragments was quanti¢ed by measuring Cerenkov radiation in a scintillation counter. 2.7. EMSA procedure EMSAs were performed according to the method of Schindler and Cashmore [52]. Each 30 Wl binding reaction contained 104 cpm of radiolabelled probe fragment (approximately 1 fmol), 2 Wg of poly(dI-dC), with or without speci¢c competitor DNA and nuclear extract (1^1.5 Wg protein) in a binding bu¡er of composition 10 mM Tris^ HCl (pH 7.5), 40 mM NaCl, 4% glycerol, 1 mM EDTA and 0.1 mM 2-mercaptoethanol. Reactions were incubated for 30 min at room temperature and then 3 Wl of 30% glycerol was added before loading on a non-denaturing 5% TGE-polyacrylamide gel. Electrophoresis was performed at 10 V cm31 for approximately 3 h (until a bromophenol blue standard loaded in a separate lane had migrated 13 cm). Gels were then transferred to ¢lter paper (Whatman 3MM) and dried before placing against X-ray ¢lm (Fuji) at 380‡C. In EMSA reactions containing speci¢c competitor DNAs, the gel-puri¢ed fragments were quanti¢ed using an ethidium bromide-stained dot protocol [53]. In the initial experiment using a mixture of all the fragments as the probe, each of the individual unlabelled fragments was used as a competitor. The molar ratio of competitor to probe was approximately 500:1, which considering that the probe was composed of 11 fragments gave a ratio of approximately 5000:1 for the particular fragment in the mixture that was being speci¢cally competed against. In all

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Fig. 2. Identi¢cation of nuclear factor binding sites within the proximal sadC promoter. (A) Diagram of the 340 to 3400 sadC promoter region showing putative cis-elements and the overlapping 60-bp fragments for use as probes in EMSA analyses, including the oligonucleotides used to create each fragment. (B) EMSA using a mixed probe composed of the 11 overlapping fragments of the sadC proximal promoter region with nuclear extracts from UV-B-treated (i) and control (ii) pea leaves. The control lanes are: probe without added nuclear extract (0) and the standard binding reaction without added unlabelled competitor fragment (3). The numbering of the other lanes (+1^+11) indicates which of the individual fragments was added as unlabelled competitor to the particular binding reaction. The free probe (F) and individual shifted species (B1^B7) are indicated.

6

the EMSAs using fragments 4 and 5 as probes the molar ratio of competitor to probe was again approximately 500:1. In the ¢nal EMSA experiment, using sf2 as the probe, the molar ratio of competitor to probe was approximately 50:1.

3. Results 3.1. Promoter fragment isolation A pair of nested oligonucleotide primers was designed using the sequence of the P. sativum sadA cDNA [36]. Because the sad genes are highly homologous, it was anticipated that these primers would anneal to sequences from multiple members of the gene family, apart from sadB which lacks the sequence to which the internal nested primer was designed [36]. Sequences £anking the 5P sad gene-coding regions were ampli¢ed by suppression/long PCR using ¢ve separate adapter-ligated DNA fragment libraries of pea genomic DNA as template and a primer speci¢c for the adapter (AP1) and the ¢rst gene-speci¢c primer (SADA-1, Table 1A). To minimise introduced errors, a thermostable polymerase mixture with proofreading activity was used. The products from this primary reaction were used as the template for a nested PCR reaction using a second adapter primer (AP2) in conjunction with a second sad gene-speci¢c primer (SADA-2, Table 1A). When analysed on an agarose gel, each of the ¢ve separate secondary PCR reactions contained at least one distinct fragment (data not shown). The two largest fragments of approximately 1.0 and 2.0 kb, which would contain the most promoter sequence, were cloned and two separate clones were sequenced. Single nucleotide variations in the codons for Ser-25 and Thr-31 indicated that the smaller fragment was derived from the sadC gene while the larger was from sadA (data not shown). There were a few single nucleotide di¡erences between the separate clones for each promoter which could be due to ampli¢cation from separate alleles or may be the result of PCR error (Fig. 1). In addition, there was a 3-bp deletion and a single nucleotide alteration in the 5P untranslated region (UTR) of the sadA sequence when compared to the cDNA for this gene (Fig. 1) [36]. This is thought to be characteristic of the sadA gene because when other smaller GenomeWalker products were sequenced, the nucleotide substitutions in the coding region were always accompanied by these changes in the 5P UTR (data not shown). These

