Planta (1994)193:57-66 P l ~ t l " l t ~

�9 Springer-Verlag 1994

Heat-inducible rice hsp82 and hsp70 are not always co-regulated Frank Van Breusegem, Rudy Dekeyser*, Ana Beatriz Garcia, Bart Claes**, Jan Gielen, Marc Van Montagu, Allan B. Caplan ***

Laboratorium voor Genetica, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Received: 2 July 1993/Accepted: 18 October 1993

Abstract. We have characterized several heat-shock-in- duced genes in rice (Oryza sativa L.) and compared their expression under a variety of conditions. Three of these genes, which are analogs of the hsp82/90 family, lie within a cloned 18-kilobase (kb) region of the genome. The mid- dle member of this cluster, designated hsp82B, has been fully sequenced. The gene uses a promoter containing six putative heat-shock elements as well as several unusual sequence motifs including a stretch of 11 thymidines al- ternating with 11 adenosines. The m R N A for this gene reaches its highest relative level of expression within 120 min after plants are shifted to 42 ~ C; no other condi- tions induce this gene. By contrast, we found that during heat stress the expression of hsp70 correlates well with increases in internal ion concentrations, and can also be induced by excess salt or ethanol at normal growth tem- peratures. These results appear to indicate that whereas hsp70 is induced by all stresses that lead to protein denat- uration including heat s t ress- HSP82 m R N A accumu- lates only upon heat stress.

Key words: Gene hsp82 - Gene hsp70 - Heat shock - Oryza

Introduction

The cell's ability to sense and respond to high tempera- tures is one of the best characterized of all the defenses an organism uses to adapt to a constantly changing

Present addresses: * Instituut ter bevordering van het Wetenschappelijk Onderzoek en Technologic in Vlaanderen, B-1000 Brussels, Belgium

** European Patent Office, D-80298 M/inchen, Germany

*** Department of Bacteriology and Biochemistry, University of Idaho, Moscow, ID 83843, USA

Abbreviations : HSF = heat shock factor; HSP = heat shock protein; kb = kilobase

Correspondence to: M. Van Montagu; FAX: 32 (9) 2645349

environment. At elevated temperatures, synthesis of most genes ceases in favor of transcription and transla- tion of a well-defined set of heat-shock proteins (HSPs; Ashburner and Bonner 1979).

The proteins induced during heat shock differ slightly in pattern and abundance between species, but fall into a few major classes distinguished by their molecular weight: H S P l l 0 , HSP90, HSP70, HSP60, and low-mo- lecular-weight HSPs. Each of the classes of heat-shock genes has been described in a wide variety of organisms including plants, but much remains to be learned about how these gene products help contribute to survival at high temperatures (Lindquist and Craig 1988; Vierling 1991).

The defensive role of the class designated HSP90 re- mains poorly understood by comparison with what is known about many of the other species despite the fact that it makes up almost 0.1% of the cellular protein (Lindquist and Craig 1988). This family encodes a set of structurally related proteins ranging from 82 to 90 kDa. They have been isolated and characterized from a variety of evolutionarily diverse species, including Escherichia coli, where the equivalent is officially named h tpG (Far- relly and Finkelstein 1984; Blackman and Meselson 1986; Bardwell and Craig 1987; Hickey et al. 1989; Conner et al. 1990). Studies of hsp82/90 and their tran- scripts have shown that plant genes, like their mam- malian counterparts, are developmentally regulated in addition to being controlled by temperature (Koning et al. 1992; Takahashi et al. 1992).

In eukaryotes, HSP82/90 is present in both nucleus and cytoplasm (Gasc et al. 1990) where it has been found associated with a very heterogeneous collection of pro- teins. In mammalian cells, HSP82/90 is most often asso- ciated with actin and tubulin (Sanchez etal . 1985; Koya- s u e t al. 1986), several different protein kinases (Zie- miecki et al. 1986; Rose et al. 1987; Miyata and Yahara 1992), and with steroid hormone receptors (Sanchez et al. 1985; for review, see Baulieu et al. 1990). In many ways, HSP82/90 appears to be behaving as a scaffold that aids steroid receptors to fold properly and form an active

58 F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes

molecule (Picard et al. 1990) and aids protein kinases to assume a structure that optimizes activity (Ziemiecki et al. 1986; Miyata and Yahara 1992). The protein contains two regions of extremely high negative-charge density which otherwise show little sequence conservation. Since the amino-terminal region is completely missing in the E. coli equivalent (Bardwell and Craig 1987), this domain may be involved in interactions with these exclusively eukaryotic proteins.

