Homeodomain Leucine Zipper Proteins Bind to the Phosphate Response

Homeodomain Leucine Zipper Proteins Bind to thePhosphate Response Domain of the Soybean VspBTripartite Promoter1

Zhijun Tang, Avi Sadka, Daryl T. Morishige, and John E. Mullet*

Genentech, Incorporated, 1 DNA Way, Mail Stop 37, South San Francisco, California 94080 (Z.T.); Institute ofHorticulture, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan 50250, Israel(A.S.); and Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77840(D.T.M., J.E.M.)

The soybean (Glycine max L. Merr. cv Williams 82) genes VspA and VspB encode vacuolar glycoprotein acid phosphatasesthat serve as vegetative storage proteins during seed fill and early stages of seedling growth. VspB expression is activatedby jasmonates (JAs) and sugars and down-regulated by phosphate and auxin. Previous promoter studies demonstrated thatVspB promoter sequences between 2585 and 2535 mediated responses to JA, and sequences between 2535 and 2401mediated responses to sugars, phosphate, and auxin. In this study, the response domains were further delineated usingtransient expression of VspB promoter-b-glucuronidase constructs in tobacco protoplasts. Sequences between 2536 and2484 were identified as important for phosphate responses, whereas the region from 2486 to 2427 mediated sugarresponses. Gel-shift and deoxyribonuclease-I footprinting assays revealed four DNA-binding sites between 2611 and 2451of the soybean VspB promoter: one in the JA response domain, two in the phosphate response domain, and one binding sitein the sugar response domain. The sequence CATTAATTAG present in the phosphate response domain binds soybeanhomeodomain leucine zipper proteins, suggesting a role for these transcription factors in phosphate-modulated geneexpression.

The soybean (Glycine max L. Merr. cv Williams 82)vegetative storage proteins VSPa and VSPb are vac-uolar acid phosphatases that accumulate in hypo-cotyl hooks and plumules during seedling develop-ment, and in leaves, stems, and pods during thereproductive phase (Wittenbach, 1982; Staswick,1989a; DeWald et al., 1992). Sink deprivation by de-podding or petiole girdling also causes a massiveaccumulation of the Vsp mRNAs and proteins (Wit-tenbach, 1982; Staswick, 1989b). The accumulation ofthe VSP in vacuoles of cells in sink tissues and inresponse to depodding led Wittenbach (1983) toidentify these proteins as vegetative storage proteins.

Vsp mRNA levels are highest in the plumule, hy-pocotyl hook, hypocotyl elongation region, andyoung leaves, whereas low levels are detected inmore mature portions of the hypocotyl, older leaves,and roots (Mason and Mullet, 1990). Vsp expressionis also high in buds and flowers of Arabidopsis(Berger et al., 1995). Tissues with high levels of VspmRNA have elevated levels of jasmonates (JAs; Ma-son et al., 1992; Creelman and Mullet, 1995), linolenicacid-derived compounds involved in plant defense(Vick and Zimmerman, 1984; Gundlach et al., 1992;

Hamberg and Gardner, 1992; Sembdner and Parthier,1993). Treatment of leaves or soybean cell cultureswith JA causes accumulation of Vsp mRNA and pro-tein (Anderson, 1988; Anderson et al., 1989; Masonand Mullet, 1990). Vsp expression and JA levels in-crease in response to wounding (Creelman et al.,1992; Albrecht et al., 1993), and Vsp expression isabsent in mutants that cannot respond (Feys et al.,1994) or synthesize JA (McConn et al., 1997).

Vsp expression is stimulated by sugars and re-pressed by phosphate and auxin (DeWald et al.,1994). Full induction of Vsp mRNA accumulation inexcised mature soybean leaves required 10 mmmethyl jasmonate (MeJA) plus illumination or 0.2 mSuc (Mason et al., 1992). In soybean cell suspensionculture, Suc, Fru, or Glc plus 10 mm MeJA wereneeded to achieve maximum induction of VspB (Ma-son et al., 1992). These experiments showed that Vspexpression is synergistically activated by a combina-tion of JA and sugars. In addition, accumulation ofVspB mRNA in soybean cell cultures was inhibitedwhen the phosphate concentration of the growth me-dium was increased from 0.31 mm to 2.5 mm (Sadkaet al., 1994). Plants fed with Man to reduce cytoplas-mic phosphate levels also showed increased expres-sion of VspB (Sadka et al., 1994).

The VspB promoter has been characterized by pro-moter deletion analysis in transgenic tobacco (Nico-tiana tabacum cv Samsun; Mason et al., 1993) and byanalysis of promoter domains in protoplasts (Sadka

1 This work was supported by the National Science FoundationGenetics Program (grant no. MCB–9514034 to J.E.M.) and by theTexas Agricultural Experiment Station.

* Corresponding author; e-mail [email protected]; fax409 – 845–9274.

