lnteraction of a developmentally regulated dna … plant cell, vol. 1, 1025-1 034, october 1989 o...

The Plant Cell, Vol. 1 , 1025-1 034, October 1989 O 1989 American Society of Plant Physiologists

lnteraction of a Developmentally Regulated DNA-Binding Factor with Sites Flanking Two Different Fruit-Ripening Genes from Tomato

Sabine Cordes, Jill Deikman, Linda J. Margossian, and Robert L. Fischer’

Department of Plant Biology, University of California, Berkeley, California 94720

To investigate mechanisms that control fruit development, we have begun experiments to identify proteins that control gene expression during tomato fruit ripening. We focused on the regulation of two different genes, E4 and E8, whose transcription is coordinately activated at the onset of fruit ripening. We report here that a DNA-binding protein specifically reacts with similar sequences flanking the E4 and E8 genes. The E4 binding site is at position -34 to -18 and, therefore, overlaps the region (TATA box) that in many eukaryotic genes serves to determine the efficiency and initiation site of transcription. In contrast, the E8 binding site is distal, located at -936 to -920 relative to the start of E8 gene transcription. Gel electrophoresis mobility retardation experiments indicate that the DNA binding activity that interacts with these two sites increases at the onset of fruit ripening. Taken together, these results suggest that this DNA-binding protein may function to coordinate E4 and E8 gene expression during fruit ripening.

INTRODUCTION

At the onset of tomato fruit ripening, many changes in metabolism occur, including increased respiration and ethylene production, chlorophyll degradation, elevated carotenoid synthesis, production of essential oils and flavor components, and increased activity of cell wall-degrading enzymes (for review see Brady, McGlasson, and Speirs, 1987). Coincident with the promotion of these metabolic pathways is the activation of gene expression. Analysis of pericarp protein extracts revealed the appearance of newly synthesized polypeptides at the onset of ripening (Biggs, Harriman, and Handa, 1986), and mRNAs that increase in concentration during tomato fruit ripening have been identified and cloned (Grierson, 1985; Mansson, Hsu, and Stalker, 1985; DellaPenna, Alexander, and Bennett, 1986; Lincoln et al., 1987). These results support the model that fruit ripening is controlled, at least in part, by the activation of gene expression resulting in the production of enzymes that catalyze diverse ripening-related processes (Brady, McGlasson, and Speirs, 1987).

To understand how gene expression is regulated during fruit ripening, we have studied two different fruit-ripening genes of unknown function, designated E4 and E8. We have found that the E4 and E8 genes are expressed similarly during plant development (Lincoln et al., 1987; Lincoln and Fischer, 1988a, 1988b; DellaPenna et al., 1989). First, both the E4 and E8 mRNAs are abundant in

’ To whom correspondence should be addressed.

ripening fruit and are not detected in leaf, root, stem, or unripe fruit. Second, both E4 and E8 gene expression is activated by ethylene, a hormone that plays an important role in controlling fruit ripening (Rhodes, 1980; Biale and Young, 1981 ; Yang, 1985). Third, the rin (ripening inhibited) mutation that blocks many aspects of ripening, including softening, ethylene production, and color development (Tigchelaar, McGlasson, and Buescher, 1978; Giovannoni et al., 1989), reduces the concentration of E4 and E8 mRNA by greater than 1 O-fold and threefold, respectively. Fourth, nuclear run-on transcription experiments indicate that the E4 and E8 genes are both regulated at the transcriptional level. However, it is important to note that certain differences in the patterns of E4 and E8 gene expression have also been detected. For example, expos- ing whole plants to exogenous ethylene results in E4 gene expression in many plant organs, whereas E8 gene expression is induced only in fruit (Lincoln and Fischer, 1988a). Taken together, these results suggest that there may be common factors involved in the control of E4 and E8 gene expression.

To investigate the mechanisms that coordinate gene expression during fruit ripening , we have initiated experiments to identify DNA sequences and nuclear factors that control E4 and E8 gene transcription. Previous results suggested that a developmentally regulated DNA-binding activity reacts with a restriction fragment flanking the E8 gene (Deikman and Fischer, 1988). We report here that

1026 The Plant Cell

th,e same DNA-binding activity reacts with DNA sequences flanking the coordinately expressed E4 gene. Methylation interference experiments indicate that the E4 binding site overlaps the TATA box region at position -34 to -18 relative to the start of transcription. In contrast, the E8 gene has a dista1 binding site located at -936 to -920 relative to the start of E8 gene transcription. The two sites share DNA sequence similarity. Gel electrophoresis mobility retardation experiments indicate that the DNA-binding activity that interacts with these two sites increases at the onset of fruit ripening. Taken together, these results suggest that the DNA-binding protein may function to coordinate E4 and E8 gene expression during fruit ripening.

