isolation and characterization of six heat shock transcription factor cdna clones from soybean

Plant Molecular Biology 29:37-51, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 37

Isolation and characterization of six heat shock transcription factor cDNA clones from soybean

Eva Czarnecka-Verner*, Chao-Xing Yuan, Paul C. Fox and William B. Gurley Department of Microbiology and Cell Science, Program of Plant Molecular and Cellular Biology, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700, USA (* author for correspondence)

Key words: HSF, DNA binding domain, oligomerization domain, stress

Received 9 December 1994; accepted in revised form 7 June 1995

Abstract

Thermal stress in soybean seedlings causes the activation of pre-existing heat shock transcription factor proteins (HSFs). Activation results in the induction of DNA binding activity which leads to the transcription of heat shock genes. From a soybean cDNA library we have isolated cDNA clones corresponding to six HSF genes. Two HSF genes are expressed constitutively at the transcriptional level, and the remaining four are heat-inducible. Two of the heat inducible genes are also responsive to cadmium stress. Comparative analysis of HSF sequences indicated higher conservation of the DNA binding domain among plant HSFs than those from yeast or other higher eukaryotes. The putative plant HSF oligomerization domain contains hydrophobic heptapeptide repeats characteristic of coiled coils and seems to exist in two structural variants. The carboxy-terminal domains are reduced in size and the C-terminal heptad repeat is degenerate.

Introduction

HSFs play a central role in the transmission of information regarding environmental stress to the nucleus where the binding of activated HSFs to the heat shock elements (HSEs) in promoters of heat shock genes results in the transcriptional induction of the heat shock response (for recent reviews see [8, 9, 18, 25, 34]). The genes for HSFs have been isolated and characterized from yeast, Drosophila, man, mouse, chicken, tomato and

Arabidopsis, and contain five functionally distinct domains: the DNA binding domain (DBD), the oligomerization domain (OD), the nuclear localization sequence (NLS), the carboxy-terminal hydrophobic domain (known as hydrophobic repeat HR3, or the '4th leucine zipper'), and the transcriptional activation domain (AD). The only highly conserved region of the protein is the DBD which consists of about 95 amino acids located within the N-terminal domain [25 ]. The second- ary structure consists of a triple helix bundle typi-

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers Z46956 (GmHSF5), Z46952 (GmHSF21), Z46951 (GmHSF29), Z46955 (GmHSF31), Z46954 (GmHSF33), and Z46953 (GmHSF34).

38

cal of the helix-turn-helix family of DBDs [12, 321.

The oligomerization domain contains at least two hydrophobic heptapeptide repeats with the characteristic 4, 3-(abcdefg)n pattern typical of bZip proteins [13]. In higher organisms HSF activation involves oligomerization of the HSF to form a trimer which is then able to bind DNA. These trimeric complexes are formed via a triple- stranded e-helical coiled coil analogous to that found in the hemagglutinin of influenza virus ([ 20 ] and references within). In addition to its role in trimerization, the OD, along with C-terminal HR3, is required to preserve cytoplasmic localization of H S F under control conditions; deletion of either of these two regions results in constitutive nuclear localization of human HSF2 [29].

The ADs are not well characterized in meta- zoan HSFs and show little conservation in sequence. Preliminary reports indicate that the ADs of mouse HSF1 and 2 are comprised of nega- tively charged clusters of amino acids at the carboxy terminus [18]. More information regarding the identify of motifs involved in transcription activation is available for plant HSFs. Recently, Treuter and colleagues determined that a small amino acid motif containing a tryptophan residue (termed the Trp repeat) located within a nega- tively charged region is involved in the transcriptional activation of three tomato heat shock factors [31].

In yeast and Drosophila, HSFs exist as a single form [3, 15, 33]. Higher eukaryotes such as man and mouse have two HSF genes, and in chicken and tomato as many as three distinct HSF genes have been found [14, 19, 21, 24, 26, 27, 28]. The finding of multiple H S Fs in vertebrates and plants has suggested the possibility that HSFs may be specialized for different stresses or different roles in development. For example, in chicken the three HSFs show differential patterns of mRNA expression. HSF1 is found in the retina and pig- mented epithelium. HSF2 is located in the brain, spinal cord and connective tissue, and HSF3 in the blood [ 19]. At present no similar information regarding mRNA distribution of plant HSFs is available, but a distinct difference can be seen in

the patterns of HSF mRNA expression between plants and other organisms in that soybean (this study), tomato, and Arabidopsis have HSFs that are transcriptionally inducible upon heat shock [14, 25].

By screening a soybean cDNA library with an oligomer probe homologous to the most conserved region of the DNA binding domain of tomato HSFs, we have isolated six individual soybean HSF cDNA clones. In this report, we describe the basic features of their structure, relatedness, genomic organization, and the transcriptional induction of their mRNAs upon heat shock and heavy metal stress.

Materials and methods

Screening of soybean cDNA library and selection of eDNA clones

A 2gtl 1 cDNA library of 6-day old soybean seedlings (unstressed shoots and leaves; Glycine max L. cv. Williams) was purchased from Clontech and used to screen for HSF sequences. About 1 X 10 6 plaques were screened at 5 x 10 4 plaques

per plate by hybridization of duplicate nitrocellulose filters with the tomato LpHSF24 oligomer probe. This synthetic 48-mer, 5 '-AATTTCTCC- A G C T T C G T T C G A C A G C T T A A C A C C T A T - GGTTTTCGAAAG-3 ' , was identical in sequence to the highly conserved region of the LpHSF24 DNA binding domain from nucle- otides 496 to 543 [27]. Duplicate filters were pre- hybridized for 1 h at 37 °C in 6 x SSC (1 x SSC is 0.15 M NaC1, 0.015 M sodium citrate), 5 × Denhardt's solution, 0.5~o sodium dodecyl sul- fate, 0.05~o sodium pyrophosphate, 100 #g of denatured herring sperm DNA per ml, and hybridized with 32p-labelled oligomer probe (2-5 x 108 cpm/#g) in the same solution at 60 °C for 16 h. Filters were then washed three times in 6 x S SC with 0.05 ~o sodium pyrophosphate at room temperature for 5 min per wash, blotted dry, wrapped in plastic wrap, and exposed to X-ray film for 24 h. Each positive phage isolate was plaque purified through three rounds of screening.

Since many of the clones contained internal Eco RI sites, the cDNA inserts were amplified from lambda DNA by the polymerase chain reaction (PCR) with high-fidelity Vent DNA polymerase (New England Biolabs) using forward and reverse 2gtll primers. PCR-generated fragments were screened for relatedness by restriction endonuclease mapping.