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et al., 1994). The VspB promoter domain from 2787 to2289 responded to JA, sugars, and phosphate. Dele-tion of the promoter to 2520 eliminated JA-modulated transcription. Gain-of-function experi-ments identified a 50-bp DNA sequence, from 2585to 2535 of the VspB promoter, that could mediate JAresponses when fused to the truncated (288) 35S-cauliflower mosaic virus (CaMV) promoter (Masonet al., 1993). Other studies showed that the promoterregion from 2536 to 2403 mediated responses tophosphate (inhibition), sugars (stimulation), andauxin (inhibition; DeWald et al., 1994; Sadka et al.,1994). Therefore, this approximately 185-bp portionof the VspB promoter provides an opportunity toidentify cis- and trans-factors mediating each re-sponse and to study how various sub-domains of thispromoter interact to regulate VspB transcription.

In this study, we further define the VspB promoterdomains, which mediate responses to phosphate andsugar using transient expression assays in tobaccoprotoplasts. Gel shift and deoxyribonuclease (DNase)-Ifootprinting assays were used to map protein-bindingsites in each of the promoter domains to obtain a moredetailed understanding of the architecture of this pro-moter. Site-directed mutagenesis of a protein-bindingsite in the phosphate response domain confirmed theimportance of the sequence CATTAATTAG in trans-factor binding. Two soybean genes were identifiedencoding homeodomain (HD)-Leu zipper proteins(ZIPs) that bind to this sequence.

RESULTS

Sugar and Phosphate Response Domains in theSoybean VspB Promoter

The VspB promoter region from 2585 to 2535 waspreviously shown to mediate responses to JA intransgenic plants (Mason et al., 1993), and the regionfrom 2536 to 2401 was shown to mediate responsesto sugars and phosphate in protoplasts (Sadka et al.,1994). To further analyze the architecture of the VspBpromoter, several additional constructs, shown inFigure 1, were made by inserting PCR-amplifiedDNA fragments of the VspB promoter in front of atruncated (288) 35S-CaMV promoter of pBI232.These constructs were introduced into tobacco leafprotoplasts by electroporation and after varioustreatments, GUS activity was assayed.

Protoplast assays were selected for this analysisbecause experimental results from each assay repre-sent an average response from many protoplasts. Incontrast, results from transgenic plants vary depend-ing on site of insertion. We note, however, that al-though the general responses observed with differentconstructs were reproducible, the magnitude of theresponses varied in different protoplast preparationsfor unknown reasons. This variation was observedeven through we used a second construct containingthe CAT reporter gene driven by the 35S-CaMV pro-moter as an internal standard (Fromm et al., 1986),and expressed results as the relative ratio of GUS/CAT activity for each treatment. Therefore, proto-

Figure 1. VspB promoter constructs and transient expression assays. The expression vector pBI232 was constructed byreplacing the 800-bp CaMV 35S-promoter of pBI221 (CLONTECH Laboratories, Palo Alto, CA) with a minimal (288)35S-CaMV promoter followed by the tobacco etch virus 59-untranslated leader sequence. Various regions of the VspBpromoter were inserted upstream of the minimal (288) 35S-promoter in pBI232 to make the constructs used in transientexpression assays (shown at the left). Protoplasts were cotransfected with a CaMV promoter-chloramphenical acetyltransferase (CAT) construct as an internal standard. The relative activity of each construct was measured after treatment ofprotoplasts for 24 h with 0.2 M Suc and 0.3 mM phosphate (b-glucuronidase [GUS]/CAT activity). The influence of Suc (60.2M Suc) and phosphate (61.25 mM phosphate) are shown at the right and expressed as fold induction.

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plast assays were useful for locating responsive do-mains in the VspB promoter but did not providequantitative information about the relative activity ofvarious promoter constructs.

The results from one set of protoplast experimentsare shown in Figure 1 to document the responsive-ness of each construct to various treatments. VspBconstructs containing the JA response domain (2580to 2535) were much more active than constructs thatlacked this part of the VspB promoter (Fig. 1,pBI232G1 versus other constructs). Constructs onlycontaining the 50-bp JA response domain fused to theminimal (288) 35S-promoter also showed high tran-scription activity in protoplasts (data not shown). Inboth cases, promoter activity was not increased fur-ther by addition of JA, suggesting that the process ofprotoplast preparation fully activated a pathway thatstimulates transcription from this domain of the VspBpromoter. It is unfortunate that this circumstancelimited the value of further analysis of the JA re-sponse domain in protoplasts.

The 2536 to 2401 portion of the VspB promoteractivates transcription from the truncated (288) 35S-promoter and this activity is responsive to sugarsand phosphate (Fig. 1, pBI232-1 versus pBI232). Sucstimulated promoter activity in the presence or ab-sence of phosphate, and phosphate repressed pro-moter activity in the presence or absence of Suc (Fig.1, data from the low-phosphate and high-Suc treat-ments are shown). This region of the VspB promoterwas further divided into two sub-domains (2536 to2484 and 2486 to 2427) and each region was fusedto the truncated (288) 35S-promoter (pBI232-11 andpBI232-5). Each sub-domain stimulated transcriptionover the basal promoter indicating that sequences ineach domain are able to activate transcription. Con-structs containing the upstream domain (2536 to2484) were not responsive to Suc, but activity wasrepressed by addition of phosphate to the medium(Fig. 1, pBI232-11). In contrast, constructs containingthe downstream domain (2486 to 2427) were acti-vated by Suc but not modulated by changes in phos-phate (Fig. 1, pBI232-5). These and earlier results(Sadka et al., 1994) indicate that the VspB promotercontains three contiguous DNA domains that canmediate responses to JA (2585 to 2536), phosphate(2536 to 2484), and sugars (2486 to 2427).