Although cells without any HSP82/90 cannot produce active steroid receptors (Picard et al. 1990; Cadepond et al. 1991) or survive high temperatures (Borkovich et al. 1989), overproduct ion of the protein is not sufficient on its own to protect yeast cells from being killed by heat (Cheng et al. 1992).

This contrasts greatly the effects of overproducing the bet ter-known protein, HSP70. Constitutive expression of this highly conserved gene enhances thermotolerance (Angelidis et al. 1991 ; Li et al. 1992). The protein HSP70 and related proteins encoded by this large gene family probably fulfill several functions in the cell, one of which is to facilitate protein folding during membrane trans- location (Chirico et al. 1988), and others might be mon- itoring the progress of these reactions and the accumula- tion of degraded or misfolded proteins (Craig and Gross 1991). Baler et al. (1992) have shown that a member of the HSP70 family binds to the heat-shock regulatory factor (HSF) and influences the mechanism that controls activity of the HSF and so plays an important role in the transcriptional regulation of heat-shock genes.

The entire set of HSPs appears to be controlled by a small family of heat-shock regulatory proteins that bind to a transcriptional control element present in the pro- moters of the different hsp genes (Pelham 1982; Scharf et al. 1990). A priori, one might, therefore, assume that all HSPs would be induced coordinately. However, some studies contradict this view. Lindquist (1980) found that HSP82/90 reaches its highest levels of expression during temperature shocks of only a few degrees whereas HSP70 synthesis increases continuously as the magnitude of the temperature shift increases. More recent studies have found that HSP90 m R N A levels declined while HSP70 m R N A levels increased when nematodes recovered f rom a quiescent dauer larva to an actively feeding form (Dal- ley and G o l o m b 1992). The relative amounts of the hsp70 and hsp90 gene products have also been seen to differ during normal growth. Rats, for example, have higher basal HSP70 levels in males than in females, yet similar amounts of HSP90 (Olazabal et al. 1992). Presumably, the differences in response to the same stress reflects dif- ferences either in the way a single signal is transduced into transcriptional activity or in the generation of multi- ple signals. In fact it is not immediately clear what these signals are. Hea t leads to the denaturat ion of proteins directly. In plants in particular, it also increases evapora- tion and thus duplicates conditions associated with drought. Either of these forms of damage could trigger product ion of a second messenger for gene induction, or alternatively, could directly activate the heat-shock regu- latory proteins by forcing a change in conformat ion or in protein-protein associations. Sudden shifts in tempera-

ture can also lead to metabolic imbalances, and perhaps these imbalances are responsible for the heat-shock- factor-independent induction of some yeast genes (Kobayashi and McEntee 1990). Similarly, the inadver- tent formation of active oxygen species as the activity of some proteins changes might account for the increased accumulation of superoxide dismutase in heat-shocked tobacco (Tsang et al. 1991).

We have isolated several heat-shock-induced genes f rom rice and compared the expression of two, hsp82 and hsp70, under a variety of physiological conditions. Our conclusion is that the two gene families are not induced by the same perturbations. Whereas hsp70 is induced by several stresses - including heat stress - that lead to protein denaturation, HSP82 m R N A accumulates only upon heat stress.

Materials and methods

Plant material. Rice plants (Oryza sativa L. var. &dica, cv. Taichung native 1) used for experiments were grown at 27 ~ C on vermiculite saturated with water for one week. Plants were then transferred to bottles or tanks and cultured hydroponically with Hoagland medium (Hoagland and Arnon 1938) for 14 d in a 12-h light/12-h dark regime. The induction studies were carried out by adding the appropriate concentrations of each product, noted elsewhere, to the medium. Conditions for heat shock (42 ~ C) were provided by a Weiss growth chamber in light.

