The EMBO Journal vol.4 no.5 pp. 11 19-1124, 1985

Thermo-induced transcripts of a soybean heat shock gene aftertransfer into sunflower using a Ti plasmid vector

Fritz Schoffi and Gotz Baumann

UniversitAt Bielefeld, Fakultfit f(ir Biologie (Genetik), D-4800 Bielefeld 1,FRG

Communicated by B.Jockusch

A genomic DNA fragment containing a soybean heat shockgene (hs6871) was inserted into the T-DNA region of theAgrobacterium tumefaciens pTiC58 plasmid. A strain carry-ing the modified Ti plasmid was used to incite tumors in sun-flower hypocotyls. The expression of the heat shock gene wasinvestigated by Northern blot analysis ofRNA and Si nucleasemapping of the transcriptional start site. Heat shock-inducedpoly(A) mRNA was detected in tumor tissue only after in-cubation at 40°C (heat shock) not at the normal growthtemperature (28°C). Transcripts from hs6871 are faithfullyinitiated in sunflower, starting at the same site on the DNAas in soybean. The low level of transcripts initiating correct-ly on hs6871 in sunflower is consistent with a general tissue-specific reduction in the expression of partially homologousnative heat shock genes in sunflower tumors.Key words: heat shock gene/promoter elements/sunflowertumor/Ti-plasmid/transcript mapping

IntroductionTemperature elevation from 280 to 40°C induces the synthesisof heat shock proteins (hsps) in soybean (Key et al., 1981). Thisresponse to the high temperature stress or heat shock (hs) hasbeen implicated as a protective mechanism to increase ther-motolerance in many organisms (Nover et al., 1984), also inhigher plants (Key et al., 1983a; Lin et al., 1984). The biologicalimportance of a group of highly abundant low mol. wt. hsps of15- 18 kd is indicated by a conservation of gene sequences(Schoffi and Key, 1982, 1983). Rapid accumulation and highsteady-state levels of hs mRNAs up to 20 000 molecules per genein the cell suggest transcriptional control of hs gene expression(Schoffi and Key, 1982). All plants tested to date also synthesizesimilar low mol. wt. hsps and hs-specific mRNAs which cross-

hybridize with soybean gene probes representing the so-calledclass 1-multigene family (Key et al., 1983b; Schoffl et al., 1984b).The soybean hs gene hs6871 used in this study is a member ofthe class I gene family which comprises at least 13 genes (Schoffiand Key, 1982, 1983; Schoffl et al., 1984a). Its protein codingsequence encompasses 459 bp translating into a protein of17.3 kd. The transcripts initiate - 100 bp upstream from thetranslational start codon. The potential promoter sequences inthe 5'-upstream region are a TATA box sequence and severalcopies of an element homologous to the Drosophila hs consen-

sus sequence 5'-CTgGAAtnTTCtAGt (Pelham, 1982; Pelhamand Bienz, 1982). This element is cis-active in regulating thermo-inducible transcription of linked genes in animal cells (Pelham,1982; Pelham and Bienz, 1982; Corces et al., 1981; Mirault etal., 1982; Burke and Ish-Horowicz, 1982; Voellmy and Runng-ger, 1982; Bonner et al., 1984).

IRL Press Limited, Oxford, England.