Identification of Protein-Binding Sites in theVspB Promoter

The location of protein-binding sites in the VspBpromoter was investigated using a combination ofgel mobility shift assays and DNase-I footprintingassays. The gel-shift assays shown in Figure 2 werecarried out using the 2611 to 2451 portion of theVspB promoter as a radiolabeled probe (Fig. 2A, p26).

This DNA fragment includes the JA response do-main, the phosphate response domain, and a portionof the DNA domain that mediates responses to sug-

Figure 2. Gel mobility shift competition assays. A, Portions of theVspB promoter used for gel-shift competition assays. Numbers indi-cate the nucleotide positions relative to the VspB transcriptionalinitiation site. DNA fragment p26 was radiolabeled and incubatedwith soybean (B) or pea (Pisum sativum L. var. Little Marvel; C)nuclear extracts. Binding reactions were performed in the absence(lane 1) or presence of 50 and 100 mass excess of p12 (lanes 2 and3), p26 (lanes 4 and 5), or p44 (lanes 6 and 7). The major DNAprotein complexes are labeled as A through D.

Homeodomain Proteins Bind to the VspB Promoter

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ars. Addition of p26 to protein extracts from soybeannuclei resulted in the formation of four major DNAprotein complexes (Fig. 2B, lane 1, bands A–D). Com-

plex formation was largely insensitive to pH (rangingfrom 6.4–8.8) and binding was not affected by MeJA(data not shown). Binding specificity was tested by

Figure 3. DNase-I footprinting assays of theVspB promoter. The radiolabeled DNA fragmentp26 (2611 to 2451) was digested with DNase Iin the presence of soybean (A) and pea (B) nu-clear extracts. After digestion, DNA fragmentswere separated on a 5% (w/v) gel and the gelregions corresponding to the free probe (lanesmarked F) and the upper bands (lanes marked B)were eluted. The purified DNAs were run on asequencing gel along with the G1A sequencingreaction (lanes marked G1A). The boxes corre-spond to regions of DNA protected from diges-tion. Numbers indicate the nucleotide positionsrelative to the VspB transcriptional initiationsite. C, Summary of the DNase-I footprintingresults using soybean nuclear extracts. Linesabove and below the sequence mark the pro-tected regions of the upper and lower strand,respectively.

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addition of unlabeled competitor DNAs to the bind-ing mixture. The results in Figure 2B show that p26(2611 to 2451) could compete for formation of com-plexes A through D (lanes 4 and 5). CompetitorDNAs containing sequences from 2536 to 2401 (p12)eliminated complexes A and B but competed to alesser extent with formation of complexes C and D(Fig. 2B, lanes 2 and 3). This suggests that complexesC and D may involve protein binding to sequencesupstream of 2536 in p26. However, DNAs contain-ing the sequence from 2585 to 2535 (p44, JA re-sponse domain), did not compete for binding (Fig.2B, lanes 6 and 7). Moreover, when this region of theVspB promoter (p44) was radiolabeled and used ingel-shift assays, no specific complexes were observed(data not shown). This suggests that formation ofgel-shift complexes C and D may require sequencesin the JA response domain (2585 to 2536) plus in-teraction with protein factors or sequences locateddownstream.

Gel-shift assays were also carried out using nuclearextracts from pea leaves to see if factors in pea wouldbind to the soybean VspB promoter. As shown inFigure 2C, gel-shift assays with extracts from peanuclei were less complex than those obtained withsoybean extracts and revealed two major gel-shiftcomplexes that showed specific binding characteris-tics (Fig. 2C, complexes A and B). The reduction incomplexity in the pea extracts was due in part topre-incubation of the extracts with poly(dI-dC)zpoly(dI-dC) for 10 min before probe addition,rather than simultaneous mixing of all binding re-agents as done in Figure 2B. This change resulted inclearer gel-shift patterns presumably by reducingnonspecific binding. Therefore, this approach wasadopted in subsequent DNA-binding experiments.

DNase-I footprinting assays were used to furthercharacterize DNA-binding sites within the soybeanVspB promoter. The p26 DNA (2611 to 2451) wasradiolabeled and incubated with protein extractsfrom soybean or pea nuclei followed by controlleddigestion with DNase I. The partially digested com-plexes were subsequently separated on acrylamidegels, the shifted band with lower mobility was ex-cised (complex A in Fig. 2), and DNA fragments wereextracted and analyzed on sequencing gels. The re-sults in Figure 3 show that four regions between2611 and 2451 in the lower strand and three regionsin the upper strand were protected from DNase-Idigestion by soybean nuclear extracts (Fig. 3A, BoxesI–IV). Similar protected regions were identified onthe upper strand of p26 using pea nuclear extracts(Fig. 3B). The footprinting results are summarized inFigure 3C. Box I was located in the JA responsedomain, Box II and Box III were both present in theDNA region that can mediate phosphate responses,and Box IV was located in the DNA domain thatmediates sugar responses.