Nucleotide sequence analysis

PCR-generated fragments were subcloned into pUC19 at the Srna I site. After purification by CsC1 gradient centrifugation and polyethylene glycol precipitation, both strands of the cDNA inserts were sequenced from two individual clones per construct by the dideoxy-chain termination method [23] (ICBR Sequencing Core Labora- tory; University of Florida). Forward and reverse pUC primers and oligonucleotides derived from internal sequences were used as sequencing primers. DNA sequences were analyzed and as- sembled with the Applied Biosystems SeqEd (version 1.0.3) sequence analysis software pack- age for Macintosh computers. Comparisons of derived amino acid sequences were performed using the GeneWorks 2.2.1 (Intelligenetics) program, and parsimony trees were constructed using the Phylogenic Analysis Using Parsimony (PAUP) software (Smithsonian Institution, 1993).

Isolation of RNA and northern blot analysis

Etiolated soybean, Glycine max cv. Corsoy, seedlings (2-3 cm long) were incubated for 2 h in 1 mM potassium phosphate buffer, pH 6.0, and 1 ~o sucrose at 28 °C (control), or 40 °C (heat shock) with shaking. Incubation media for heavy-metal treatment (2 h) included, in addition, 500 #M CdC12. Total RNA was extracted by the TNS/ PAS method [4]. Poly(A) + RNA was recovered by oligo(dT) cellulose chromatography and quan- tified by two means: optical density (OD26o) de- terminations, and electrophoresis on 1.4~o agar-

39

ose gels followed by ethidium bromide staining. For northern blot analysis, 5/~g (20 #g for Gm- HSF31) of poly(A) + RNA was fractionated on 2~o agarose gels containing formaldehyde and capillary transferred to either nitrocellulose (Schleicher & Schuell) or nylon Hybond-N membranes (Amersham). Filters were incubated indi- vidually with six radiolabelled cDNA probes: Eco RI fragments for GmHSF34 (876 bp), GmHSF5 (1203 bp), GmHSF21 (671 bp), GmHSF31 (965 bp), and GmHSF33 (1067 bp); and a Pst I-Barn HI fragment for GmHSF29 (1211 bp) where the Barn HI site was supplied by the vector since the cDNA clone was missing its 3' Eco RI site. Only GmHSF5 and GmHSF21 probes contained sequences corresponding to DNA binding domains. A Hind III-Hinc II (2.0 kb) fragment from the soybean actin gene was isolated from the pSAC3 plasmid kindly provided by Joe L. Key and Ron T. Nagao (The University of Georgia, Athens, GA). Specific activities of radiolabelled probes were 2-4 x 108 cpm//~g of DNA. Northern blot hybridizations were carried out overnight at 42 °C in standard hybridization solutions containing 50~o formamide, 5x SSC, 5x Den- hardt's solution, 0.1~o SDS, 50 mM sodium phosphate buffer pH 7.5 and 100 #g/ml of denatured herring sperm DNA. Filters were washed at room temperature 3 times for 5 min in 2 x SSC and 0.1~o SDS followed by one 5 min rinse in 0.2 x SSC and 0.1~o SDS. The filters were then blotted dry, wrapped in plastic wrap and exposed to X-ray film at -80 °C for 2 to 7 days. For hybridizations with the actin probe, filters were exposed to X-ray film from a few hours to overnight.

Southern blot analys&

Genomic DNA was isolated from 7-day old etiolated soybean plumules according to Jofuku and Goldberg [ 16]. A 10/~g portion of soybean DNA was digested with restriction endonuclease Eco RI or Hind III, electrophoresed on a 0.8~o agarose gel and transferred to a Hybond-N nylon membrane (Amersham). The hybridizations and

40

probes were as described for the northern blots. The filters were exposed to X-ray film at -80 °C for 4 to 7 days.

Protein expression of GmHSF34

For protein expression a PCR-generated fragment spanning the coding region of the GmHSF34 cDNA clone was cloned in-frame into the pET 15b expression vector (Novagen) between the Nde I and Barn HI sites. HSF protein was expressed in the lambda DE3 lysogen of strain BL21 according to the pET System Manual. Induction was carried out at 37 °C with 1 mM IPTG for 3 h. HSF protein was found predominantly in inclusion bodies and was isolated according to Frankel et al. [ 11 ] using the 1 ~o sarcosyl extrac- tion method with minor modification. Washed inclusion bodies were incubated in lysis buffer (50 mM Tris pH 8.0, 2 mM EDTA) containing 1 ~o sarcosyl for 1 h at room temperature. The samples were centrifuged at 11 000 rpm for 20 min in order to remove membranes and then diluted 10-fold. The final buffer content was as follows: 50 mM Tris pH 8.0, 6 mM MgC12, 50 mM NaC1, 5~o glycerol, 1.5 mM DTT, 0.1~o sarcosyl, 2 mM EDTA. Aliquots of recombinant HSF protein were frozen in liquid N2 and stored at -80 °C until further use.

Electrophoretic mobility shift assay (EMSA) of recombinant GmHSF34 and competition of HSE binding with synthetic oligomers

Assays for DNA-protein binding and gel retar- dation were extensively described previously [5, 6]. The DNA-protein binding assays (20 #1) included 20 mM HEPES pH 7.9, 70-100 mM NaC1, 0.1 mM EDTA, 0.5 mM DTT, 1.2 mM MgCI2, 1 ~/o glycerol, 0.25 #g/#l BSA, 0.25-0.5 ~g poly(dI-dC) and 40 pg (1 x 105 cpm) of 32p_ labelled HSE1 oligomer probe. Double stranded HSE1 (5 ' - tcgacGTTAGGATTTTTCTGGAA- CATACAAg-3' ) oligomer was 3' end-labelled by fill-in with Klenow large fragment [5]. HSE1 and

HSE2 (5 ' - tcgacATGGTGTGGAGAATTCA- ACCAAg-3') double-stranded oligomers were used as specific competitors of recombinant HSF binding activity, while the AT composite (5' - t c g a c A A A A A T A A T A T T A A T A T T A T A T T - GAAAg-3') oligomer served as the nonspecific competitor [5, 6]. Bases in lower case indicate linker sequences not present in the Gmhsp17.5E promoter.

Results

Isolation of six soybean HSF cDNAs

A total of 34 positive clones were plaque purified and grouped into six families based on similarities in restriction endonuclease digestion patterns. Seven clones were < 200 bp in size and were not analyzed further. DNA sequences were determined for the longest representatives of each group and their derived amino acid sequences are shown in Fig. 1. Only two of the cDNAs (Gm- HSF34 and GmHSF5) appeared to be full-length. The nucleotide sequence of GmH SF34 contained a single open reading frame of 282 amino acids beginning with ATG at nucleotide 300 and terminating with TGA at nucleotide 1146 resulting in a derived protein having a predicted molecular weight of 31 194 Da. The open reading frame of GmHSF5 included 370 amino acids (42 112 Da), starting at nucleotide 46 and terminating with TAA at nucleotide 1156. Clone GmHSF21 was missing the 3' half of the transcript, but contained the entire DBD and a portion of the OD. The remaining three partial cDNAs (GmHSF31, GmHSF29 and GmHSF33)represented 5' trun- cations, two of them terminating at the same position (Eco RI site) within the N-terminal portion of the DBD (Fig. 1).