To test the functional significance of the proposed soybean hspromoter elements, we have exploited the natural gene transfersystem ofAgrobacterium tumefaciens (for reviews, see Van Mon-tagu and Schell, 1981; Bevan and Chilton, 1982; Caplan et al.,1983) to introduce hs6871 into the genomes of otherdicotyledonous plants. The Agrobacterium tumor-inducingplasmid (pTi) transfers a specific section of its DNA (T-DNA)to plant cell nuclei where it is stably integrated and maintained.Plant vectors based on the Ti-plasmid have been used to studyexpression of foreign genes introduced into higher plant cells.Genes of non-plant origin are not likely to be expressed proper-ly by their own promoters as has been shown for rabbit globin(Shaw et al., 1983), yeast alcohol dehydrogenase (Barton et al.,1983), chicken actin and ovalbumin genes (Koncz et al., 1984).The promoter regions of T-DNA genes can, however, be usedfor efficient constitutive expression of foreign genes fused tothem. This has been demonstrated for the nopaline synthasepromoter and bacterial antibiotic resistance genes (Herrera-Estrella et al., 1983a, 1983b; Bevan et al., 1983; Bevan, 1984;Fraley et al., 1983). The expression of plant genes in transformedplant cells does not necessarily require the assistance of a T-DNApromoter for correct initiation of transcription. Storage proteingenes of dicots (phaseolin) and monocots (zein) are constitutive-ly transcribed in sunflower callus tissue (Murai et al., 1983; Mat-zke et al., 1984). Light-regulated expression of a pea ss-Rubiscogene in petunia (Broglie et al., 1984) and of a chimeric geneconsisting of the 5'-flanking region of another pea ss-Rubiscogene linked to the coding region of a bacterial chloramphenicolacetyl transferase gene in tobacco tissue (Herrera-Estrella et al.,1984) suggest recognition of conserved plant promoter elementsacross phylogenetic barriers, at least for certain biologically im-portant genes. The implications of a common functional and generegulatory concept in plant hs response makes genes belongingto the class I multigene family in soybean favourable candidatesfor investigating thermo-regulation of gene expression inheterologous genetic systems. Here we report the first evidencefor thermo-induced and faithfully initiated transcription of a soy-bean hs gene in sunflower tumor cells, under conditions whichalso induce the expression of highly homologous sunflower hsgenes.

ResultsConstruction ofa T-DNA vector and transfer ofhs6871 into thegenome of sunflowerThe strategy for inserting hs6871 into the T-DNA of pTiC58(Depicker et al., 1980) was very similar to that used by othergroups for insertion and transfer of foreign genes. The construc-tion of plasmid pGGB1005 (Figure 1) is based on the plasmidpSUP201-3 (Simon et al., 1983) which replicates autonomouslyin Escherichia coli but not in other Gram-negative bacteria towhich it can be transferred by mobilization in the presence ofa chromosomally integrated broad host range plasmid. To pro-vide an efficient target site for homologous recombination withpTiC58 in A. tumefaciens, the EcoRJ14 fragment of the T-DNA

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F.Schoffl and G.Baumann

Km[

/Ap

/AP

Fig. 1. Schematic diagrams of the vector plasmids pGGB1005 andpGGB1017. The approximate location of genes for kanamycin resistance(Kin), ampicillin resistance (Ap) and the site for plasmid mobilization (mob)is indicated.

region was spliced into the respective site on pSUP201-3. TheEcoR114 fragment also contained a TnS element (KmR) insertedinto the center by transposon mutagenesis in E. coli (Schroderet al., 1981). The Tn5 marker served for selection of transcon-jugants in matings between E. coli donor and Agrobacterium reci-pient cells and it also provided a single BamHI restriction sitefor cloning of the hs gene. pGGB1005 was stably inherited byAgrobacterium only after co-integration with pTiC58 as was in-dicated by a 103-fold higher yield of KmR transconjugants incrosses between E. coli (pGGB1005) and A. tumefaciens(pTiC58) compared with crosses with Ti-plasmid-less A. tume-faciens recipient strains. KmR transconjugants derived from pTi-less recipient strains contained only TnS sequences of the vectorplasmid at different chromosomal positions, resulting from trans-position events (data not shown). pTiC58: :pGGB1005 co-integrates were found to be very stable with a reversion rate of< 10-3. Homogenization by double cross-over did not arisespontaneously and could not be examined genetically since thesecond plasmid marker of pGGB1005 (ApR) is not expressedsufficiently highly in Agrobacterium.The hs gene hs6871 was integrated into the BamHI site of

pGGB1005 by linkage of a 1800-bp Sau3A fragment from thegenomic subclone phs68lO (Schoffl and Key, 1983). This frag-