RESULTS

E4 Gene Structure and Organization

Previously, we described the isolation of a cDNA clone, pE4, representing a 0.9-kb mRNA that accumulates during tomato fruit ripening and when unripe fruit are exposed to ethylene (Lincoln et al., 1987). To analyze the E4 gene, a bacteriophage X library of tomato leaf nuclear DNA was screened by hybridizing plaques with labeled pE4 DNA, a genomic clone was isolated, and a map of its restriction endonuclease sites was determined (Figure 1A). To investigate E4 gene organization further, labeled pE4 DNA was hybridized with restriction endonuclease-digested tomato genomic DNA that had been transferred to nitrocellulose. The molecular mass of the hybridizing genomic restriction fragments was consistent with the restriction map of the genomic clone shown in Figure 1A (data not shown), suggesting that there is one E4 gene present per haploid tomato genome.

To investigate the structure of the E4 gene, we determined the DNA sequence of both genomic and cDNA clones. The 3.8-kb EcoRl restriction fragment that hybridized with pE4 was subcloned (designated pE4RR3.8, Fig- ure 1 B), and the sequence of 2797 bp (EcoRI to Ncol site) was determined (Figure 1C). In addition, the 790-bp DNA sequence of pE4FL, a putative full-length E4 cDNA clone, was determined (Figure 1C and Methods). Comparison of the cDNA and genomic DNA sequences showed that the E4 gene is composed of two exons and one intron that displays consensus splice junction sequences (Brown, 1986). The 3'-end of the mature message is preceded by a consensus poly(A) addition signal. The longest open reading frame encodes a polypeptide of 25 kD, which agrees with the result of in vitro translation of E4-selected mRNA (Lincoln et al., 1987). The transcription start site was defined by S1 -nuclease protection and primer extension experiments (data not shown), and the location determined using these techniques coincided with the 5'-end of the cDNA clone pE4FL (Figure 1C). TATA and plant -80 consensus sequences (Heidecker and Messing, 1986)

I h b A. H

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ATTGGATCCGGATCTGGACAGCCCGGATCAGCCGGCTCTU;AGmGCCCMmGCTGCC~CTGC~GG~AGT L D P O L D S P D Q P G L E F A Q F A A G C F U G V

CCMTTTGGC~CCACAGGCTTGGAGGAGTAGTGMCAC~A~TTGGGTACTCTCA~MTGTCCATGACCCGM E L A F Q R V G G V V K T E V G Y S Q G N V H D P N

CTACMGCTTAmCCTCCGGAACMCCGMCATGCCGAffiCCATTCU;ATCCAG~GACCCGMTGTCTGCCCGTA Y K L I C S G T T E H A E A I R l Q F D P N V C P Y

L t c a t r L t a t g t g a t t a B ~ B t r a n a a a t t ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ g ~ ~ ~ ~ g ~ ~ g ~ ~ K g t t g ~ ~ g ~ ~ = K f f i T A A T G A T G T G G G G W D V G

AMGCMTACCGCTCAffiMTATATTACTATMTGATCCTCAGGCTCMCTGGCMGGGAGTCGTTAGMGCTMGCA K Q Y R S C I Y Y Y H D A O A Q L A R E 9 L E A K Q

GMGGM~ATGGATMGMMTTCTCACTGUUTTCTTCCTGCTMGAGA~TATACAGCTGMGAGTATCACCA K E F M D K K I V T E I L P A K R F Y R A E E Y H Q

GCMTATCTAGACMGGCTGCAGA~TTGTMGCAGTC~CTGCUUGGGCTGCMTGACCCMTMGGTGCTA Q Y L E K G G G R G C K Q S A A K C C N D P I R C Y

C G G T T G A C A C C A G A T C ~ G M T G T C A T A G C M C T A C ~ G M C T T G T T A G A C A ~ G C T G T C T T G C T T C ~ h M T G rPoly A

TTGMThMCATGACMTGATCTTATMCTACTTGCTCTCTTGGATCGMTMCTAGTTGTCGTAMGTATTCTCCT

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Figure 1. E4 Gene Structure.