DNA binding domain and nuclear localization sequences

All six soybean HSFs share extensive identity in the region of the DBD among themselves and

GmHSF21 GmHSF34 GmHSF5 GmHSF31 GrnHSF29 GmHSF33

MY MERIRVKEEE AVTCGGGSSS SSSSSSSFSP QPMEGLHEVG MSQRSV

M ALLLDNCESI LLSLDTHKSV

GmHSF21 GmHSF34 GmHSFE GmHSF31 GmHSF29 GmHSF33

PPPFLSKIFD MVEDSSTDSI VSWSMARNSF WWDSHKFSA DILPR~KHG pAPFLTKTYQ LVEDQGTDQV ISWGESGNTF WWKHADFAK DLLPKYFKHN pAPFLTKTYQ LVDDPSTDHI VSWGEDDTTF VVWRPPEFAR DLLPNYFKHN

EFAR NLLPNYFKHN EFAR DLLPKYFKHN

EFRF IVWRPAEFAR DLLPKYFKHN

GmHSF21 GrnHSF34 GmHSF5 GmHSF31 GmHSF29 GmHSF33

110 120 130 140 150

T NFSSFIRQLN AYGFRKVDPD RWEFANEGFL AGQRHLLKTI KRRRNVSQSL NFSSFVRQLN TYGFRKIVPD KWEFANEHFK RGQKELLSEI KRRKTVPQSS NFSSFVRQLN TYGFRKIVPD RWEFANEFFK KGEKNLLCEI HRRKTHHQHH NFSSFVRQLN TYGFRKIVPD RWEFANEFFK KGEKHLLCEI HRRKTAQPQQ NYSSFVRQLN TYGFRKVgPD RWEFANDCFR RGERALLRDI QRRKLLPVPP NFSSFVRQLN TYGFRKVVPD RWEFANDCFRRGERALLRDI QP~qKLLPVPP

GmHSF21 GmHSF34 GmHSFS GmHSF31 GmHSF29 GrnHSF33

1~0 170 180 190 200

QQKGGSGACV EVCEFGLEGE LE~LKRDP~NI I~4AEIVP~RH QQLNSREQLN- AHPPEAGKSG GDGNSPLNSG SDDAGSTSTS SSSSGSKNQG SVETNTTPSH QQVQA~/NHH HHNKFGLNVS SIFPFHNNRL SVSPSHDSDE VIIPNWCDSP GIMNHHHHHA HSPLGVNVNV PTFFPFSSRV SISTSNDSDD QSNWCDSPPR AAAAPTAVTA NTVTVAVAAP AVRTVSPTTS CDEQVLSSNS SPIAGNNNNT AAAAPAAVTA NTVTVAVAAP AVRTVSPTTS GDEQppavrl lhkrcedrag

GmHSF21 - 3 ' truncation GmHSF34 QLSSENEKLK KDNETLSCEL ARARKQCDEL VAFLRDRLMV GPDQIDRI~ GmHSF5 PRGVACVNNN NSSSNNyNTV TALSEDNERL RRSNNMI~4SE LAHMKKLYND GrnHSF31 GATSLVNGAA AANYNTSVTA LSEDNERLRR SNNMLMSELA HMXKLYNDII GmHSF29 VHRTTSCT%'A PELLDENERL RKENMQLSNE LSQLKGLCNN ILALMTNYAS GmHSF33 agreg&aga f aret cfig

GmHSF2t GmHSF34 QGSCGSENW GEGCGGDCLK LFGVWLKGDT LTDKRNNHKR GBED~MGFGG GmHSFS IIYFVQNHVK PVAPSNNNNp PSFLLCSDNS NINNNTSPQT QTQAQASPMS GmHSF31 YFVQNHVKPV APSNSYSSSL LLCNTPSATP ISSANNVS~4 QRPMNQLLGY GmHSF29 GFSRQQLESS TSAARTVpVP EGKAALELLP AKHVSSADEA GHVGGAAPCA GmHSF33 -RQQLESS TSAVRTVPVP DGKAPLELLP AKHVSSADDA LHVGGAAGAA

GmHSF21 GrnHSF34 PRLKESKPVV DFGAVNIMMK SNRVCN GmHSF5 NVSTVQRQLK QFVGCYSNNT KQARAVNSPT NSSITIVEEE ANSNSCKTKL GmHSF31 YS3~PKQGAT QITQPQTYW NSPTNTSRSS ITIFE~PASS NINSCKTKLF GmHSF29 %ANAGEAEVP KLFGVSIGLK RCRTECEGEA ECEDQNQMQT RAQA~TQTQS GmHSF33 ACATGNAAEA EVPKLFGVSI CLKRCRTECE AEPEGEDQNQ MQTRAQTQSQ

GmHSF21 GmHSF34 GmHSF5 FGVSLQSKKR VHPEYGSNNV LQSSETNKAR LALENDELLG LNLMPPSTC GmHSF31 GVSLQSKKRV HPDCGSNPET NRARLVLEKD DLGLNLMPPS TC GmHSF29 SQEPDHGSDV KSEPLDGDDS DDQDHDPRWL ELGK GmHSF33 SSQEPDHGSD VKSEPLDGDD SDYQDHDPHW LEL

Fig. 1. Amino .acid sequences of six soybean HSF cDNA clones. The sequences of cDNA clones are aligned by the similarities in their DNA binding domains (positioned from 51 to 145). Clones GmHSF31, GmHSF29 and GmHSF33 are 5'-truncated, while GmH S F21 is truncated at the 3' terminus (past HR1). The sequence written in lower case for pseudogene-like clone GmHSF33 represents the shifted open reading frame after the assumed deletion of 69 amino acids (entire OD) at position 185 (amino acid 109) (compare with clone GmHSF29 and see text). The amino acids written in bold case in GmHSF29 and GmHSF33 sequences are not present in the reciprocal clone. The triangle indicates point of deletion of 11-12 amino acids from plant HSF DBDs as compared to yeast or other higher eukaryotes. Potential NLSs (DBD proxi- mal) are underlined.