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Fig. 2. Cellular levels of hs-specific poly(A) RNAs in different tissues ofsunflower and soybean tested by Northern blot hybridization. 1 ygRNA/lane was loaded in A and 2 lig RNA/lane in B. The radioactiveprobes were specific for the coding region (A) and for the 5'-non-translatedRNA (B) of hs6871 represented by a 878-bp and a 208-bp TaqI fragment,respectively (see Figure 4). The RNAs were from sunflower hypocotyl (1),sunflower tissue transformed by pGGB1005 (2), both after 40°C heat shock,sunflower tissue transformed by pGGB1017, cultivated at 25°C (3) and aftera 40°C heat shock (4), soybean hypocotyl grown at 25°C (5) and after40°C heat shock (6).

ment contained the entire gene coding region and > 1 kb of 5'upstream DNA including the proposed gene regulatory promoterelements (Schoffl et al., 1984a). The resulting vector plasmidpGGB1017 contained the hs gene and the KmR gene in oppositeorientations within TnS, the expression of neither one seemedto be affected.

After transfer to Agrobacterium, a single transconjugant (strainPC4817) containing the pTiC58::pGGB1017 co-integrate was us-ed to transform sunflower hypocotyl according to Murai et al.(1983). Incited crown gall tissue was harvested after 2 weeksand DNA was prepared according to Nagao et al. (1981) to testthe integration of the vector plasmid along with the T-DNA bySouthern blot hybridization. Using pGGB1017 as a radioactive-ly labelled probe in hybridization with EcoRI/HindIll-digestedgenomic DNA, the presence of the entire plasmid in the plant

Regulated transcription of a soybean heat shock gene in sunflower twnors

genome was detected. Control hybridizations of identical blotswith non-T-DNA probes, clone pGVO414 (Depicker et al., 1980)derived from pTiC58 were negative (data not shown). Bacterialcontaminations were therefore not likely to interfere with thetranscriptional analysis in plant cells.Copy number estimates of pGGB1017 in sunflower DNA

revealed less than one molecule per cell, ranging from 0.1 to0.5 copies in different DNA preparations. This number is-10-fold lower than that reported for fully transformedsunflower callus derived from single cloned cells (Matzke et al.,1984), indicating a low number of transformed cells in primarytumor tissue used in our experiments. However the advantageof this method is the fast generation of sufficient amounts oftransformed tissue to test expression of transferred genes in thenew genetic background.

The analysis of hs induced transcripts in sunflower tumor tissueTo examine the induction of hs6871-specific transcripts inprimary tumors incited by pGGB1017, we have isolated poly(A)RNA from tissues before and after a 40°C heat shock. Northernblots of these RNAs and appropriate control RNAs (see Figure 2)were hybridized with radioactive probes representing the codingregion (Figure 2A) and the 5'-non-translated region (Figure 2B)of the hs gene. The map of the respective restriction fragmentsof hs6871 is depicted in Figure 4. The control RNAs used inthese experiments were isolated from pGGB1005-transformedtissue after heat shock (lanes 2), from untransformed sunflowerhypocotyl after heat shock (lanes 1), soybean hypocotyl before(lanes 5) and after heat shock (lanes 6). A strong conservationof hsp coding sequences between soybean and sunflower is de-tectable by cross-hybridization with the probe (Figure 2A).However, there are significant differences in intensities ofhybridization signals between soybean and sunflower hypocotylhs RNAs (lanes 1 and 6) and even between sunflower hypocotyland tumor hs RNAs (lanes 1, 2 and 4). pGGB1017 transformedtissue (lane 4) and control tumors (lane 2) contain equal amountsof hs-specific poly(A) RNAs.The presence of the soybean gene does not significantly in-