(A) Restriction endonuclease site map of XE4-3. B, BamHI; Bg, Bglll; H, Hindlll; K, Kpnl; N, Ncol; P, Pstl; R, EcoRI; X, Xbal. (B) Strategy for determining DNA sequences. pE4RR3.8 was subcloned from hE4-3. Horizontal arrows indicate the extent and direction of DNA sequence determinations. Structure of E4 tran- script: filled box, untranslated exon sequences; open. translated exon sequences; cross-hatched, intron sequences. (C) DNA sequence of the E4 gene and predicted amino acid sequence of the polypeptide encoded by cDNA clone pE4FL. TATA, plant -80, and poly(A) addition consensus sequences are underlined. Lower-case DNA sequences represent introns. Start, transcription start as determined by SI protection and primer extension experiments and by pE4FL sequence data. Poly A, polyadenylation site. Amino acid sequences are in single-letter code.

Regulation of Gene Expression during Fruit Ripening 1027

are present 28 bp and 87 bp, respectively, 5' to thetranscription start site.

The E4 gene and predicted polypeptide sequences werecompared with sequences compiled in the National Insti-tutes of Health GenBank, the European Molecular BiologyLaboratory Nucleotide Sequence Library, and the NationalBiomedical Research Foundation Protein Identification Re-source. As shown in Figure 2, the homology search indi-cated a distant relationship with the 29-kD polypeptideencoded by the EIP28/29 gene from Drosophila melano-gaster (Cherbas et al., 1986; Schulz, Cherbas, and Cher-bas, 1986). Optimal alignment of 141 amino acids revealed37% amino acid sequence identity with only three gaps.In addition, 35% of the nonidentical amino acids repre-sented conservative substitutions. Expression of theEIP28/29 gene has been shown to be rapidly induced bythe steroid hormone ecdysone in Drosophila cell lines(Savakis, Koehler, and Cherbas, 1984). However, thefunction of the EIP28/29 and E4 genes remains to beelucidated.

In Vitro Binding of Nuclear Proteins from Tomato Fruitto E4 and E8 5'-Flanking Sequences

Previously, we isolated a cDNA clone, pE8, representinga 1.4-kb mRNA that accumulates during fruit ripening(Lincoln et al., 1987) and showed that multiple DNA-bindingfactors react in vitro with end-labeled restriction fragmentsflanking the E8 gene (Deikman and Fischer, 1988). Inparticular, an activity that increased at the onset of fruitripening reacted specifically with the E8-1 restriction frag-ment (see Figures 3A and 3B for restriction fragmentsflanking the E4 and E8 genes, respectively). However,adding molar excess quantities of unlabeled competitorE4-4 restriction fragment abolished the interaction be-tween the factor and end-labeled E8-1 DNA, suggestingthat the same DNA-binding activity reacted with the DNAsequences flanking the E4 and E8 genes.

31 AQFAAGCFWGVELAFQRVGGVVKTEVGYSQGNVHDPNYKLICSGTTE pE4I h I l l l h l ' : I I " N I I ' |: : hh : ••42 ATFGMGCFWGAESLYGATRGVLRTTVGYAGGSSDLPTYRKM——GD pE28

A.-400 -ZOO 0 +100

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Figure 3. In Vitro Binding of Nuclear Proteins to DNA SequencesFlanking the E4 Gene.(A) Schematic representation of the E4 5'-flanking region. Positionof restriction fragments is relative to the start of E4 gene tran-scription. E4-4, -403 to +65; E4-4a, -293 to -85; E4-4b, -85to +65. A, Alul; Av, Aval; Y, Rsal; S, Sstl; A44 and A6, endpointsof deletions. ~*, transcription initiation site.(B) Schematic representation of the E8 5'-flanking region. Positionof restriction fragments is relative to the start of E8 gene tran-scription. E8-1, -1088 to -682; E8-1a, -1088 to -886; E8-1b,-1027 to -886; D, Oral; Y, Rsal; X, Xbal; A2, endpoint of deletion.v~, transcription initiation site.(C) Influence of unlabeled homologous and heterologous DNAson DNA binding. Binding reactions were carried out with end-labeled E4-4b DNA using nuclear proteins isolated from 30% redtomato fruit. The specific unlabeled competitor DNA and the molarratio of unlabeled to labeled DNAs in the binding reaction areindicated above the lanes. N, control reaction without addition ofprotein; F, free DNA that is not bound to protein; B, DNA boundto factor(s).

78 HAEAIRIQFDPNVCPYSNLLSLFWSRHDPTTLNRQGNDVGKQYRSGI pE486 HTEVLEIDYDPTVISFKELLDLFWNNHE-YGLT---TPIKRQYASLI pE28

125 YYYNDAQAQLARESLEAKQKEFMDKKIVTEILPAKRFYRAEEYHQQY pE4129 LYHDEEQKQVAHASKLEEQERRAPEIITTEIASKENFYPAEAYHQKY pE28Figure 2. Alignment of the Amino Acid Sequences Predicted fromthe E4 mRNA and the EIP28/29 mRNA.