41

with tomato HSFs (Fig. 2). The eukaryotic consensus sequence shown in Fig. 2 is from Scharf et al. [25] and shows identity between all known HSFs including those from yeast, Drosophila, vertebrates and tomato. As expected, conservation of the D N A binding domain is higher within the plant HSFs than between eukaryotes in general. In the conserved central portion of the DBD, limited homology can be seen to the helix-turn- helix D N A binding motif common to the bacterial counterpart of HSF, sigma factor 32, and to sigma 70 [3] (Fig. 2, boxed). The invariant and conserved amino acids of the D B D are clustered predominantly at the N-terminus and in the C-terminal 39 amino acids from residues 41 to 79. Out of 51 conserved amino acids, approximately

AtHSF1 LpHSF24

GrnHSF21 GmHSF34 GmHSF5 GmHSF31 GrnHSF29 GmHSF33

Plant

Euk~yote

PPPFLSKTYD MVEDPATDAI VSWSPTNNSF IVWDppEFsR D ~ PAPFLLKTyQ LVDDAATDDV ISWNEIGTTF VVWKTAEFAK PPPFLSKTYE MVEDSSTDOV ISWSTTRNSF IVWDSRKFST PPPFLVKTYD MVDDPSTDKI VSWSPTNNSF VVWDPpEFAK PPPFLSKIFD MVEDSSTDSI VSWSMARNSF VVWDS~KFSA PAPFLTKTYQ LVEDQGTDOV ISWGESGNTF VVWKHADFAK PAPFLTKTYQ LVDDPSTDHI VSWGEDDTTF VVWRPPEFAR

EFAR EFAR

EFRF IVWRPAEFAR

P~aPFL-Kty - ~D--TD- i ~SW .... F vVW .... Fs!- dlLP-yFKHn

P--F--K .... v- ......... W ..... sf -%,- .... F .... LP-yfKH-

AtHSF1 LpHSF24 koHSF30 LpMSF8 GmHSF21 GmHSF34 GmHSF5 GmHSF31 GmHSP29 GmHSF33

Plant

Euka~yote

7 .;° ;° ~FSSFVRQLN TYGFRK~DPD RWEFANEGFL RGQKHLLKKI SRRKS ~FSSFVRQLN TYGFRKGFRK~VPD KWEFANENFK RGQ/(~LLTAI RRBKT ~FSSFIRQLN TyGFRK~PD RWEFANEGFL GGQKHLLKTI KRRRN ~FSSFVRQLN TYGFRK~DPD RWEFANEG~L RGQKHLLKSI SRRKP ~FSSFIRQLN AYGFRK~PD R~FANEGFL AGQRHLLKTI KRRRN ~FSSFVRQLN TYGFRK~VPD KWEFANEHFK RGQKEL~SEI KRRKT ~FSSFVRQLN TYGFRKI~D RWEFANEFFK KGE~NLLCEI NRRKT ~FSSFVRQI~ TYGFRK~D RWEFANEFFK KGEKHLLCEI HRRKT ~YSSFVRQLN TYGF D RWEFANDCFR RGEP~LLRDI QRRKL ~FSSFVRQLN TYGF D RWEFAND~_~RGERALLRDI QRRKL

NfSSFvRQLN tYGFRK-~PD rWEFANe-F- -Ger~k LL--I -RRk-

N--SF-RQLN -Yg--Kv ..... eF .... F ...... LL--I ---k-

Fig. 2. Amino acid comparison of DNA binding domains of Arabidopsis, tomato and soybean HSFs. AtHSF1 is from Htlbel and Sch6ttt [14]; and LpHSF24, LpHSF30 and LpHSF8 sequences are from Scharfetal. [26, 27]. GmHSF29, GmHSF31 and GmHSF33 sequences are from partial cDNA clones. A putative bipartite nuclear localization sequence is underlined and the homology to bacterial sigma factor 32 (amino acids 253-278, LQELADRYGVSAERVRQLE- KNAMKKL) and sigma 70 (573-598, LEEVGKQFDVTRE- RIRQIEAKALRKL) is boxed [ 3 ]. Triangle between residues 67 and 68 indicates position of 11-12 amino acid insertion in animal and yeast HSFs. Plant consensus sequence was ex- trapolated to show invariant (capital letters) and conserved residues (lower-case letters). The allowance of two amino acid variability in assigning conserved residues was maintained as in the eukaryotic HSF consensus derived by Scharfet al. [25].

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28 are hydrophobic and 18 are charged. The charged amino acids are primarily located at the C-terminal half of the DBD and confer an overall basic charge for the DBD.

A feature that distinguishes the DNA binding domain of plant HSFs from other eukaryotes, including yeast, mammals and Drosophila, is the deletion of 11 to 12 amino acids between position 67 and 68 (Fig. 2; marked by triangle). In addition to soybean, this feature has been conserved in tomato [27] and Arabidopsis [14] (Barros, Czarnecka-Verner, Baldwin and Gurley; unpublished data). Also interesting is the location of the single intron in the tomato LeHSF8 (K.-D. Scharf, unpublished, GenBank accession number X67599) and Arabidopsis AtHSF1 [14] genes 5 amino acids away between Tyr-62 and Gly-63.

Oligomerization domain

A compilation of OD s of all published plant H S F s is shown in Fig. 3. From this analysis two structural variants of the OD are evident. Type I in-

cludes the animal and yeast HSFs in addition to AtHSF1, LpHSF8, LpHSF30, and GmHSF21. Type II appears at present to be unique to plants and consists of LpHSF24, GmHSF5, Gm- HSF29, GmHSF31 and GmHSF34. Type I is distinguished from Type II by the inclusion of a glutamine-rich region between hydrophobic repeats 1 and 2 (HR1 and HR2). In Type II, HR1 and HR2 are adjacent. A second difference between plant Types I and II is the spacing between the OD and the DBD; Type I is separated from the DBD by only 12 to 26 amino acids, whereas this distance is approximately 50 to 74 amino acids in Type II HSFs. The spacing between the OD and the DBD in other eukaryotes (Type I) varies from 13 amino acids for hHSF2 to 66 amino acids for yeast ScHSF.

Plant HSFs show considerable amino acid identity among themselves within HR1, but dis- play only limited conservation in this area with yeast, Drosophila and human HSFs (Fig. 3, shaded boxes). There is no detectable amino acid similarity to the N-terminal portion of the HR1 repeat from yeast, or with HSFs from higher eu-

a d a d a d a d hHSF1 (137) ~T KLL T DVQI~KG~E C ~ S K~ImMI~

I hHSF2 (126) ~TKI I S SAQKVQIKQE TIE S P~S ELKS DmHSF (166) ~s KT LT DVIC3/MRGRQDNLD S RF SAMKC ScHSF (344) ~TAILGELEQI KYNQTAIS KDLLRIN~

a d GmHSF5 (170) SPPRGVAGVNNN~SSSNNYN~

GmHSF31 (111") SPPRGATSLVNGAAAANYNT~T~E

II ~., .s,=. ,1o:¢) NSSPIAGNHNNTVHRTTSCT~D GmHSF34 (134) STSSSSSGSKNQGSVETNTTP~H~S LpHSF24 (131) STSSPDSKNPGSVDTPGKLS~

a d a LpHSF30 (120) RRRNVGQSMNQQGS~

I LpHSF 8 (148) AQQQHQP~>GHSASVpAC'VgVGKF~E GmHSF21 ( 135 ) RRNVS QSLQQKGGSpACVEVGEF~G AtHSF1 (157) SQQLS QGQGSMAAL!S SCVEVGKFG~_.. ~