crease the signal intensity in lane 4. The intensity differencesof the hybridization signals between soybean and sunflower areprobably caused by the lower sequence homology and/or copynumber of hs genes in sunflower. The differences betweensunflower hypocotyl and tumor tissue cannot be explained in thisway. The much lower steady-state levels of hs RNAs in tumortissue may be caused by tissue-specific components which af-fect transcription or stability of hs mRNAs.No sequence homology can be detected between soybean and

sunflower hs genes when the 5'-specific hybridization probe ofhs6871 is used (Figure 2B). The probe used in this experimentwas a 208-bp TaqI DNA fragment (see Figure 4), which coversonly 47 bp of the 5'-translated but 103 bp of 5'-non-translatedRNA and additional 58 bp of the flanking promoter DNA (Schofflet al., 1984a). The lack of hybridization with sunflower RNAindicates that sequence conservation of plant hs genes was pro-bably confined only to regions in the protein coding sequence.Even the members of the class I multigene family in soybeandiverge in 5'-terminal DNA sequences (Schoffi et al., 1984aand unpublished data). Since sunflower tumors containing thesoybean hs gene hs6871 do not also produce sufficient hs-specificRNA to score positive in Northern blot hybridization (seeFigure 2B, lane 4), we had to apply a more sensitive test to iden-tify transcription of this gene.The same poly(A) RNAs as used for the Northern blot analyses

N

- - " -*<~0s 208

*4 150A

Fig. 3. SI nuclease mapping of the 5' start site of hs6871 transcripts insunflower and soybean. The schematic diagram (B) outlines the principle ofthe method. The poly(A) RNAs tested by this assay for the protection of thecharacteristic 150 nucleotide fragment (A) were from sunflower hypocotyl(1), sunflower tissue transformed by pGGB1005 (2), both after 40°C heatshock, sunflower tissue transformed by pGGB1017 and cultivated at 25°C(3) and after 40°C heat shock (4), soybean hypocotyl grown at 250C (5)and after 40°C heat shock (6).

were also tested by the Si-nuclease mapping technique (Berk andSharp, 1977). From previous work it was known that hybridiza-tion of hs-poly(A) RNA from soybean with the 5'-32P-end-labelled208-bp TaqI fragment protects 150 nucleotides from subse-quent digestion by SI-nuclease (Schoffl et al., 1984a, see alsoFigure 3B). Significant amounts of the characteristic 150 nucleo-tide long DNA are only detectable when RNA from pGGB1017-transformed tissue which has been subjected to a 40°C heat shockis used (Figure 3A, lane 4). Negative results were obtained withRNAs from sunflower hypocotyl, 40°C (lane 1), control tumorsinduced by pGGB 1005, 40°C (lane 2) and by pGGB 1017, 25°C(lane 3). These data suggest thermo-induced transcription ofhs6871 in sunflower tumors, with a faithful initiation at the samesite as used also in soybean (lanes 5, 6). The applied techniqueproved to be very sensitive since it was also possible to pick upthe low level of basal transcription of this gene in soybeanhypocotyl at 25°C (lane 5). The different intensities of the new-ly generated bands at the 150 nucleotide position in Figure 2should directly correlate with the relative amounts ofhs6871-specific transcripts, since identical, non-saturating con-

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F.Schffl and G.Baumann

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ditions were used for hybridization. The low level of this tran-script in sunflower tumors compared with that in soybeanhypocotyl (lanes 4 and 6) is consistent with a low steady-statelevel of partially homologous native hs-mRNA (Figure 2A,lane 2) and the low copy number of the transformed gene intumor tissue.

DiscussionWe have shown for the first time that transcription of plant hsgenes may occur in heterologous plant cells in a thermo-regulatedfashion with faithful initiation. The conserved promoter elements,present in the 5'-upstream (Schoffl et al., 1984a, see Figure 4)region of soybean class I hs genes are probably the key elementsfor controlling transcription. They are possibly recognized bytranscription factors which regulate the hs response in soybeanand in sunflower by similar mechanisms. At least two proteinfactors which interact by sequential binding with DNA regionscovering the TATA box sequences and the upstream control ele-ment of Drosophila hs genes have already been identified (Wu,1984). The resemblance of the respective binding sites in animalsand plants suggests a common mechanism for the activation ofhs genes in nature. Other sequences may also be involved in trans-criptional control of hs genes, but all 5'-upstream regulatory se-

quences at least, should be present within the 1-kb region ofDNAflanking hs6871 in the vector construct used in this study.