sequences have been deposited in GenBank with accession numbers AF242183 (sadAP) and AF242182 (sadCP). 3.2. Promoter sequence analyses The sadA (1625 bp) and sadC (749 bp) promoter fragment sequences were analysed to identify general transcription elements and potential regulatory cis-elements using the longest corresponding cDNA sequences [36] to de¢ne the putative RNA initiation sites for each gene (Fig. 1). In agreement with this assignment, TATA boxes are positioned 33 and 31 nucleotides upstream of these sites and consensus CCAAT boxes are positioned at 96 and 94 nucleotides upstream of these sites in the sadA and sadC genes, respectively. In parallel with the high degree of identity in their coding nucleotides (99% identity), the sadA and sadC promoters share 85^98% identity in four 66^153-nucleotide regions within the 749 nucleotides available for the sadC promoter. Within these four regions, only two insertion/deletion events are needed to accommodate the alignment providing further evidence of the degree of sequence identity. Between these four regions, multiple insertion/deletion events and sequence variations have occurred presumably in regions not needed for coordinate transcriptional activation of the sadA and sadC genes. The most conserved of these sadA and sadC promoter regions (98% identity) spans the proximal promoter sequence from 3136 to +1 (relative to the sadC transcription start). Included in this region are a TGACG motif [21,54], which is present in a variety of plant promoters including that of the wounding- and jasmonate-responsive lipoxygenase 1 gene of barley [55]; multiple WRE2 and WRE3 elements conserved in several wound-responsive pea cytochrome P450 monooxygenase promoters (CYP73A9, CYP82A1) [49]; MRE and ACE elements identi¢ed in the UV-B- and UV-A/blue light-responsive parsley and Arabidopsis CHS promoters [25,32], an Abox identi¢ed in elicitor-induced parsley PAL and 4CL promoters [44], as well as CCAAT and TATAAA boxes needed for basal transcription. The next most conserved sequences (93% identity) are the 3408 to 3472 region that contains no previously identi¢ed regulatory elements and the 3244 to 3396 region that contains a SBF-1 binding site identi¢ed in the silencer region of the French bean CHS promoter [47] within the sadA copy of which is a consensus binding site for factor GT-1, involved in lightand non-light-responsive transcription [46,56,57], the WRE3 element described above [49], and a GC-rich ele-

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[41]. Other potential regulatory elements in the 3475 to 3607 region of the sadC promoter are not present in the sadA promoter, including the WRE1 and WRE3 elements [49], the G-box present in many stimulus-responsive plant promoters [42], and the A-boxes previously found in elicitor-induced promoters of the early phenylpropanoid pathway [44]. Additionally, a MYB.Ph3 element identi¢ed in many CHS promoters [48] and an H-box identi¢ed in promoters for genes in the early phenylpropanoid pathway [43] exist in the most distal promoter sequences represented only in the sadA promoter (Fig. 1). 3.3. EMSA analysis of the proximal sadC promoter EMSA was used to determine which of the predicted cis-elements could act as binding sites for pea leaf nuclear factors in vitro. The sadC proximal promoter was chosen

Fig. 3. Localisation of nuclear factor binding sites within the proximal sadC promoter. (A) Diagram of the overlapping 60-bp fragments around the fragment 4/5 overlap region showing the oligonucleotides used to create each fragment and how they can be annealed to produce half-fragments to act as competitor DNAs. (B) EMSA using the sadC promoter fragments 4 and 5 as probes with nuclear extracts from UVB-treated (i), wounded (ii) and control (iii) pea leaves. The control lanes are: probe without added nuclear extract (0) and the standard binding reaction without added unlabelled competitor fragment (3). The numbering of the other lanes indicates the unlabelled competitor fragments or half-fragments added to each particular binding reaction. The free probe (F) and individual shifted species (B1^B4) are indicated.