a

iii~ii a !/~ RRSNNN

RRSNN~ !::~ =

i!i!~i;i

...... t t~RDRNI

HR1

a d d a ~ a d a

~--~---~ ~o~umooo~z~ r.ZS~.VQS,RZ~.GWr~ZPU~NDSGS~SMPKYSRQFS ~re, svs~ ~ o ~ q v z ~ z v ~ ZVTr.VQm~Q,.~.Sr.r,~.~.r.,~NGAQK~LFQazvKE WI~I~ ~.QKI-IAKQQqIVNKLI( LITTVQPSRN~SGVIG~VQLMINNTPEIDRARTTSETES WQENM~ ~EP.HRTQQQ~LEKMFI LTSIVPHLDPK~IMDGLGDPKVNNEKLNSANNIGLNP, DN

a d a d a

3N ~QLKGLCNN~TJ

~SS rQAKKQCNEL~

~Q

~A ~Q

H R 2

d a d a d

~ SNNNNFP S FLLCSDNSN INNNT SPQTQTQ

VQNHVKPV~SNSYS SSLLLCNTPSATP I S SANNNPKQG

MTNYASGF ~QQLES S TSAARTVPVP EGKAALELLPAKH

LRDRLMVGP DQIDR~QGSCGSENVVGEGGGGDCLKLF

LSOYVKVAPDMINR~MSNGTP S GSS LEELVKEVGGVKDL

d a d a d a d a d a d

~L~QQQQS T D. G~QGMVQRLGGMELRQQQ~MS~SAKAVN S p GF LAQF~QQQ

'~1 ~ ° ° ~ " s ~ " . . . . . . . . . . . . . . . I ' " l i : : l . . . . . . . . . . . . . "'1"" ~___~QQQQTTDN~Q__VM~___LQ__V~_Q_R_Q MS: V NP F KQ

HR2

Fig. 3. Amino acid sequence comparison of oligomerization domains of plant and other selected eukaryotic HSFs. LpHSF24, LpHSF30 and LpHSF8 sequences are from Scharf et al. [26, 27] and AtHSF1 is from H~ibel and Schtiffl [14]. Human HSF1 sequences are from Rabindran et al. [21], human HSF2 from Schuetz et al. [28], Drosophila HSF from Closet al. [3], and Sac- charomyces cerevisiae HSF from Wiederrecht et al. [33]. Sequences are aligned by the conserved residues (stippled). Hydropho- bic repeats 1 (HR1) and 2 (HR2) are boxed. Positions a and d mark the hydrophobic amino acid 4,3-heptad pattern (abcdefg), characteristic of coiled-coil proteins. GmHSF21 cDNA is a partial clone truncated at the 3' terminus at the Eco RI site. The numbers in parenthesis indicate the starting amino acid of the H S F sequences counting from the first methionine with the exception (numbers with the asterisk) of GmHSF31 and GmHSF29 which are 5'-truncated clones.

43

karyotes. The HRls of plant HSFs seem to conform to generally derived rules which govern the leucine zipper formation in bZip proteins [ 1, 13]. The hydrophobic amino acid 4,3-heptad pattern (abcdefg)4_ 5 is clearly distinguishable, with conserved leucines at position d and other amino acids at the alternate hydrophobic position a. The preference for t -branched amino acids (valine, isoleucine, and threonine) is not strong in plant HSFs (Fig. 3). The e and g positions are thought to be the dominant determinants of multimeriza- tion specificity in coiled-coil interactions. In plant HSFs, as with other leucine zipper proteins, these positions are frequently occupied by charged amino acids: lysine, arginine, aspartic acid and glutamic acid. Although asparagine is known to destabilize coiled-coil interactions, it is often found in bZip proteins located at the a position of the third heptad repeat [ 13]. The destabilizing effect is thought to ensure correct alignment of repeats, preventing the less stable, partially over- lapping configurations from occurring. In yeast and other eukaryotic HSFs this asparagine residue is also highly conserved; however it is positioned at the a of the fifth heptad, while the a position of the third heptad is occupied by the invariant glutamine which functions likewise as a leucine zipper destabilizer (for compilation of HSF sequences see also Scharfet al. [25]). Sev- eral generalizations regarding HR1 structure in plant HSFs can be drawn from a comparison with those from other eukaryotes: (1) plant HRls have one heptad less than other eukaryotes; (2) hydrophobic amino acids other than leucine are frequently found at the d position of the fourth and fifth heptad; and (3) zipper destabilizing amino acids are often present in the a position of the first and/or second heptad in addition to those present in the third and fifth heptad of all characterized HSFs. Each of these differences would tend to make the plant HR1 interaction less stable than its counterpart in yeast, animal, or insect HSFs.

In contrast to the clear potential of HR1 to form coiled-coils, HR2 does not easily conform to the general description of leucine zippers (Fig. 3). It consists of only two to three 4,3-heptad

repeats in all characterized HSFs, and there is only limited use of leucine at the d position. The presence of two hydrophobic amino acids at the a and d positions (aa, dd; 'double zipper'), characteristic of other eukaryotic HSFs, is also seen in plant HR2s. The centrally located phenylala- nine residue is conserved, but the e and g positions show only limited occupation by charged amino acids. A further indication of low potential to form conventional zipper is the frequent oc- currence of the or-helix-breaking amino acids pro- line and glycine.

HSF carboxy-terminal heptad repeat

The C-terminal halves of HSF proteins show very little conservation between species; however, an additional hydrophobic repeat (HR3) is nearly always present (Fig. 4A). In higher eukaryotes this structural feature is thought to be involved in maintaining cytoplasmic localization of the HSF under basal conditions (basal repression) via a masking mechanism involving folding of the H S F protein [22, 29]. In yeast the role of the C-terminal heptad repeat (zipper C) appears to be different; HR3 (zipper C) overlaps the C-terminal activator but seems not to play a role in basal repression [2]. In soybean HSFs, HR3 is reduced in size, encompassing two to four heptad repeats which are destabilized by the presence of polar un- charged, or even hydrophilic, amino acids at the a positions (Fig. 4A). There is also some ten- dency for the short HR3s to exhibit the double- zipper configuration seen for HR2. The potential for 0~-helix formation of HR3s seems very low in plant HSFs due to the frequent presence of pro- line and glycine. Another unusual feature of many plant HSFs is the location of HR3 at the extreme C-terminus. A C-terminal HR3 implies that in many plant HSFs the activator must be N- terminal to HR3 as was shown in tomato [31 ], or overlap HR3 as is the case in yeast [2]. In other higher eukaryotes the activator is located between the C-terminus and HR3.