Amino acids 31 to 171 of E4 and 42 to 174 of EIP28/29 areshown. The symbol, |, indicates amino acid sequence identity; acolon (:) indicates a conservative substitution.

To localize the E4 DNA binding site, nuclear proteinswere extracted from ripening fruit and reacted with DNAsequences flanking the E4 gene. As described in Methods,all reactions included poly(dl-dC)-poly(dl-dC) duplex DNAto eliminate nonspecific protein-DNA interactions. Thepresence of DNA-binding factors was assayed by the DNAgel electrophoresis mobility retardation assay (Singh et al.,1986). As shown in Figure 3C, the mobility of the E4-4brestriction fragment was retarded when reacted with nu-clear extracts isolated from ripening tomato fruit. To ana-lyze the specificity of binding, molar excess quantities ofunlabeled competitor DNAs were added to the reactions.

1028 The Plant Cell

Whereas unlabeled fragment E4-4b eliminated the bindingof protein to end-labeled E4-4b DNA, addition of pUC18plasmid DNA, an adjacent restriction fragment E4-4a, or5'-flanking sequences (-463 to +61) of a light-regulatedtomato gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase failed to abolish binding (Figure3C). However, addition of molar excess quantities of un-labeled E8-1 prevented the formation of the more slowlymigrating complex with end-labeled E4-4b. These resultsare consistent with those observed in analogous bindingcompetition experiments using end-labeled E8-1 DNA(Deikman and Fischer, 1988), and suggest that a factorextracted from ripening tomato fruit nuclei forms a specificcomplex with the E4-4b and E8-1 restriction fragmentsflanking the E4 and E8 genes.

To identify specific guanine residues involved in thebinding reaction, methylation interference experimentswere performed. Methylation of guanine residues in themajor groove has been shown to disrupt the interaction offactors with DNA either by steric hindrance or by disruptingthe contact between specific guanine residues and thebinding factor (Otwinowski et al., 1988). To this end, theE4-4b and E8-1 restriction fragments were end-labeled,partially methylated, and reacted with nuclear protein ex-tracts isolated from ripening tomato fruit. The resultingDNA-protein complexes were isolated, cleaved at methyl-ated guanine sites, and fractionated by gel electrophoresisas described in Methods. As shown in Figure 4, analysisof restriction fragment E4-4b indicated that methylation ofguanine residues at position -21 of the noncoding strandand position —30 of the coding strand interfered withbinding. As shown in Figure 5, analysis of restriction frag-ment E8-1 revealed that methylation at position -923 ofthe noncoding strand and position -931 and -932 of thecoding strand interfered with binding. These results showthat an essential component of both binding sites is guan-ine residues separated by 8 bp on opposite DNA strands.

The DNA sequences surrounding the guanine residuesthat interfere with binding are identical at 11 of 17 positions(Figure 6). To determine whether these regions are suffi-cient for binding to nuclear factors, complementary oligo-nucleotides 27 residues in length and spanning a pre-sumptive binding site, E4 (-38 to -12) and E8 (-940 to—914), were chemically synthesized, annealed, and ligatedas described in Kadonaga and Tjian (1986). Each wasinserted into pUC119, and recombinant plasmids contain-ing three adjacent binding sites were identified. The sub-cloned binding sites were then isolated and end-labeled.As shown in Figure 7, gel electrophoresis mobility retar-dation experiments indicate that each binding site formeda DNA-protein complex when reacted with nuclear extractsisolated from ripening tomato fruit. This result indicatesthat the E4 and E8 oligonucleotides contain sufficientsequence information for binding factors in nuclear ex-tracts isolated from ripening tomato fruit.

To determine whether the E4 and E8 binding sites

compete for the same factors, molar excess quantities ofunlabeled oligonucleotides were added to the binding re-actions. As shown in Figure 7, addition of the unlabeledE4 binding site E4 (-38 to -12) abolished the interactionbetween factors and the end-labeled E8 binding site E8(—940 to —914). Conversely, adding unlabeled E8 bindingsite E8 (-940 to -914) eliminated binding to the end-labeled E4 binding site E4 (-38 to -12). In contrast,addition of excess unlabeled control oligonucleotide (seeMethods for DNA sequence of the control oligonucleotide)

A.CODINGG F B

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5 T T T C T A T A T A A A © A A A C A T A C AA A A © A T A T A T T T C T T T G T A T G T

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Figure 4. Identification of a Factor Binding Site Proximal to theE4 Gene Transcription Initiation Site.(A) Methylation interference analysis. The coding and noncodingstrands of double-stranded E4-4b DNA were labeled, partiallymethylated with dimethyl sulfate, and reacted with nuclear pro-teins isolated from 30% red tomato fruit. Free (F) and bound (B)DNA were separated by nondenaturing acrylamide gel electropho-resis, eluted, cleaved with piperidine, and analyzed by denaturingpolyacrylamide gel electrophoresis. G, partial chemical degrada-tion products of the probe cleaved at guanine residues; —»,methylated guanine residues that interfere with binding.(B) Location of DNA binding region relative to the start of E4 genetranscription. DNA sequence from —33 to -12 is shown. Methyl-ated guanine residues that interfere with binding are circled, v-.,E4 gene transcription start site.