ment whose binding properties will be described later in this paper. The most distal and least conserved region (85% identity) is the 3625 to 3749 region that contains a TCA motif identi¢ed in many stress-inducible promoters

Fig. 4. Further localisation of the factor binding sequence within the proximal sadC promoter fragment 4/5 overlap region. (A) Diagram showing the sequence of the 4/5 overlap and 18^19-bp sub-fragments covering this region. The three sub-fragments (sf1, sf2, sf3) which were created by annealing oligonucleotides carry the 5P extension 5P-AATT-3P to permit radiolabelling using Klenow polymerase. (B) EMSA using the sadC promoter fragments 4 and 5 as probes with a nuclear extract from UV-B-treated (6 h) pea leaves. The control lanes are: probe without added nuclear extract (0) and the standard binding reaction without added unlabelled competitor fragment (3). The numbering of the other lanes indicates the unlabelled fragment, half-fragment or sub-fragment added to each particular binding reaction as competition. The free probe (F) and individual shifted species (B1^B4) are indicated.

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for this analysis because it shares the same elements as the corresponding sequence of the sadA gene and in a parallel study it was found to be su⁄cient to drive wound-responsive reporter gene expression in transgenic Arabidopsis (Gittins and Strid, unpublished data). Overlapping 60-bp probes were generated using oligonucleotides for high-resolution EMSA analysis of this region. Eleven fragments overlapping one another by 30 bp were produced to cover a region of 360 bp extending from position 341 to 3400 relative to the proposed transcription start site for sadC and not including the putative TATA box element (Figs. 1B and 2A). Each fragment was cloned and its sequence con¢rmed. A radiolabelled equimolar mixture of the 11 di¡erent sadC promoter fragments was used as the probe for EMSAs with nuclear extracts prepared from UV-B-irradiated and control pea leaves (Fig. 2B). It was noted that despite gel puri¢cation, the probe contained an additional contaminant band (lane 0). In the absence of any speci¢c competitor fragment, the banding pattern was very similar when the mixed probe was incubated with nuclear extracts from UV-B-irradiated (i) or control (ii) pea leaves (lanes 3). Seven separate bands were recognised and named B1^ B7. With the UV-B-irradiated nuclear extract, bands B1 and B7 were more distinct than with the control extract, although this is probably because the former gel was run for slightly longer and so the resolution was better. Band B5 co-migrated with the probe contaminant band, although its higher intensity suggested that it contained more than one molecular species. To determine which of the bands was the product of speci¢c fragment association with factors in the nuclear extracts, an excess of each of the unlabelled fragments was added to 11 separate binding reactions. In these competition reactions (Fig. 2B, lanes +1 to +11) the highermobility bands, B5^B7, were largely unchanged by the addition of the speci¢c competitors. The fact that their intensity is slightly reduced by the addition of more poly(dI-dC) (data not shown) suggests that they result from association with non-speci¢c DNA binding factors, so these bands were not considered further in this study. In all lanes, addition of the unlabelled fragments caused a slight overall reduction in the intensity of bands B1^B4 and produced an intense slower-migrating species above them. The former phenomenon may be attributed to non-speci¢c DNA binding while the latter is less easy to explain. Of the speci¢c competitor fragments, only two produced consistent changes in the banding pattern that were reproducible in separate experiments. Addition of either fragment 4 or 5 caused a substantial reduction in the intensity of bands B2 and B3 and the complete disappearance of B1 and B4 (Fig. 2B, lanes +4 and +5). This was seen with both the UV-B-treated and control nuclear extracts. A sequence shared by these overlapping fragments contains elements that appear to be responsible for most of the speci¢c nuclear factor binding in the prox-