In GmHSF29 an additional 4,3-hydrophobic heptad repeat is located five amino acids down-

44

A B a d a d a d a d a d a d

hHSF1 (384) H LS D~AMD S~NLQT~ S HGF S~T SAL~F LpHSF24 (281) DYNG~ hHSF2 (360) [!l LLD~SIDC~DFQ~GRQF~DPDLL~LF LpHSF30 (I) (292) VADDJ DmHSF (578) MAS~ELHG~SMQD~TLKDL~GDGV~QNMLM~F LpHSF30 (2) (332) VKTP~ LpHSF30(316) ~QP~ '~ ' v~KTP]~ ;EELQE~)QLG~. LpHSF8(1) (447) GADI[ LpHSF8 (402) ~ MIMPE~SQLQG~ENNTD~GCDS~DTIAVE~M LpHSF8(2) (466) VGDP~

GmHSF34 (226) KLFG\ a d d a a d d a a a GmHSF29 (288~ DHDPI

GmHSF5 ( 3 4 9 ) ~LEND~GLNIAe~PST~ GmHSF33 (230~ DHDPr GmHSF31 (28~ ~EKD~GLNLMPPSTC GmHSF5 (162) VIIP} LpHSF24 (212) LE~EVG~LEEQGSYND GmHSF31 (i0~ DDQS~

GmHSF29 (204~ AG~GAAP~ANAGE~EVPKL~S Trp repeat

GmHSF34 (252) MGF G GP R~KE SKP V~F GA~S NR~CN d a d a d a a d a d

a d d a

MR3

IKMSSPA :ELLSED ~ELQDL )SGLLDE ~KFLQSP ~KGDTLT ~ELG~

~DSPPRG ~DSPPRG

C mHSFI (425) P~AENS~DS~QLVHYTAQ~L~LLDPD~SE~LFELGES GmHSF34 (236) T~TDKRN~K~D~MGFG~SK~SNRVC_N I

mHSF2 (473) DPEPT~LVI~LEPLTE~~LD SD~LLD S

GmHSF5 (324) VS. r .~VHPEYGS~t~ I~DI~LGLNr . I~PSTC_ . I GmHSF31 (26d) KTKLFGVSL~KRVHPDCGSNP~T~EKDDLGL~STC I Gm~F2~ (25~) MQTRAQAQTQTQSSQEPDHGSDVKSEPLDGDDSDDQDHDPRWLELG_K

AD

Fig. 4. Amino acid sequence comparison of the third hydrophobic repeat (HR3) (A), tryptophan repeat (B), and putative HSF activation domains in plant and other selected eukaryotic HSFs (C). Positions a and d, and numbers in parenthesis are as described for Fig. 3. The tryptophan residue and d positions are stippled. Tomato LpHSF30 and LpHSF8 contain two consecu- tive Trp repeats in their C-terminal domain designated [1] and [2]. Mouse HSF1 and 2 activation domains were reported by Morimoto and colleagues [ 18]. Position of first amino acids of selected sequences are indicated in parenthesis with an asterisk indicating numbering from the beginning of the partial clones. C-terminal amino acids in HSF proteins are underlined. In C, boxes indicate amino acids having similar properties and the locations of HR3 within putative C-terminal activators.

stream from the DBD. This repeat also seems to possess a very reduced potential to form a coiled- coiled structure since it does not contain leucine residues but is enriched for alanines and valines instead.

Trp repeat motifs

The Trp repeat was shown by Treuter and colleagues to be involved in transcriptional activation of three tomato HSFs [31]. This motif is comprised of a single tryptophan residue which is usually embedded in a region of negative charge. In tomato HSFs the Trp repeat is found in one to two copies within the C-terminal half of the protein. Similar sequences are present in soybean GmHSF34, GmHSF29, and in a nontranslated portion of the pseudogene-like GmHSF33 (Fig. 4B); however, in soybean HSFs the Trp

motifs are not repeated. In the two closely related GmHSF5 and GmHSF31 proteins, a putative Trp repeat is located between the DBD and HR1 instead of in the more common C-subterminal position. The significance of the Trp motif in soybean HSFs has not been determined, although it seems likely that some may be transcriptional activator motifs as in tomato.

Morimoto and colleagues report that in mouse HSF1 and 2 the activation domains are C- terminal to the fourth leucine zipper, acidic in nature, and function as constitutive activators when juxtaposed to the Gal4 DBD [18]. When the C-terminal domains of soybean and mouse HSFs are compared, only slight similarity in amino acid sequence can be seen between mouse HSF1 and GmHSF34, and between mouse HSF2 and GmHSF5 and 31 (Fig. 4C). As with mammalian HSFs, the activation domains of the three tomato HSFs contain a concentration of

negative charge. In contrast, the C-terminal regions (from HR2 to the C-terminus) of soybean HSFs, although enriched for charged amino acids, are not strongly acidic overall. The net charge is negative only in the putative AD of Gm- HSF29, nearly neutral in GmHSF5, weakly basic in GmHSF31, and highly basic in GmHSF34.

HSF relatedness

The six soybean HSFs can be placed into four groups based on amino acid sequence comparisons. The relationship between these groups is illustrated in the phylogram shown in Fig. 5. Overall, the parsimony analysis suggests that all characterized HSFs from plants can be assigned to two major groups. These groups correspond exactly to the Type I and Type II classification proposed for the OD based on the organization of HR1 and HR2 (Fig. 3). In order to see the two major groups of relatedness, the parsimony analysis was conducted using the bootstrap method with 100 repetitions at the 50~ confidence level. A total of 69 positions (amino acids plus gaps in the case o fhHSF1) within the DBD (positions 37 to 94, Fig. 2) were used in the analysis to enable a comparison to be made that included both full length and partial cDNA clones.

LpHSF30 SF21

I 1 ~ AtHSF1

j z ~ G m H S F 2 9 I 21 i GmHSF33

6 ~ GmHSF5 20 ~ GmHSF31 I I

3 ~ GmHSF34 4 LpHSF24

hHSF1 Fig. 5. HS F consensus tree from parsimony analysis of partial DNA binding domains. The analysis was conducted using the PAUP software with 100 repetitions according to the bootstrap method at the 50% confidence level. Human HSF1 was included as a point of reference. Amino acid positions 37 to 94 (Fig. 2) within the DBD were used to allow comparison of partial cDNA clones. Numbers indicate differences in amino acid identity.

45

It is clear that cDNAs GmHSF29 and 33 are closely related as well as cDNAs GmHSF5 and 31. In addition, clone GmHSF34 can be grouped with LpHSF24, and GmHSF21 is related to LpHSF30. The GmHSF5 and GmHSF31 amino acid sequences not only share identity in the DBD, but several blocks of 100 ~o identity can be found within the remaining portion of both proteins, including the C-terminal 11 amino acids (Fig. 1). A similar analysis conducted at the 80~o confidence level (not shown) maintained the same assignment of the 6 soybean HSFs into four relatedness groups, but information regarding the existence of the major types I and II was lost.