A.CODING

G F BNONCODING

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see Methods for description of stages). To determinewhether binding and gene transcription coordinately in-crease, oligonucleotide probes representing the E4 and E8binding sites were reacted with nuclear protein isolatedfrom fruit at the mature green 1 and 4 stages. As shownin Figure 8, gel electrophoresis mobility shift experimentsindicate that DMA-binding activity was significantly less inunripe mature green stage 1 fruit than in ripening maturegreen stage 4 fruit. It is unlikely that the unripe maturegreen 1 stage fruit extract was inactivated or degradedduring isolation because equal levels of a DNA-bindingprotein activity were detected when mature green 1 and 4stage fruit extracts were reacted with 5'-flanking se-quences from another fruit-ripening gene, polygalacturon-ase (J.R. Montgomery and Ft.L Fischer, unpublished re-sults). We conclude from these results that, at the onsetof ripening, DNA-binding activity and gene transcription ofthe E4 and E8 genes coordinately increase.

B.5 ' A T T C C C A A C A A T A © A A A 3 '

T A A G © £ ) T T G T T A T C TTT

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Figure 5. Identification of a Factor Binding Site Flanking the E8Gene.

(A) Methylation interference analysis. The noncoding strand ofdouble-stranded E8-1a DNA and the coding strand of double-stranded E8-1b were labeled, partially methylated with dimethylsulfate, and reacted with nuclear proteins isolated from 30% redtomato fruit. Free (F) and bound (B) DNA were separated bynondenaturing acrylamide gel electrophoresis, eluted, cleavedwith piperidine, and analyzed by denaturing polyacrylamide gelelectrophoresis. G, partial chemical degradation products of theprobe cleaved at guanine residues; —>, methylated guanine resi-dues that interfere with binding.(B) Location of DNA binding region relative to the start of E8 genetranscription. DNA sequences from —936 to —920 are shown.Methylated guanine residues that interfere with binding are circled.~, E8 gene transcription start site.

did not abolish the interaction between the factor and end-labeled E4 or E8 binding sites. This result suggests thatthe E4 and E8 binding sites react with the same factor.

Regulation of DMA-Binding Activity during FruitDevelopment

E4 and E8 gene transcription is activated at the onset ofripening, between the mature green 1 and mature green 4stages of fruit development (Lincoln and Fischer, 1988a;

DISCUSSION

Comparing the E4 (Figure 1) and E8 (Deikman and Fischer,1988) genes reveals little DNA sequence identity in theiramino acid coding regions, as well as in their respective5'-flanking regions. However, transcription of these twogenes is coordinately activated at the onset of tomato fruitripening in response to ethylene (Lincoln et al., 1987;Lincoln and Fischer, 1988a). To elucidate mechanisms thatmay coordinate gene expression during fruit ripening, wehave begun to analyze DNA-binding proteins that specifi-cally react with the E4 and E8 genes.

E4 -03A ATTTCTATATAAAGAAA -018TAAAGATATATTTCTTTi i i i i i i i i i i

E8 -936 ATTCCCAACAATAGAAA -920TAAGGGTTGTTATCTTT

CONSENSUS ATT-C-A---A-AGAAATAA-G-T---T-TCTTT

Figure 6. Comparison of Binding Sites Flanking the E4 and E8Genes.

DNA sequences are numbered relative to the transcription startof the E4 and E8 genes. Underlined G represents guanine residuesthat interfere with binding when methylated (see Figures 4 and 5and text). Overlined sequence represents putative E4 gene TATAbox. Vertical lines, DNA sequences identical in E4 and E8; -, DNAsequences not identical in E4 and E8; », DNA sequences identicalin E4, E8, and C (control) oligonucleotide.

1030 The Plant Cell

E4 C EB C E4 C E8 CN 0 10 30100 100 N 0 10 30100 10O N 0 10 301OO 1OO N 0 10 30 100100

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Figure 7. In Vitro Interaction of Factors with OligonucleotideBinding Sites.