Further Analysis of Protein Binding to Box II

DNase-I footprinting assays revealed protein bind-ing to Box II that is located immediately downstreamfrom a G-box sequence (CACGTG; Fig. 4A). G-boxsequences in numerous promoters are known to bindbasic ZIPs in higher plants (e.g. Donald et al., 1990;Schindler et al., 1992; Shen and Ho, 1995). To testwhether the G box influences protein binding to BoxII, and to further investigate binding to Box II, gel-shift assays were carried out using a 32-bp oligonu-cleotide that spans these sequences (Fig. 4A, GAT2B).In the absence of competitor DNA, radiolabeledGAT2B formed two major complexes with proteinsextracted from soybean nuclei (Fig. 4B, lane 1). Ad-dition of unlabeled GAT2B oligo to the binding reac-tion eliminated complex A but not B (Fig. 4B, lanes 2and 3). In a similar manner, a modified form of

Figure 4. Sequences involved in protein binding to Box II. A, OligoGAT2B corresponds to a 32-bp region of the VspB promoter thatincludes a G-box sequence and the Box-II sequence. Oligo AT wasderived from oligo GAT2B by mutation of sequences in the G box(stars correspond to altered bases). Oligo G was derived from GAT2Bthrough mutation of sequences in Box II. B, The 2611 to 2451region of the VspB promoter was radiolabeled and used with soybeannuclear extracts to carry out gel-shift assays. Binding reactions wereperformed in the absence (lane 1) or presence of 50 or 100 massexcess of oligonucleotides GAT2B (lanes 2 and 3), AT (lanes 4 and 5),and G (lanes 6 and 7). The major DNA protein complexes are labeledA and B.




GAT2B containing a mutated G box (labeled AT; seeFig. 4A) also effectively competed for complex-Abinding (Fig. 4B, lanes 4 and 5). In contrast, oligoscontaining sequence changes in Box II (Fig. 4A, la-beled G) were not as effective in binding competitionassays (Fig. 4B, lanes 6 and 7). These results indicatethat the sequence TTAATT in Box II plays a role inprotein binding and that the G-box sequence is notrequired for the interaction observed in gel-shiftassays.

HD-ZIPs Bind to Box II

Genes encoding proteins that interact with the2611 to 2451 portion of the VspB promoter wereidentified by screening a cDNA expression libraryfrom 10-d-old soybean seedlings (Vinson et al., 1988).Two of the clones isolated using this technique, Gm-Hdl56 and GmHdl57, showed 40% sequence similarityoverall, and both encoded HD-ZIPs (GenBank acces-sion nos. AF184277 and AF184278). The sequence ofGmHdl56 is shown in Figure 5A. The proteins en-coded by GmHdl56 and GmHdl57 contain anN-terminal HD, followed by a ZIP, and a variablelength C-terminal sequence (Fig. 5A; HD is boxed,Leu in the ZIP domain are circled). The C-terminaldomains of both proteins are negatively charged andcontain large numbers of Ser and Thr residues (36 outof 151 residues for GmHDL56 and 32 out of 163residues for GmHDL57). Each of the proteins con-tained numerous potential recognition sequences forPKC, CK2, cAMP-dependent protein kinases, andTyr kinases.

A comparison of the amino acid sequences of Gm-HDL56, GmHDL57, and HDs from other organismsis shown in Figure 5B. The six plant HDs used in thiscomparison share over 70% sequence identity in pair-wise comparison and 30% identity when comparedwith the HDs of human HEX and Drosophila Antp.GmHDL56 and GmHDL57 contain 11 out of the 12amino acids conserved in most HDs (Fig. 5B, markedwith stars; Scott et al., 1989). The ZIP present inGmHDL56 and GmHDL57 is characteristic of manyplant HD proteins (Ruberti et al., 1991; Shena andDavis, 1994). The ZIP domain of the plant HD-ZIPproteins consists of up to six heptad repeats, with aLeu residue at every seventh position (Fig. 5).

DNase-I footprinting assays were performed to de-termine if GmHDL56 had a specific binding site onthe 2611 to 2451 portion of the VspB promoter. A63-His-tagged form of GmHDL56 was prepared andpurified for footprinting assays to eliminate back-ground from other DNA-binding proteins that mightbe present in bacterial extracts. The results shown inFigure 6 demonstrate that GmHDL56 binds to Box IIin the VspB promoter. The DNase-I footprinting re-sults obtained with GmHDL56 were similar to thoseresults obtained in Figure 3 using extracts of soybeannuclei.