An examination of open reading frames of clones GmHSF29 and GmHSF33 suggests that these two genes have diverged recently. Within the DBD sequences available for analysis they differ only by one amino acid, and amino acid sequences of the OD are identical, with one exception, up to amino acid 108 of GmHSF33. At this position a deletion of 69 amino acids seems to have occurred in GmHSF33 which changed the reading frame resulting in a truncation of the C-terminal half of the protein (Fig. 1). The original reading frame past the point of deletion is still present in the untranslated 3' tail of the transcript and is very similar (including the subterminal Trp repeat) to that of GmH S F29. Differences between GmHSF29 and the silent C-terminal portion of GmHSF33 include 12 substitutions, one 3 amino acid deletion, and one 2 amino acid insertion. Despite the strong overall amino acid identity between GmHSF29 and GmHSF33, the DNA sequences differ in the codon wobble position. This pattem of relatedness suggests that the original gene was duplicated and functioned long enough to pick up point mutations at the wobble base positions. At some later time GmHSF33 acquired a series of deletions and insertions that prevented translation of the OD and C-terminal domain, but maintained the DBD. We have not designated GmHSF33 as a pseudogene since the derived protein may still be able to bind the promoter of heat shock genes and influence transcription.

46

Characterization of HSF gene organization and expression

The genomic organization of soybean H SF genes was analyzed by Southern blot hybridizations (Fig. 6). Six replicate filters containing soybean genomic D N A digested with restriction enzymes Eco RI or Hind III were hybridized with cDNA probes as described in Materials and methods. The restriction digest patterns were different for each clone with the exception of the closely related G m H S F 2 9 / G m H S F 3 3 pair. In view of strong DBD conservation between HSFs, some of the weaker bands may reflect cross-hybridization since most probes, with the exception of Gm- HSF29, contained at least a portion of the DBD. Although the H S Fs constitute a gene family, these results indicate that the individual members represented by the cloned H S F cDNAs are present in low copy number within the soybean genome.

The levels of the respective H S F mRNAs were examined in 3-day old soybean seedlings which

Fig. 6. Genomic analysis of six soybean HSF genes. Soybean genomic DNA was digested with Eco RI (E) or Hind III (H). HSF probes used for Southern hybridizations were as described in Materials and methods. The band sizes below each lane were estimated by mobility extrapolation of molecular weight markers (lambda DNA Hind III digest and ~X174 DNA Hae III digest).

were either kept under control conditions, or were heat- or cadmium-stressed (Fig. 7). Individual northern blots were hybridized with H S F probes (Fig. 7A), then stripped and rehybridized with the soybean actin probe as a loading accuracy control (Fig. 7B). H S F m R N A expression patterns were consistent with our previous classification into four relatedness groups. G m H S F 5 and Gm- HSF31 mRNAs were very low in abundance and constitutively expressed. In addition, both heat and cadmium treatments resulted in a decline in m R N A levels. GmHSF29 and GmHSF33 mRNAs were detectable under control conditions, heat-inducible, and not affected by cadmium treatment. GmHSF21 mRNA was not detectable in control tissue, and was fairly well induced by heat and to a slight extent by cadmium. GmHSF34 mRNA seemed to be the most abundant of all tested HSFs in the control seedlings and was readily induced by heat and cadmium shocks. In addition, both stresses caused the appearance of a higher-molecular-mass RNA homologous to the GmHSF34 probe. Since all three tomato L p H S F genes and AtHSFI from Arabidopsis contain introns, it is possible that a similar situation exists in soybean with the upper bands representing unspliced m R N A [14, 26]. The inhibition of intron processing by cadmium treatment was found previously in other stress- induced soybean genes [7]. From northern blot analyses we conclude that the phenomenon of heat inducibility of H S F mRNAs is not unique to tomato and Arabidopsis, but seems to be a common feature of plant HSFs.

To establish whether the cDNA clones did in- deed code for functional HSFs , the open reading frame of GmHSF34 was overexpressed and purified from Escherichia coli cells. The ability of the recombinant protein to bind the HSE was tested in the EMSA using HSE1, HSE2, and AT composite double-stranded oligomers as specific or nonspecific competitors of binding (Fig. 8). The size of the recombinant G m H SF34 was estimated by SDS-PAGE to be 37.4 kDa which is slightly higher than the calculated molecular mass of 31 194 Da with allowances for the histidine tag (2 kDa) introduced by the pET15b vector (Fig. 8A).

47

Fig. 7. Levels of HSF mRNAs in soybean seedlings. Panel A shows the results of hybridizations to six HSF probes and panel B to a soybean actin clone. Poly(A) RNAs are as follows: control (lane 1), heat-shocked (lane 2), and cadmium chloride treated (lane 3). Blots in panel A were aligned according to the position of actin RNA as indicated; hence, the positions of HSF RNA bands reflect their respective size. The amounts of poly(A) RNA loaded in all three lanes were identical as judged by optical density estimation and ethidium bromide staining of ribosomal RNA bands. Cadmium treatment caused a decline in the synthesis of actin RNA.

Fig. 8. SDS-PAGE and electrophoretic mobility shift assays with E. coli expressed GmHSF34. A. SDS-PAGE was done according to Laemmli [ 17]. An aliquot of approximately two micrograms of recombinant GmHSF34 (lane 1)was electrophoresed on a 12% polyacrylamide gel with Rainbow markers (Amersham; myosin 200 kDa, phosphorylase b 92.5 kDa, bovine serum albumin 69 kDa, ovalbumin 46 kDa, carbonic anhydrase 30 kDa, trypsin inhibitor 21.5 kDa, lysozyme 14.3 kDa) (lane 2). Gel was stained with Coomassie blue for 30 min, destained overnight, dried, and photographed. B. EMSA. After a 15 min incubation carried out at room temperature, recombinant GmHSF34 complexes with HSE1 probe were resolved on 4% polyacrylamide gels. Nonradioactive DNA competitors were included in the reactions at 10- and 100-fold molar excess: HSE1 (lanes 2 and 3), HSE2 (lanes 4 and 5) and AT composite (lanes 6 and 7). Lane 1 contained no oligomer DNA as competitor. F is free probe and B is probe bound by HSF.

Similar anomalous SDS-PAGE migration patterns were noted for most of the HSFs studied and are thought to be due to the unusual, elon- gated, non spherical shape of H S F molecules [ 20 ]. Recombinant GmHSF34 was capable of specific HSE binding as shown in Fig. 8B since only HSE1 (lanes 2 and 3), and to lower extent HSE2 (lanes 4 and 5), oligomers competed binding. The AT composite oligomer, known to be the target of AT-rich sequence binding proteins (ATBPs) and high-mobility-group proteins (HMGs), did not cause a decrease in HSE-HSF complex formation (lanes 6 and 7) [6]. As with Drosophila HSF, bacterially expressed GmHSF34 did not require heat shock activation in order to form a complex with DNA indicating that it also has an intrinsic affinity for DNA [3].