DNA sequence and preparation of oligonucleotides are describedin Methods. Binding reactions were carried out with nuclearproteins isolated from 50% red tomato fruit. End-labeled oligo-nucleotides are indicated below the lanes. The specific unlabeledcompetitor Oligonucleotide and the molar ratio of unlabeled tolabeled DMAs in the binding reaction are indicated above thelanes. E4, E4 (-38 to -12) Oligonucleotide; E8, E8 (-940 to-914) Oligonucleotide; C, control Oligonucleotide; N, control re-action without addition of protein; 0, no unlabeled Oligonucleotidecompetitor DNA added; F, free DNA that is not bound to protein;B, DNA bound to factor(s).

Delineation of Shared Binding Sites

binding of this factor to the E4-4b and E8-1 restrictionfragments occurs only at the sites delineated by the meth-ylation interference experiments.

Common Binding Sites Have Similar DNA Sequences

Comparison of the E4 and E8 binding sites revealed sev-eral conserved features. First, the binding sites share DNAsequence identity at 11 of 17 positions, with similar DNAsequences flanking a nonconserved AT-rich core. Second,methylation of guanine residues on opposite DNA strandsin each binding site interferes with binding. Because theyare separated by 8 bp, the two guanines are on the sameface of the DNA helix and within one helical turn of eachother in each binding site.

That these conserved features are sufficient for bindingof the same factor was shown by competition experiments(Figure 7). Addition of a molar excess of the unlabeled E8binding site abolished the interaction with the factor andthe end-labeled E4 binding site, whereas adding unlabeledE4 binding site interfered with factor binding to end-labeledE8 binding site. Taken together, these results indicate thatthe chemically synthesized E4 and E8 binding sites reactwith the same factor(s) present in ripening tomato nuclei.The factor did not bind to a control Oligonucleotide (Figure

Ripening tomato fruit nuclei contain several DNA-bindingactivities that interact in vitro with sites flanking the E4 andE8 genes. Some of these activities are unique to the E4gene (data not shown) and to the E8 gene (Deikman andFischer, 1988). However, binding experiments in the pres-ence of unlabeled competitor DNAs indicated that a DNA-binding protein reacts with restriction fragments E4-4b andE8-1 flanking the E4 and E8 genes, respectively (Figure3C; Deikman and Fischer, 1988). The specificity of theinteraction was demonstrated in control experiments,where the factor was shown not to bind with pUC118plasmid DNA, 5'-flanking sequences of the polygalactu-ronase fruit-ripening gene, or 5'-flanking sequences of thelight-regulated tomato gene encoding the small subunit ofribulose-1,5-bisphosphate carboxylase, (Figure 3C; Deik-man and Fischer, 1988; J.R.Montgomery and R.L. Fischer,unpublished results).

Methylation interference experiments localized bindingto sites flanking the E4 (Figure 4) and E8 (Figure 5) genes,respectively, that share DNA sequence similarity (Figure6). Several lines of evidence indicate that these are thecorrect binding sites. First, the factor binds directly tochemically synthesized oligonucleotides spanning the re-gions delineated by the methylation interference experi-ments (Figures 7 and 8). Second, addition of a molarexcess of chemically synthesized unlabeled E4 binding siteE4 (-38 to -12) abolished the interaction between thefactor and the end-labeled restriction fragments E4-4b andE8-1 (data not shown). These results suggest that the

Figure 8. Regulation of a DNA-Binding Activity during FruitDevelopment.

DNA sequence and preparation of oligonucleotides are describedin Methods. End-labeled oligonucleotides used in binding reac-tions are indicated below the lanes. E4, E4 (-38 to -12) Oligo-nucleotide; E8, E8 (-940 to -914) Oligonucleotide; F, free DNAthat is not bound to protein; B, DNA bound to factor(s). Lane N,control reaction without addition of protein. Nuclear proteins iso-lated from the following tomato fruit cultivars at the indicatedstage of development: lane 1, mature green stage 1 fruit; lane 2,mature green stage 4 fruit.


7) containing 6 of the 11 nucleotides conserved in the E4 and E8 sites, including the guanine residues separated by 8 bp (Figure 6). This result suggests that binding involves more than the interaction between the factor and the guanine residues in the configuration described above, and that the additional conserved DNA sequences in the E4 and E8 sites are also important for binding site recognition.

Placement of E4 and E8 Binding Sites 1s not Consewed

The position of binding sites relative to the start of E4 and E8 gene transcription differs significantly. The E8 site is far upstream from the E8 transcription initiation site, at position -936 to -920. Whether the factor binding to the E8 site regulates E8 gene transcription is unknown. How- ever, its distal placement, and the positive correlation between factor binding and E8 gene transcription (Figure 8; Deikman and Fischer, 1988), are consistent with the hypothesis that the E8 site is an enhancer-like region for tíigh-leve1 gene expression.