Figure 5. Soybean HD Leu zipper sequence analysis. A, The nucle-otide and deduced amino acid sequences of GmHdl56. Boxes de-limit the HD. The periodic Leu forming the ZIP are circled. B,Comparison of HD and ZIP sequences from different organisms. Adash indicates identity with GmHDL56. The three predicteda-helices, turn, and HD-binding domain are indicated. The 12 in-variant and highly conserved amino acids in the HDs are marked bystars. Within the ZIP domain, dots denote heptad Leu repeats andconserved amino acid positions (Ruberti et al., 1991). The chargedamino acid residues at positions a and d of the GmHDL ZIPs are inbold letters. CHB6 and CHB3 (Kawahara et al., 1995), Athb-6 andAthb-5 (Soderman et al., 1994), HEX (Bedford et al., 1993), and Antp(McGinnis et al., 1984) were selected for this comparison.

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Figure 7 shows gel-shift assays using extracts of E.coli cells expressing GmHDL56 or GmHDL57. In theabsence of competitor DNA, E. coli extracts contain-ing either GmHDL56 or GmHDL57 form two majorcomplexes with the 2611 to 2451 portion of the VspBpromoter (Figure 7, lanes 1 and 7). Unlabeled probeDNA (2611 to 2451) can compete with complex-Aformation in extracts containing GmHDL56, andcomplexes C and D in extracts containing GmHDL57(Figure 7, lanes 2 and 8). In contrast, a nonspecificchloroplast DNA probe did not compete significantlyfor binding (Fig. 7, competitor psbA, lanes 3 and 9).Oligos containing Box II and the G box and similaroligos with modified G-box sequences (GAT2B, AT)were able to compete for binding (Fig. 7, lanes 4, 5,10, and 11). In contrast, oligo G that contains muta-tions in Box II (TTAATT) did not compete for binding(Fig. 7, lanes 6 and 12). These results are similar tothose obtained with nuclear extracts (Fig. 4).

DISCUSSION

VspB expression is regulated during plant develop-ment and by wounding, water deficit, light, metabo-

lites such as phosphate, carbon, and nitrogen, and bythe plant regulators jasmonic acid and auxin (Masonand Mullet, 1990; Staswick, 1990; Mason et al., 1992;DeWald et al., 1994; Creelman and Mullet, 1997). Thisinvestigation focused on portions of the VspB pro-moter that respond to at least three primary agents:jasmonic acid, phosphate, and sugars. Previous stud-ies showed that modulation of VspB transcription byJA, sugars, and phosphate is mediated by an approx-imately 185-bp DNA domain located between 2585and 2401 in the VspB promoter. The results pre-

Figure 7. Gel mobility shift competition assays of GmHDL56/57from Escherichia coli. DH5a cell extracts that contain GmHDL56(lanes 1–6) and GmHDL57 (lanes 7–2) were incubated with theradiolabeled DNA fragment from 2611 to 2451 of the VspB pro-moter in the absence (lanes 1 and 7) or presence (lanes 2–6, 8–12) of100-fold mass excess of various competitor DNAs as indicated on topof the autoradiogram (p26, psbA, GAT2B, AT, and G).

Figure 6. DNase-I footprinting assays with purified GmHDL56. Theend-labeled upper or lower strand of the DNA fragment from 2611to 2451 of the VspB promoter was digested with DNase I in theabsence (lanes marked F) or presence (lanes marked B) of purified63-His tagged GmHDL56. Lanes marked G1A refer to Maxam-Gilbert sequencing reactions of the same DNA fragments. The pro-tected DNA sequences are boxed.




sented in this paper provide evidence that this pro-moter domain is composed of three contiguous re-gions that mediate responses to JA (stimulation),phosphate (repression), and sugars (stimulation).

JA Response Domain

Mason et al. (1993) previously demonstrated that a50-bp DNA region of the VspB promoter, locatedbetween 2585 and 2535, could mediate responses toJA in vivo when fused to a truncated (288) CaMV35S-promoter. In the current study using protoplasts,the JA response domain alone, or part of larger seg-ments of the VspB promoter, conferred high expres-sion on the truncated (288) 35S-CaMV promoter.However, constructs containing the JA response do-main were not responsive to JA in protoplasts. Thissuggests that the JA response pathway or anotherparallel pathway that acts through the JA responsedomain is fully activated during the preparation ofprotoplasts. It is also possible that JA cannot mediateresponses in protoplasts.

Previous comparisons of the JA response domainwith other promoters, and specifically to otherwound- and JA-responsive promoters, suggestedthat a C-rich sequence in the JA response domainmight help mediate JA responses (Ryder et al., 1984;Schulze-Lefert et al., 1989; Creelman et al., 1992). Inthe current study, DNase-I footprinting analysis us-ing the 2611 to 2451 promoter region provided pre-liminary evidence that proteins bind to the C-richsequence in a region of the VspB promoter labeledBox I (ACCCTAGAACCTTC). The evidence is con-sidered preliminary because footprints in this regionwere weak and only observed on one DNA strand. Inaddition, the JA response domain in isolation did notform stable sequence-specific gel-shift complexes,suggesting that binding to Box I may involve inter-action with factors that bind outside of this domain.We reported previously that fusion of the VspB JAresponse domain to 35S-promoters truncated to 246did not respond to JA, whereas fusion of this domainto 35S-promoters truncated to 288 activated tran-scription in the presence of JA 7-fold compared withthe basal promoter (Mason et al., 1993). The 288construct contains the as-1 cis element and bindsASF1 (Xiang et al., 1996). This complicates interpre-tation of the results because as-1 and other similarelements (nos-1) can mediate responses to severalhormones including JA (salicylic acid, auxin, etc.;Kim et al., 1993; Xiang et al., 1996). We conclude thatfactors binding to the truncated (288) 35S-promotertake the place of factors that help mediate JA re-sponses from the VspB promoter in vivo. Furtherstudies will be required to test if the Box-I sequenceis important for JA-modulated transcription in vivo,and to identify the endogenous sequences and trans-factors that help mediate JA-induced transcriptionfrom the VspB promoter.