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imal region of the sadC promoter. Similar experiments were performed using a nuclear extract prepared from wounded pea leaves and the competitor analyses results were identical to those seen with the UV-B-treated extract (data not shown). To con¢rm that fragments 4 and 5 share the major site of factor binding, and to demonstrate that the 30-bp overlap region contained the critical cis-elements, these fragments were individually radiolabelled and used as probes in further competition EMSA reactions (Fig. 3B). In these reactions, nuclear extracts from UV-B-irradiated (i), wounded (ii) and control (iii) pea leaves were used in identical sets of reactions. In reactions without competition (lanes 3), four predominant bands were observed corresponding to bands B1^B4 in Fig. 2B. With fragment 4 as the probe, the intensity of band B1 appeared to be greater than with fragment 5 as the probe. The pattern of bands was similar with each of the three nuclear extracts although the resolution of bands B1/B2 on the gel for the wounded extract reactions was poorer, probably because this gel was run for a slightly shorter time (Fig. 3B, ii). Also, the intensity of bands in the B3/B4 region appeared greater with the control extract, as did the intensity of a less distinct band located below B4 (Fig. 3B, iii). Lastly, from the less intense banding with the UV-B extract reactions it appeared that B3 was in fact composed of a doublet of bands (Fig. 3B, i). As competition, the unlabelled fragments 4 and 5 were added to binding reactions containing their respective probes (Fig. 3B, lanes 4 and 5). In the other competition reactions, four half-fragments, 2b/3a, 3b/4a, 4b/5a and 5b/ 6a, produced by annealing of the relevant oligonucleotide pairs (Table 1B) were used to represent sub-sections of the 60-bp fragments between positions 3340 and 3221 (Fig. 3A). The fragment 2b/3a acted as a negative control because it represents sequence outside fragments 4 and 5. Factor binding by probes 4 or 5 to produce bands B1, B3 and B4 was almost completely abolished when the same unlabelled 60-bp fragment was used as competition. Of the four half-fragment competitors, only 4b/5a, representing the 4/5 overlap region, was able to substantially reduce factor binding, although the extent of this reduction, particularly to the B1/B2 bands, was not as great as seen with the complete 60-bp fragments (Fig. 3B). It was noted that the intensity of B2 was also diminished by addition of the other competitor fragments (2b/3a, 3b/4a and 5b/6a) which suggests that this band is mainly the result of interaction with a non-speci¢c DNA binding factor. The intensity of the other three bands (B1, B3, B4) was diminished by competition with 4b/5a although not all to the same extent. Bands B3/B4 were more readily competed away than B1. Possibly the weaker competition by the half-fragments and the variable degree of reduction in the intensity of the di¡erent bands re£ect the lower a⁄nity of binding factors for shorter DNAs and the di¡erent a⁄nities of separate factors for their relevant binding sites,

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respectively. In respect of the former hypothesis, it has previously been noted that in in vitro assays, cis-elements are more e¡ectively bound if they are located on longer DNA fragments [46,58]. In the separate binding experiments using the three di¡erent nuclear extracts, no major di¡erences were seen in the pattern of competition other than with the band beneath B4 seen in reactions with the control extract. This was more intense and appeared to be more e⁄ciently competed away by speci¢c probes than in reactions with the stressed leaf nuclear extracts (Fig. 3B, iii). The sequence of the 30-bp half-fragment 4b/5a, which appeared to contain the major site of factor binding in the proximal sadC promoter, was examined to identify cis-elements known to bind transcription regulatory factors. No perfect matches to recognised motifs were observed although there was one copy of a WRE3 element with a single mismatch [49]. This overlaps with an interesting sequence element, designated the GC-rich region in Fig. 1, which consisted of a palindromic GTGG repeat separated by a GC-rich triplet. This sequence is £anked by the same 4-bp repeat (TTTT). To localise the binding site further and see if binding by separate factors could be attributed to di¡erent sequences, three sub-fragments of the 4b/5a fragment were produced by annealing of oligonucleotides (Table 1C, Fig. 4A). These sub-fragments of 18 or 19 bp, named sf1, sf2 and sf3, and overlapping one another by 12 bp, were used as competitors in further EMSA reactions. Again fragments 4 and 5 were used as the radiolabelled probes but this time only the UV-B-irradiated pea leaf nuclear extract was used (Fig. 4B). As previously, unlabelled fragments 4 and 5 showed almost complete competition with the relevant probes for factor binding, although again, band B2, while reduced in intensity, was still present. Competition by unlabelled 4b/5a was less e⁄cient, particularly with bands B1/B2. When the unlabelled subfragments sf1, sf2 and sf3 were added to separate binding reactions, sf1 had no e¡ect on the pattern of bands, sf3 caused minor reduction in the intensity of bands B3/B4, while sf2 totally competed away bands B3/B4 and caused a slight reduction in the intensity of bands B1/B2. These results indicate that the main element(s) with factor binding activity reside on sf2 although the incomplete competition, particularly for the binding of the factors responsible for bands B1 and B2, suggests less e⁄cient factor binding to the short competing fragments and variation in the a⁄nity with which separate factors bind to their target sequence(s). The slight competition for bands B3/ B4 shown by sub-fragment sf3 suggested that some factors could still bind to elements carried by this competing fragment, although they may be incomplete. It should be noted that sf3 carries a centrally located copy of a putative WRE3 element [49] and so the failure of this sub-fragment to act as an e¡ective competitor indicates that this element is not involved in factor binding. As a ¢nal experiment, sub-fragment sf2, shown to con-