It is not known whether the sequences located furtherdownstream from the 3' end of the protein-coding region ofhs6871 are required for transcriptional efficiency or mRNAstability. The present construction does not contain any of theas yet identified signal sequences for 3'-splicing and polyadenyla-tion in other systems (for review, see Proudfoot, 1984). Thepresence of sequences, such as the canonical polyadenylationsignal AATAAA, is implicated in correct termination and instability of globin miRNA (Higgs et al., 1983; Whitelaw andProudfoot, 1983).

Similar sequences are also contained in many plant genes;absence, however, may not necessarily cause inefficient transcrip-tion or mRNA instability. This is indicated by a high steady-statelevel of mRNA from chimeric gene lacking a polyadenylationsignal (Schreier et al., 1985). Our failure to detect hs6871-specific

transcripts in sunflower tumor tissue by Northern blot hybridiza-tion may be due to a random termination of the mRNA and toa reduced steady-state level in this tissue. The low level offaithfully initiated hs6871 transcripts detected by SI nucleasemapping can be explained by the tissue-specific expression ofhs genes which is high in leaf, lower in epicotyl and hypocotyl(Schoffl, 1984) and minimal in tumor tissue (this paper). Thelow copy number of the transformed gene in primary tumors aswe have observed, may contribute additionally to an apparentlylow expression of this gene in the heterologous plant. The light-regulated pea ss-Rubisco gene transcripts appear also at muchreduced levels when induced in heterologous plant callus tissues(Broglie et al., 1984; Herrera-Estrella et al., 1984). This mayindicate that the activation of environmentally regulated genesis generally less effective in heterologous plant systems. How-ever, there is hope that the very conserved hs response may allowhigh levels of transcription of hs6871 and other hs genes in thetissue of transformed plants obtained by using disarmed T-DNAvector systems (Zambryski et al., 1983; Bevan, 1984).We do not know yet whether hs6871-specific mRNAs are

translated in sunflower into a functional protein. This can be testedupon availability of specific antibodies or high level transcrip-tion of the gene. The advantage of using hs promoters as buildingblocks in chimeric gene construction should be a fast initiationand high level of transcription under conditions which ultimate-ly block or reduce temporarily the expression of most other genes.It is important to note that T-DNA gene transcripts for theoctopine synthase are also reduced by hs (Gurley et al., in prep-paration).

Materials and methodsBacterial strains and plasmidsA. tumefaciens C58 (RifR) and the EcoRI14 T-DNA fragment (see Depicker etal., 1980) containing a TnS insertion were kindly provided by Dr. W. Klipp(Universitat Bielefeld). The plasmid mobilization system with E. coli S 17-1 andthe vector plasmid pSUP201-3 was kindly provided by Dr. R. Simon (Univer-sitiit Bielefeld) and its genetical use is described by Simon et al. (1983).Bacterial conjugation and transfonmationConjugations involving E. coli and A. tumefaciens strains were carried out asdescribed by Van Haute et al. (1983). Transfonnations of E. coli were as describedby Maniatis et al. (1982).

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Regulated transcription of a soybean heat shock gene in sunflower tumors

DNA manipulationsAll DNA manipulations (restriction endonuclease digestion, ligation, dephosphory-lation and gel electrophoresis) were performed according to Maniatis et al. (1982).DNA and RNA isolationPlasmid DNA was isolated by the sarcosyl lysis method (Bazaral and Helinski,1968) and purified by CsCl ethidium bromide density gradient centrifugation(Radloff et al., 1967).