A G-box motif, which is characterized by the dyadsequence CACGTG, is also located in the JA responsedomain of the VspB promoter and other JA-responsive promoters (Bell and Mullet, 1991; Creel-man et al., 1992; Kim et al., 1992; Mason et al., 1993).A large number of related basic ZIPs have been re-ported to bind to the G box and related sequences(i.e. Donald et al., 1990; Zhang et al., 1993; Hong etal., 1995; Lu et al., 1996). In potato (Solanum tubero-sum), the G-box motif in the PinII promoter wasrequired for JA-mediated expression (Kim et al.,1992). However, another study concluded that the Gbox was not required for JA-mediated responses(Lorbeth et al., 1992). In the current study, proteinbinding to the G box was not observed in DNase-Ifootprinting analysis. Therefore, additional in vivoanalysis will be required to determine if the G box isinvolved in mediating JA or other responses of theVspB promoter.

Sugar Response Domain

VspB transcription is activated by sugars and re-pressed by phosphate. The region of the VspB pro-moter mediating these responses was previously lo-calized between 2536 and 2401 (Sadka et al., 1994).In earlier work, it was not clear whether these me-tabolites acted through the same or different pro-moter elements. In this study, the DNA region from2536 to 2484 was found to mediate responses tophosphate but not Suc, whereas the region from2486 to 2427 was able to mediate responses to Suc,but not phosphate. Therefore, although addition ofsugars to plant cells can alter phosphate levelsthrough the formation of sugar phosphates, the twoeffectors mediate their responses through two differ-ent domains in the VspB promoter.

The sugar response domain of the VspB promoter(2486 to 2427) enhanced transcription from the basal(288) 35S-promoter in the presence of Suc. DNase-Ifootprinting assays of the region 2611 to 2451 re-vealed a protein-binding site, labeled Box IV, locatedin the sugar response domain (2474 to 2488). Box IVcontains the sequence GAAATAAATTG that, likeother sugar response elements, is AT rich (for review,see Smeekens and Rook, 1997). Although the regionfrom 2486 to 2427 can mediate responses to chang-ing sugar levels, other portions of the VspB promotermay also be involved in this response. For example,preliminary DNase-I footprinting assays suggest thatthere are additional AT-rich protein-binding sites im-mediately downstream of 2427 that may also beinvolved in this response (data not shown).

Phosphate Response Domain and the Role ofHD-ZIP Proteins

The 2536 to 2484 domain of the VspB promoterstimulated transcription from the truncated (288)

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35S-promoter in protoplasts suspended in low-phosphate concentrations. Therefore, this domain ofthe VspB promoter is able to activate transcription atlow phosphate, at least when combined with a trun-cated (288) 35S-promoter. At high phosphate con-centrations, transcription from this domain de-creased nearly to basal levels. Gel-shift and DNase-Ifootprinting assays revealed the presence of two ad-jacent protein-binding sites (Box II and Box III)within the phosphate response domain. Mutation ofthe sequence CATTAATTAG located in Box II re-duced protein binding to this domain in gel-shiftassays. Trans-factor binding to Box II may help me-diate inhibition of VspB transcription at high phos-phate. Analysis of mutations in Box II in the contextof an intact VspB promoter in transgenic plants willbe needed to better define the role of this sequence invivo.

Two soybean genes encoding HD-ZIPs were iden-tified by screening expression libraries with the 2611to 2451 portion of the VspB promoter. Gel-shift as-says showed that E. coli extracts containing Gm-HDL56 or GmHDL57 formed specific complexeswith the 2611 to 2451 portion of the VspB promoter.Moreover, these protein DNA complexes could becompeted with oligos that contain Box II, but not byoligos containing mutated Box-II sequences. DNase-Ifootprinting assays using purified soybean Gm-HDL56 showed that this protein can protect Box II invitro. This is consistent with the presence of HD-binding sites in Box II (core sites contain TAAT;Wolberger, 1996). The organization of TAAT se-quences in Box II (TAATTAAT) is similar to thebinding site for the HD protein even-skipped, wheretwo HD proteins bind on opposite sides of the DNA(Wolberger, 1996). It is more important that the VspBBox-II sequence, CATTAATTAG, is similar to se-quences previously shown to bind HD-ZIP proteins(Sessa et al., 1993; Meijer et al., 1997; Sessa et al.,1998). Studies of this class of transcription factors inArabidopsis revealed the existence of four differentgroups of HD-ZIP proteins that can be distinguishedin part based on their binding site specificity (Sessa etal., 1994). Box II is similar to sequences that bind tomembers of the first class of these proteins [HD-ZIPI; binds to CAAT(A/T) ATTG]. One member of thisclass of genes is activated by abscisic acid and waterdeficit (Soderman et al., 1996) and ectopic expressionof Athb-1 alters leaf cell fate (Aoyama et al., 1995). Itis interesting that a member of the second class ofHD-ZIP proteins, ATHB-2, functions as a negativeregulator of gene expression and is involved in me-diating specific auxin responses (Steindler et al.,1999).