tain the major factor binding element in the proximal sadC promoter, was radiolabelled and used as the probe in a competition EMSA (Fig. 5). In order to be able to directly compare the results obtained with the 60-bp probe fragments, the same gel system was used to analyse the binding reactions. The migration of DNA binding factors in non-denaturing polyacrylamide gels is not greatly a¡ected by the size of DNA fragment to which they are bound [59], and so the use of the same gel conditions permitted a direct comparison of the gel migration of protein species binding to the di¡erent probes. In this experiment, the three di¡erent nuclear extracts, from UV-B-irradiated, wounded and control pea leaves, were again compared (Fig. 5). With the very small sf2 probe, the e⁄ciency of factor binding was reduced and this necessitated a long exposure, which produced poor resolution. However, it was still clear that multiple species bound to the probe (Fig. 5, bracket). Although it is di⁄cult to distinguish separate bands in the smear, the species appeared to migrate to the same position in the gel as the B1^B4 bands seen in the EMSAs with the longer probe fragments (Fig. 5, lanes 3). One other lower- and one higher-mobility band were also seen in the absence of any speci¢c competitor fragments (Fig. 5, arrows). No major di¡erences could be detected when the three nuclear extracts were used. As speci¢c competition, unlabelled sf1, sf2 and sf3 were added to separate reactions with each extract. It

Fig. 5. Con¢rmation of the presence of a factor binding sequence within the proximal sadC promoter sub-fragment sf2. EMSA using sub-fragment sf2 as the probe with nuclear extracts from control, UV-B-treated and wounded pea leaves. The control lanes are: probe without added nuclear extract (0) and the standard binding reaction without added unlabelled competitor fragment (3). The numbering of the other lanes indicates the unlabelled competitor sub-fragment added to each particular binding reaction. The free probe (F), speci¢c (bracket) and non-speci¢c (arrows) shifted species are indicated.

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should be noted that due to the small size of the probe the molar excess of competitor to probe which could be achieved was only around 50:1 compared to 500:1 in the earlier experiments. Upon addition of the unlabelled fragments, both the lower- and the higher-mobility bands £anking the smear disappeared indicating that they were formed by non-speci¢c DNA binding factors. At the same time another extremely low-mobility band appeared in these lanes. The origin of this band, and why its intensity varies with the competitor fragment used, is uncertain. Addition of the competitor fragment sf1 had no e¡ect on the pattern of the smeared bands. Competitor sf3 caused a reduction in their intensity, while sf2 produced a substantial reduction in all shifted species. This result con¢rms that the major site of factor binding in the proximal sadC promoter is located on sf2 and is likely to be centred about the 11-bp GC-rich sequence GTGGCGCCCAC.