In contrast, the E4 site is at position -34 to -18 and spans the presumptive TATA box region (-29 to -23; Joshi, 1987). Studies in vitro (Grosveld et al., 1981; Hu and Manley, 1981; Mathis and Chambon, 1981; Sasylyk and Chambon, 1981; Zarucki-Schulz et al., 1982; Concino et al., 1984) and in vivo (Benoist and Chambon, 1981; Grosveld et al., 1982; McKnight and Kingsbury, 1982; Dierks et al., 1983; Charnay, Mellon, and Maniatis, 1985) studies have shown that for many genes, although not all, the TATA motif is critical for both promoter activity and for determining the precise site of transcription initiation. The TFllD protein (also known as BTFl and DB) interacts specifically with the TATA motif. In conjunction with two other constitutive factors (TFIIB, TFIIE) and proteins that bind to specific distal enhancer sequences, it directs the initiation of transcription approximately 30 bp downstream (Sawadogo and Roeder, 1985; Buratowski et al., 1988; Horikoshi et al., 1988a, 1988b; Nakajima, Horikoshi, and Roeder, 1988). However, severa1 lines of evidence suggest that the factor reacting with the E4 TATA region (-34 to -18) is not a ubiquitous TATA-binding factor. First, we do not detect this DNA-binding activity in unripe mature green stage 1 fruit (Figure 8), when presumably many thousands of genes (Goldberg, 1986) are undergoing TFIID-mediated transcription. Second, the factor we observe binding to the E4 TATA region does not react with the TATA region of other tomato fruit-ripening genes, e.g., E8 and polygalacturonase, or the light-regulated gene encoding the small subunit of ribulose-1 ,Bbisphosphate carboxylase (Deik- man and Fischer, 1988; data not shown). In this regard, it is important to note that most of the DNA sequences that are conserved between the E4 and E8 sites flank the sequence TATATAA (Figure 6), suggesting that the TATA motif may not play an important role in the in vitro binding observed.

Recent studies suggest that DNA-binding proteins that react with distal sites can functionally replace proximal TATA-binding factors such as TFIID. In yeast the GCN4 protein stimulates transcription of amino acid biosynthetic genes by binding to enhancer sequences upstream from TATA elements (Hope and Struhl, 1985; Arndt and Fink, 1986). However, it has recently been shown that replacing a TATA element with a GCN4 binding site permits GCN4- mediated gene transcription (Chen and Struhl, 1989). In addition, a naturally occurring GCN4-responsive gene, TRP3, has a GCN4 binding site instead of TATA sequences (Aebi et al., 1984; Zalkin et al., 1984). These results suggest that DNA-binding proteins that act at distal sites can in some cases also functionally interact with the RNA polymerase II transcription complex at a proximal site, approximately 30 bp upstream from the start of transcription. We speculate that, in an analogous fashion, factor binding to both the distal E8 site (-936 to -920) and to the proximal E4 TATA region (-34 to -18) might play a role in the coordinate activation of E4 and E8 gene transcription at the onset of fruit ripening. In this regard it is interesting to note that gel retardation and footprinting experiments suggest that the factor may also react with a more distal E4 binding site at position -749 to -728 relative to the start of E4 gene transcription (data not shown) that has DNA sequence similarity with the binding sites shown in Figure 6. However, competition experiments suggest that another class of factors uniquely binds the distal E4 site as well (data not shown). Experiments designed to test the significance of factor binding in vivo are in progress that involve mutagenesis of binding sites, followed by analysis of promoter function in transgenic tomato plants.

METHODS

Plant Material

Tomato plants were grown under standard greenhouse condi- tions. For studies on the regulation of DNA-binding activity during fruit development, nuclear proteins were isolated from tornato fruit (Lycopersicon esculentum cv VFNT Cherry). Fruit maturity stage and ethylene evolution rates were determined as described previously (Lincoln et al., 1987). Mature green stage 1 fruit were full size, and evolved ethylene at a low leve1 (0.6 rf: 0.2 nL/g/hr). In mature green stage 4 fruit carotenoid pigrnents were observed in the interior of the fruit, and elevated ethylene levels were observed (3.5 rf: 1 .O nL/g/hr).

lsolation of Clones

A library of tomato (L. esculentum cv VFNT Cherry) genomic DNA in the Charon 35 vector was screened by plaque hybridization with labeled pE4 to obtain a genomic clone, XE4-3, containing an E4 gene. Endpoints for subclones were generated by digesting

1032 The Plant Cell

DNA with either restriction endonucleases or with exonuclease 111 (Henikoff, 1984). The name of each subclone indicates its flanking restriction endonuclease or exonuclease 111 site and molecular mass. A cDNA library enriched for full-length cDNA clones of tomato ripe fruit mRNAs (DellaPenna, Alexander, and Bennett, 1986) was screened by colony hybridization with labeled pE4 to obtain a full-length E4 cDNA clone, pE4FL.