A rice (Oryza sativa) HD-ZIP protein of the HD-ZIPII class that binds to the sequence CAAT(G/C) ATTGalso functions as a negative regulator of transcription(Meijer et al., 1997). In a similar manner, in this paperwe report that HD-ZIP proteins bind to a domain of

the VspB promoter that mediates reduction in tran-scription when phosphate levels are high. Althoughgel-shift and DNase-I footprinting assays demon-strate that GmHDL56/57 can bind to the VspB pro-moter in a sequence-specific manner, Southern anal-ysis shows that like other genomes (Shena and Davis,1994), soybean encodes numerous HD-ZIP pro-teins (data not shown). Therefore, it is not clear ifGmHDL56/57 are the only HD-ZIPs that can interactwith the VspB promoter in vivo. Northern analysisdid not clarify this question because mRNA hybrid-izing to these genes is present in most tissues anddevelopmental stages, and RNA abundance showsminimal change in response to MeJA and phosphatetreatments (data not shown). Moreover, it is possiblethat other HD-proteins bind to Box II or Box IIIbecause both sites contain the core TAATNN se-quences required to bind these proteins. Systematicexamination of VspB promoter activity in plantsoverexpressing each HD-ZIP protein and plants withmutations in the genes encoding each HD-ZIP pro-tein in a plant like Arabidopsis will be required toidentify the specific HD-ZIP proteins involved inregulation. Even so, the identification of this class ofproteins as likely candidates involved in phosphate-mediated regulation of the VspB promoter will helpfocus this analysis.

MATERIALS AND METHODS

Preparation of Nuclear Extracts

Soybean (Glycine max L. Merr. cv Williams 82) plantswere grown in a growth chamber as previously described(Mason et al., 1992) until the seventh trifoliate was about 1cm long. The third and fourth trifoliates were excised un-der water. Individual leaflets were incubated in the lightwith their cut ends in 10 mm 6 MeJA for 18 h. Leaf nuclearextracts were prepared as described by Jacobsen et al.(1990). Pea (Pisum sativum L. var Little Marvel) plants weregrown in constant light at room temperature for 10 d.Approximately 1 kg of shoots was harvested for prepara-tion of nuclear extracts (Green et al., 1989).

Preparation of Competitor DNA Fragments and Probes

DNA fragments of the VspB promoter (p26 [2611 to2451], p12 [2536 to 2401], and p42 [four concatenatedcopies of 2585 to 2535]) were excised from vectors and gelpurified. Complementary oligonucleotides (GAT2B, G, andAT) were annealed to prepare competitor DNAs. The DNAfragment, p26, was 39-end labeled with the Klenow frag-ment of DNA polymerase I, gel purified, and used as aprobe in gel mobility shift and DNase-I footprinting assays.

Gel Mobility Shift Assays

Binding reactions (10 mL) contained 2.5 mg of poly(dI-dC)zpoly(dI-dC); 40 mm KCl; 20 mm HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.5;




0.1 mm EDTA; 5% (v/v) glycerol; 0.5 mm dithiothreitol; 10ng mL21 leupeptin; 10 ng mL21 antipain; and 2.5 mg ofsoybean nuclear extracts. Competitor DNA fragments wereincluded in the binding reactions as indicated in the figurelegends. For the binding reactions using pea nuclear ex-tracts, the amount of poly(dI-dC)zpoly(dI-dC) was adjustedto 10 mg. After 10 min of pre-incubation at room tempera-ture, 5 3 104 cpm of radiolabeled probe was added andincubation was continued for another 10 min. Afterward,the reactions were loaded onto a 5% (w/v) polyacrylamidegel (in 0.53 Tris-borate/EDTA) and electrophoresis carriedout at approximately 10 V cm21 in a cold room.