4. Discussion The promoters of two stress-responsive pea sad genes were isolated and fully sequenced. As would be expected for related and co-ordinately regulated genes, the sequences share a high degree of homology and they contain a number of putative cis-elements, which may be related to their function. To move the study a step closer to the situation in vivo, high-resolution EMSA analysis, using 11 overlapping 60-bp fragments, was conducted to examine the nuclear factor binding potential of the proximal region of the sadC promoter known to contain cis-elements involved in stress-responsive expression. Through competition, substantial factor binding activity was found to reside within the 30-bp overlap between fragments 4 and 5, containing a GC-rich element. Multiple shifted species (at least four) were seen in EMSA analyses with promoter sub-fragments covering this region suggesting that more than one protein factor binds to this sequence. The pattern of nuclear factor binding with test (UV-B or wounding stress) and control leaf nuclear extracts appeared to be practically identical, as has been seen in a number of other studies in which EMSA analysis has been used to examine stimulus-induced gene expression [57,60^ 62]. Di¡erential gene expression may not simply be explained by the availability of a speci¢c trans-acting factor binding to a promoter element, but rather it may require modi¢cation of ubiquitous factors or changes in their interactions. It is also possible that our mixed-probe strategy masks faint stress- or control-speci¢c bands produced by non-abundant binding factors or that the experimental conditions used were unfavourable for the binding of such factors. To rule out these possibilities, further experiments will include separate EMSA reactions using each of the 11 individual fragments and test a range of binding reaction and gel electrophoresis conditions. Although the EMSA banding patterns appeared to be

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qualitatively identical, there were slight quantitative di¡erences in band intensities in reactions using the stressed and control nuclear extracts. In the EMSA analyses using fragments 4 and 5 as probe there was more binding activity in the control extract than in the stressed extracts, particularly in the B3/B4 region and the less distinct band just below this (Fig. 3B, iii). This may indicate that, among the factors binding to this fragment, a repressor is present in non-stressed cells. Upon a stress stimulus the hypothetical repressor would be inactivated in some way to allow transcriptional activation to occur. Additional support for the involvement of a repressor in sad gene activation may be drawn from the ¢nding that the sad genes and other stressinduced genes from pea are up-regulated by treatment of young plants with low doses of cycloheximide (Brosche¤, Schuler and Strid, unpublished observations). Inducibility by cycloheximide is recognised as a feature of immediate early genes responding to a particular stimulus [63] and one explanation for its e¡ect is the degradation of a labile repressor following inhibition of protein synthesis. Although there are a number of other modes by which cycloheximide can induce gene expression [64^66], at least one recently described repressor, designated AtMYB4, plays a role in UV-B-induced expression in Arabidopsis [35]. A comparable transcription factor in pea may exert a similar ability to repress sad gene expression. Unexpectedly, none of the motifs identi¢ed by in silico sequence analysis of the sad promoters, including the fairly well conserved ACE and MRE elements, showed signi¢cant factor binding potential in EMSAs. Instead, the major site of factor binding in the proximal sadC promoter identi¢ed by the method used here involved the GC-rich sequence GTGGCGCCCAC. A one-nucleotide variant of this novel motif (GTGGTGCCCAC) is present in the sadA promoter but no other perfect matches to this binding element were found in searches of sequence databases. The motif shares the sequence GTGG which forms the half-site for di¡erent classes of plant cis-acting element. The importance of GTGG half-sites was ¢rst recognised in the CaMV 35S promoter where three copies are present in an activator element within the distal region [67]. This sequence has also been identi¢ed as forming the half-site of the palindromic G-box (CCACGTGG) element, which is involved in regulation of gene expression in response to many stimuli [42]. Other sequences that are related to the G-box and contain the GTGG half-site are abscisic acidresponsive elements (ACGTGGC) involved in the abscisic acid-induced expression of genes following water stress, cold stress, salt stress and wounding [68,69]. The notable di¡erence between the arrangement of the sad promoter motif and the previously characterised G-box and related elements is that in the former the two copies of GTGG sequence converge on opposite strands while in the latter they diverge, which is likely to have implications for factor binding. The GC-rich sequence of the sad promoter is similar to