Analysis of Nucleic Acids

The 3.8-kb EcoRl restriction fragment from XE4-3 that spans the E4 gene was subcloned in both orientations into pUC118 to enable both sense and antisense single-strand template preparation (Vieira and Messing, 1987). Deletions were generated using exonuclease III (Henikoff, 1984). Nucleotide sequences were determined using the dideoxy chain-termination method (Sanger, Nick- len, and Coulson, 1977). DNA sequence analysis and searches of the NIH GenBank, EMBL Nucleotide Sequence Library, and NBRF Protein ldentification Resource were performed using the Bionet National Computer Resource for Molecular Biology. S1 -nuclease protection assays and primer extension analysis were performed as described in Deikman and Fischer (1988).

Preparation of Nuclei and Nuclear Extracts

Nuclei were isolated as described by Walling, Drews, and Gold- berg (1986). Proteins were extracted from the nuclei by the procedure of Miskimins et al. (1985). To inhibit protease activities, 0.8 mM phenylmethylsulfonyl fluoride and 1 O pM leupeptin were added to all solutions.

Preparation of Oligonucleotide Templates

Oligonucleotides that span presumptive binding sites E4 (-38 to

-91 4), TTTTATTCCCAACAATAGAAAGTCTTG; a control oligonucleotide designated C, GTACATTAAAAAGTACAATCGTCAAC; and their respective complimentary sequences were chemically synthesized, annealed, and ligated as described in Kadonaga and Tjian (1 986). Each was inserted into pUCll9, and recombinant plasmids containing three adjacent binding sites were identified.

DNA Gel Electrophoresis Mobility Retardation Assay

-1 2), ATCCATTTCTATATAAAGAAACATACA; E8 (-940 to

End-labeled DNA restriction fragments used in the binding reactions were isolated from low-melting-point agarose gels. Binding was carried out in 15-pL reactions containing 0.25 ng of end- labeled DNA restriction fragment, 1.5 pg of nuclear protein, 10 mM Tris (pH 7.5), 50 mM NaCI, 1 mM DTT, 1 mM EDTA, 10% glycerol, at 3OoC for 30 min. In addition, 4.4 pg of poly(d1-dC). poly(d1-dC) duplex DNA was included in all reactions. Titration experiments with end-labeled E4 restriction fragments indicated that this amount of poly(d1-dC). poly(d1-dC) was sufficient to eliminate nonspecific protein-DNA interactions. The protein-DNA complex was separated from unbound DNA by electrophoresis on nondenaturing 4% acrylamide gels as described by Singh et al. (1 986). Following electrophoresis, the gel was dried and exposed to x-ray film with an intensifying screen for 12 hr to 24 hr.

Methylation lnterference Analysis

End-labeled DNA was partially methylated with dimethyl sulfate (Maxam and Gilbert, 1980) and reacted with nuclear proteins as described for the DNA gel electrophoresis mobility retardation assay, except that the binding reactions were 1 O-fold larger. The DNA-protein complex and unbound DNA were separated by electrophoresis on nondenaturing 4% acrylamide gels (Singh et al., 1986), eluted from the gel, cleaved at the methylated G residues with piperidine, and analyzed on denaturing 8% polyacrylamide gels (Maxam and Gilbert, 1980). DNA methylation and piperidine cleavage reactions were carried out with reagents and procedures supplied by Clontech (Palo Alto, CA).

ACKNOWLEDGMENTS

We express our gratitude to Dr. Alan Bennett for a cDNA library representing ripe fruit mRNAs, Dr. Wilhelm Gruissem for the tomato RuBP carboxylase gene, and to Alex Federman for con- tributions to this work. We also thank Barbara Rotz and John Franklin for providing excellent greenhouse services. This research was supported by a National lnstitutes of Health grant (GM33856). Computer resources used to carry out our studies were provided by the National lnstitutes of Health-sponsored BIONET National Computer Resource for Molecular Biology.

Received August 21, 1989; revised August 30, 1989.

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DOI 10.1105/tpc.1.10.1025 1989;1;1025-1034Plant Cell

S Cordes, J Deikman, L J Margossian and R L Fischerfruit-ripening genes from tomato.

Interaction of a developmentally regulated DNA-binding factor with sites flanking two different

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