DNase-I Footprinting Assays

Binding reactions were scaled up 15 times from the gelmobility shift assay with the following modifications. Thebinding reaction also included 10 mm MgCl2 and 1 mmCaCl2. After the binding reaction, 0.15 units of DNase I(Boehringer Mannheim, Indianapolis) were added to thebinding mixture and incubation continued for another 1.5min. The reaction mixture was loaded onto a 5% (w/v)polyacrylamide gel. After electrophoresis, the gel wastransferred to one chromatography paper (Whatman,Clifton, NJ) and exposed to a film. The exposed film wasaligned with the gel and the low mobility bands and thebands corresponding to the free probe were cut out andtransferred to a tube (Eppendorf Scientific, Westbury, NY).The gel slices were soaked in 1 mL of elution buffer (50 mmTris-HCl [pH 8.0], 1% [w/v] SDS, 2 mm EDTA, and 10 ngmL21 tRNA). After boiling for 5 min, the paper shreds wereremoved. Another 300 mL of elution buffer was added. Thetube was incubated at 55°C overnight. The supernatant wasextracted once with phenol and once with CCl4. DNA wasprecipitated with ethanol. Chemical sequencing reactionswere performed as described by Ausubel et al. (1991). DNAsamples were denatured and loaded onto an 8% (w/v)sequencing gel.

Plasmid Construction and Site-Directed Mutagenesis

The vector plasmid pBI232 was constructed by replacingthe 800-bp CaMV 35S promoter of pBI221 (CLONTECH)with a minimal (288) 35S-CaMV promoter followed by thetobacco etch virus 59-non-translated leader sequence (Car-rington and Freed, 1990). VspB promoter fragments wereprepared by PCR. The PCR-amplified promoter fragmentswere designed with flanking restriction endonuclease rec-ognition sequences and inserted in pBI232. Site-directedmutagenesis was performed as described by Deng andNickoloff (1992).

Protoplast Isolation and Transient Expression Assays

Protoplasts were obtained from leaves of 4- to 6-week-old tobacco (Nicotiana tabacum cv Samsun) essentially asdescribed by Sadka et al.(1994). Protoplasts were trans-fected with GUS and CAT constructs (Fromm et al., 1986)

following the gene pulser electroprotocols (Bio-Rad, Rich-mond, CA) for Nicotiana plumbaginofolia. After electropora-tion, protoplasts were transferred to medium containingosmoticum (mannitol) with or without 0.2 m Suc (at con-stant total molarity of Suc plus mannitol), in the presenceor absence of 1.25 mm phosphate (pH 7.0), and incubatedfor 24 h in constant light (150 mE m22 sec21) at 23°C. Theprotoplasts were divided into two parts. One part wasassayed for GUS activity (Mason et al., 1993). The otherpart was assayed for CAT activity (Seed and Sheen, 1988).

Construction of a Soybean cDNA ExpressionLibrary in lZAP

Soybean plants were grown in growth chambers as pre-viously described (Mason and Mullet, 1992). Total RNAwas prepared from 10-d-old soybean seedlings using themethod described by Chirgwin et al. (1979) and Glisin et al.(1974). Polyadenylated mRNA was isolated from totalRNA using an mRNA isolation system (PolyAtract, Pro-mega, Madison, WI). cDNA was synthesized using theZAP-cDNA synthesis kit (Stratagene, La Jolla, CA). ThecDNA was ligated into the Uni-ZAP XR vector and packedin vitro using Gigapack II Gold packaging extracts (Strat-agene). The primary phage library contained 6 3 106 re-combinant plaques.

Screening of the cDNA Expression Library

The 32P-radiolabeled DNA fragment from 2611 to 2451of the VspB promoter was prepared by PCR. The PCRmixture (100 mL) contained 10 ng of the template plasmid,13 reaction buffer, 50 mm deoxynucleotides, 50 mCi of[a-32P]dCTP, 50 mCi of [a-32P]dTTP, and 4 units of TaqDNA polymerase (Promega). The amplified probe was pu-rified by passing through the G-50 column twice. ThecDNA expression library was screened for proteins, whichspecifically interacted with the probe as described by Vin-son et al. (1988).

DNA Sequence Analysis

Sequence data was generated by using a DNA sequencer(ABI 373a, Applied Biosystems Inc) with samples preparedwith the ABI Dye Terminator Cycle Sequencing ReadyReaction Kit (Perkin-Elmer, Foster City, CA).

DNase-I Footprinting Assays with Purified GmHDL56

The coding sequence of the GmHdl56 gene was amplifiedand cloned into the BamHI and KpnI site of pQE-30 (QiagenUSA, Valencia, CA). Protein expression and purificationwere performed using the Qiaexpress system (Qiagen). Thefusion protein was isolated under denaturing conditionsusing a spin column (Ni-NTA, Qiagen). Binding reactions(60 mL) contained 6 mg of poly(dI-dC)zpoly(dI-dC), 40 mmKCl, 20 mm HEPES (pH 7.5), 0.1 mm EDTA, 5% (v/v)glycerol, 0.5 mm dithiothreitol, 10 mm MgCl2, and 1 mm

Tang et al.



CaCl2. The reactions were started by the addition of 4 mg ofpurified 63-His-tagged GmHDL56, and incubated for 10min at room temperature. DNase I (0.15 units; BoehringerMannheim) was added to the binding reactions and incu-bation continued for 1.5 min. The reactions were stoppedby phenol extraction. Chemical sequencing reactions wereperformed as described by Maxam and Gilbert (1980).DNA samples were denatured and loaded onto an 8%(w/v) sequencing gel.

Received June 28, 2000; returned for revision August 29,2000; accepted October 7, 2000.

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