Discussion

We have cloned a total of six HSF cDNAs from soybean. Although four out of the six represent partial clones, we have been able to establish their relatedness to each other and to HSFs from tomato, Arabidopsis and man. From parsimony analysis plant HSFs are not closely related to any

48

of the HSF forms present in other eukaryotes (HSF1, 2, or 3). Figure 5 indicates that five HSF relatedness groups can be seen in tomato, Arabi- dopsis and soybean with each group containing two members at present. None of the plant species characterized appear to contain representatives for all of the classes suggesting that the specialization of HSFs may have been relatively recent. Unpublished data from our laboratory indicate that two HSFs from Arabidopsis are in the group containing GmH S F34 and LpH S F24, with more similarity to LpHSF24 in the DBD (Barros, Czarnecka-Verner, B aldwin and Gurley, in prepa- ration). Arabidopsis AtHSF1 is most closely related to LpHSF8 [ 14].

Several distinctions between plant and other eukaryotic HSFs can be drawn based on amino acid sequence analysis. The most characteristic feature appears to be the presence of 11 additional amino acids in the DBD of animal and Saccharomyces cerevisiae HSFs, and 12 amino acids in the case of Schizosaccharomyces pombe. The absence of these amino acids in plant HSFs suggests that this region may not be critical for DNA binding in animal HSFs. The deletion is positioned immediately C-terminal to the region of sigma 3°-7° homology and may demarcate the boundary between the DNA binding domain proper and the adjacent conserved region. Recent studies by Flick et al. suggest that part of the conserved region C-terminal to the point of the deletion/insertion may not function directly in DNA binding [10]. Mutation experiments reduced the definition of the DNA binding domain in yeast ScHSF from 110 amino acids to 89, excluding 21 amino acids located at the C- terminal end which correspond to positions 78 to 98 in Fig. 2 [ 10]. In a truncated protein containing exclusively the DBD (forms monomer only) these amino acids were not necessary for DNA binding activity, but were indispensable for DNA binding by the trimeric form of full-length HSF. This region may function as a flexible linker between the DBD and OD allowing for proper ori- entation when the trimeric form is bound to DNA [10]. Other evidence suggests that this potential linker domain may also be specialized for nuclear

localization. Mutational studies with human HSF2 indicate that all, or part, of this region may be required for transport to the nucleus [29]. The split configuration of clustered basic charges suggests that the NLS associated with the DBD may be bipartite in the case of LpHSF24, GmHSF34, GmHSF5, GmHSF31, GmHSF29 and Gm- HSF33 (underlined in Fig. 2). The presence of other regions of bipartite positive charge suggests that additional NLSs may be located in the HR1 of the oligomerization domain and in the C- terminal domain of soybean HSFs.

The oligomerization domains in plants and other eukaryotes show two general patterns of organization. Both configurations are bipartite, but in one, the two regions of hydrophobic repeats (HR1 and HR2) are separated by intervening amino acids enriched for glutamine. In the other, HR1 and HR2 are juxtaposed (Fig. 3). In most eukaryotes HR1 and HR2 are separated by intervening amino acids (type I), whereas tomato and soybean HSFs show both types of organization. The functional significance of these two configurations is not known.

In general, the size of plant HSF proteins ranges from 30 to 50 kDa which is smaller than those of yeast, Drosophila, or chicken HSFs (60 to 110 kDa) [3, 19, 26, 27, 33]. The most dras- tic reductions in plant HSFs are seen in the C-terminal domains (with the exception of LpHSF8) which terminate immediately after HR3. The large reduction in this region raises questions regarding the location of transcriptional activation motifs. Regions of similarity with mammalian activation domains overlap the C- terminal heptad repeat of soybean HSFs which suggests that some plant activation domains may be similar in organization to the C-terminal activator of yeast H S Fs where zipper C overlaps the activator [2]. Although the Trp repeat has been identified as the activation motif for tomato HSFs, it only exists in a single copy in GmHSF29 and GmHSF34, and is not present in the C-terminal domains of GmHSF5 and Gm- HSF31. Therefore, it seems likely that additional amino acid motifs may function as transcriptional activators in soybean HSFs. Another possibility

is that some plant HSFs may not have an activation domain and function as repressors by competing with activator HSFs for binding sites in heat shock promoters.

We have examined the patterns of expression of the six soybean HSFs under basal, heat shock, and heavy metal stress conditions. The general conclusions being that most HSF genes are heat inducible, and that some can be elevated in expression by cadmium stress. The expression of the two constitutive soybean HSFs (GmHSF5 and GmHSF31) is generally very low compared to the heat inducible ones. Similarly, in tomato tissue culture, mRNA for constitutively expressed LpHSF8 was not detectable after 2 h of heat shock at 39 °C, and was 10-fold less abundant than mRNA corresponding to the two heat inducible HSFs [25].

For the thermally inducible HSFs of tomato, the transcriptional response is similar to that of HSP genes, showing a rapid and transient induction [25]. However, expression at the transcriptional level does not necessarily imply transla- tional expression. In tomato tissue culture cells an increased production of H S F mRNA during heat shock did not correlate with an increase in HSF protein synthesis [25]. Generally accepted mod- els regarding regulation of the heat shock response by HSF have the levels of the receptor of heat stress (the HSF) constant between basal and heat shock conditions. Upon heat shock a change oc- curs in the activation state of the pre-existing HSF(s), not in HSF abundance. In plants this condition may still be met if the induction of H S F mRNA by high temperature does not lead to increased translation of HSF protein as suggested by the observations in tomato. If this is generally true, plants may be similar to animals in that the levels of HSF proteins do not vary substantially between basal and stress conditions; however, the significance of HSF mRNA induction still remains to be determined.

The significance of multiple HSFs in plants is not clear since yeast and Drosophila perform well with only a single HSF and mammals with two. In human cells there is clear specialization ofH SF function; HSF2 is specifically induced (DNA

49

binding) by hemin treatment of human erythroleukemia cells, and HSF1 is primarily activated by heat shock [30]. Although in plants there is no information regarding developmental specialization in HSF expression, in tomato the three HSF genes showed differing degrees of induction to heat stress (zero to five-fold) and exhibited promoter specificity in the level of reporter gene activation in transient assays when various heat shock promoters were tested [25]. Soybean has more HSF genes than previously reported for other organisms which may be partially explained by its tetraploid genomic composition. Multiple HSFs may have become adapted for particular roles in the heat shock response by specializing in terms of: (1) heat shock promoter specificity, (2) strength of the activation domains, and (3) timing of expression before and during stress. There is already evidence supporting differential promoter specificity in tomato [31 ], and differential patterns of expression in tomato and soybean; however, a comparative analysis of activation domains has not been exhaustively conducted. Although each of the characterized tomato H S F s appears to have transcriptional activation motifs in the C-terminal domain, the situation in soybean is not as clear.

Acknowledgements

We express our appreciation to Archana Nair and Bat Cai Tan for assistance in screening cDNA libraries, and we thank Don Baldwin for critical review of this manuscript. We also thank Dr. Robert Ferl for assistance with the parsimony analysis. This project was supported by NIH grant RO1 GM39732 to WBG. Florida Agricul- tural Experiment Station Journal Series number R-04279.

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isolation and characterization of six heat shock transcription factor cdna clones from soybean

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