Total DNA from A. tumefaciens was isolated as described by Meade et al.(1982). High mol. wt. DNA from sunflower tumors was isolated from purifiednuclei essentially as described by Nagao et al. (1981).RNA isolation and poly(A) RNA purification was carried out according to

Schoffi and Key (1982). Standard RNA preparations were made from 20 g sun-flower tumor tissue. The average yield of total RNA was 8- 10 mg containing-100 ug poly(A) RNA.Inoculation of sunflower hypocotyls by recombinant bacteriaSunflower seeds (Helianthus annus var. Spanners) were germinated at 250C inplant growth chambers and hypocotyls were infected after 1 week by needle punc-ture with a drop of an overnight culture of engineered Agrobacteria (Murai etal., 1983). Incited tumors were harvested after 2 weeks. The wet weight of tumortissue per tray ( -250 plants) was 20 g in average.Heat shock conditionsA heat shock temperature of 400C was applied for 45 min to tumors or hypocotylaccording to Key et al. (1981). An additional incubation was carried out at roomtemperature for 30 min before grinding of the tissues.Southern blot hybridization of DNADNA from bacteria or plants was digested to completion with restriction endo-nucleases as indicated. Electrophoresis, transfer and hybridization of DNAfragments was carried out as described by Schoffl and Key (1983). 32P-labelledhybridization probes were generated by nick translation of purified plasmid DNAto a specific activity of - 108 c.p.m./4g DNA.Copy number reconstitutions of the vector plasmid pGGB1017 for quantita-

tion in primary tumors was made with the calculated amounts of plasmid DNAwith 10 jAg/lane digested calf thymus DNA and 10 ytg/lane plant DNA digestedwith the same restriction enzymes. The copy number determination was basedon 9.8 pg DNA per diploid sunflower genome (Smith, 1977). The modified T-DNA (co-integration with pGGB1005 and pGGB1017) of the pTiC58 was deter-mined in A. tumefaciens total DNA (2 tg/lane) in comparison with slandard plasmidDNA digests (0.1 tg/lane) and hybridizations with appropriate DNA-probes.Northern blot analysis of RNAPoly(A) RNAs (1 yg or 2 Ag/lane as indicated) were electrophoresed in 2%agarose/6% formaldehyde gels and blotted to nitrocellulose sheets. Immobiliza-tion of the RNA and hybridization was performed according to Baulcombe andKey (1980) with the following modifications. Lack of formamide in the hybridiza-tion solution and the hybridization was carried out at 600C. 32P-Labelled DNAwas added to the hybridization mixture. Nick-translated DNA (Figure 2B) ora single-stranded DNA (Figure 2A) have been used as probes. The latter wasprepared by primer extension (Hu and Messing, 1982) of an 878 nucleotide Ta-qI fragment (see Figure 4) cloned into mp9.

SI nuclease napping5 Ag total poly(A) RNA were hybridized for 18 h at 42°C with the 5' 32P-end-labelled DNA fragment (Maniatis et al., 1982), treated with S1 nuclease (1000U/ml, 30 min, 37°C) followed by electrophoresis and autoradiography of theprotected DNA fragments on a sequencing gel (Schoffl et al., 1984a). The frag-ment sizes were determined by a sequencing standard running on the same gel.

AcknowledgementsWe thank Dr. W. Klipp (Universitat Bielefeld) for helpful discussion and pro-viding Agrobacterium strains and T-DNA clones, Dr. R. Simon for supplyingthe pSUP201-3mob vector system, Sieglinde Angermuller for technical assistance,Hanne Schoffl for help in the preparation of this manuscript and Dr. M. O'Con-nell and M. Hynes for critical reading and suggestions. The research was sup-ported by grants from the Deutsche Forschungsgemeinschaft and the AgrigeneticsResearch Associates.

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Schell,J. (1983) EMBO J., 2, 2143-2150.

Received on 24 January 1985; revised on 25 February 1985

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