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a number of other recognised classes of plant cis-elements including the GC repeat element (e.g. ACCGGCCCACTT) thought to be a positive regulator of photoregulated gene expression [70], the GCC box element that is involved in the expression of plant defence factors [71] and the anaerobic responsive element (GCg/cCC) found in the promoter of the maize Adh1 promoter [58]. These apparent homologies to characterised cis-acting elements may be simply the result of the high GC content of this sequence in the sadC promoter or they may have functional signi¢cance that awaits con¢rmation. In the EMSA analysis aimed at localising the factor binding sites within the fragment 4/5 overlap region, incomplete competition was seen for two of the shifted species, B1 and B2 (Figs. 3B and 4B). Sub-fragment sf2 was a less e⁄cient competitor than fragment 4b/5a, particularly when fragment 4 was the probe (Fig. 4B). It is thought that band B2 probably represents the DNA binding activity of a non-speci¢c factor, and so would not be subject to speci¢c competition. The failure to compete away the B1 band is most likely due to the reduced binding e⁄ciency of short competing fragments and variation in the a⁄nity with which di¡erent transcription factors bind to their target sequences. However, e⁄cient binding by the B1 factor may also require sequences outside fragment sf2, either to form the binding site or to act as sites for additional factors which may act in a co-operative manner to assist factor binding to the 11-bp GC-rich element. Outside the sf2 fragment, there are some interesting sequences, which may include speci¢c factor binding sites involved in cooperative binding. The sequence TTTGTTG at the right hand end of the fragment 4/5 overlap region resembles the GAmyb protein binding element (TTTGTTA) in the promoter of the gibberellin-responsive high-pI K-amylase gene of barley [72]. Although the SAD proteins share a high sequence similarity with a GA-regulated protein from tomato [36], there is no evidence that the sad genes are induced by gibberellin. The putative myb binding element may be shared by similar factors involved in di¡erent stimulus^response pathways. Another sequence outside sf2, ACTTTT, which is located at the right hand end of the GC-rich core, is identical to the OBP1 binding site in the Arabidopsis thaliana GST6 gene promoter [73]. OBP1 is a Dof domain protein (DNA binding with one finger) which plays a role in expression of the GST6 gene induced by auxin, salicylic acid or oxidative stress. The Dof factors have been shown to enhance the binding of a number of bZIP transcription factors to their target sequences [74]. With their role in expression in response to stimuli (some of which also induce the sad genes), and in promoting the binding of other factors, the Dof factors are possible candidates as binding factors to the identi¢ed sad promoter element. The presence of multiple cis-elements within the 30-bp overlap between fragments 4 and 5 may explain the responsiveness of the sad genes to a wide variety of stresses. This short sequence could represent the hub through

which the signals from diverse stress response pathways converge and where shared or speci¢c transcription factors interact to regulate transcription by a combinatorial mechanism, as has been suggested for other plant genes regulated by environmental stimuli [74]. The sad genes are constitutively expressed in pea roots [36], and so root-speci¢c factors may be involved in combinatorial control in response to developmental signals centred on the same sequence. The ability of transcription factors to enhance the DNA binding by other factors (e.g. Dof factors) may contribute to a combinatorial control mechanism. We are currently attempting to con¢rm that the sad promoter element identi¢ed in this study is necessary for stress-induced expression. In addition we plan to identify the pea nuclear factors that bind to this sequence and con¢rm that they play a role in the expression of the sad genes. Due to its small size it would be surprising if all the cis-elements required to direct stress-speci¢c expression were con¢ned within the fragment 4/5 overlap region. However, if this were the case, this sequence could prove extremely useful as the basis of a minimal promoter to drive the expression of genes to disclose or combat the e¡ects of environmental stress.

Acknowledgements We would like to acknowledge the support of the Strategic Network for Swedish Plant Biotechnology, the Swedish Natural Science Research Council (NFR), the Swedish Research Council for Engineering Sciences (TFR), the Carl Trygger Foundation and the Crafoord Foundation. We also thank Dr Mikael Brosche¤ for useful help and advice.

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