Construction and screenino of cDNA libraries. A cDNA library was prepared from poly(A) + RNA isolated from rice roots stressed with 1% Murashige and Skoog salts (Murashige and Skoog 1962) for 3 d using kits obtained from Pharmacia (Uppsala, Sweden) and Prome- ga (Madison, Wis., USA). After second-strand synthesis and methylation, EcoRI linkers were added. The cDNA was purified from excess linkers according to the manufacturer. The cDNA was cloned into the EcoRI site of pSP65 (Melton et al. 1984) and transformed into SC5-1 bacteria. The cDNA library was analyzed by differentially screening with labeled first-strand cDNA obtained with mRNA from control plants versus RNA from plants treated for 4 d with 1% Murashige and Skoog salts.

Fig. 1. Southern hybridization of nuclear rice DNA. Rice DNA (cv. Taichung native 1) was digested with BamHI, EcoRI, and HindIII and probed with the 21G3 cDNA (see Materials and methods). Sizes of hybridizing bands are indicated (kb)

F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes 59

TCTAGATTGTA•TAAATGTTT•GTT•CAAATTAGTTTTGCTGAC•AACGAA•CTAGGC•GTGTTCTTTG•AATACTATTC•AACTTCAATAACTCATAAC 100 TCATTTGTCACACGCACATTT~TCAAACTATC~ACGTAATTTGTTTCTATGAAGTTTGATAAAAATATTGTTTTA~AATCATATTAATATGTATATAT 200 ATATATATATATATAAGTTTTTTAATTGATACTTAATTAATCGTGTCAATAGGTTGTTTTGTTTTGCGTGTTACGAGGAAAGGCTTCC~CCTTAACCAA 300 ATAACATAACCCTAAAGATAAAGA~CACAAGT~TTcA~TCG~TTCCAGAACCTT~TTGCATCTAAT~G~GATGTGCAGTTGAA~TTCCAAAATTCATC 400 CTGTAATTAAGTTGAACCTTCTTGCATCTAATTTG~GATTTTTTCTATTTTGcA~GATGCTATTCGAATGACAGTTTTAGTGATATTTTTGT~TTTTT 500 ~AGTGG~AGTTTTACAATAACGATTTAAAGCAGTGGTAAATTTATAATTGccc~TTA~AAGAATTATAGTATT~AcTATT~CCATACAGTA~TAGACcAT 600 AGAGATGATGGTTTTCTTAATTTAAACcGACTCCAAGCCTCTCCA~TCCTCCAGAAC~TTCCAAAATATAAAAA~ATAACCAACCGACTGACATGTGGG 700 CCCGACGACTCGTCAAGTCGTCCATCTCCCAACTC~CCCG~TGGTTCGA~CCCCTGGTAGAACGTTCGAGAACCCCCATCTCCC~CTTATATATCGCCCC 800 CTTTTTCCCCATCTTTCT~CAATCGAATCGCACACGCCACGCGAGCTCCCTCTCCTAAAGcC~TAACCCTAGCCTCCAAGTCGTCGTCCTCCGGAAGCTT 900 CCGGCCTCCGCCGCCGCCGCAGCGCCATGGCCTCGGAGACGGAGACGTTCGCCTTCCAGGCGGAGATCAACCAGCTGCTGTCGCTCATCATCAACACGTT 1000

M A S E T E T F A F Q A E I N Q L L S L I I N T F CTACTCCAACAAGGAGATCTTCCTCCGCGAG~TCATCTCCAACTCCTCCGATGTAAGTGCCCTCGGTTTACTGGTTCAGATCTGCACGCG~GCGCGTTAG 1100

Y S N K E 1 F L R E L I S N S S D AAGGGCTTTTTTTTGGTCCGATTGGTAGATcGGTTTGTTcACCC~TCTGGTTCGAGcTGA~TCGTGTGTGTGTTGTTATGTGTGTGGGTGTGTGAGGGA 1200 GTTTGCACTGcAGcACTGGTTA~TGGTTAcTGAGAcATcATcCcAACATAAATCTGCGATGTGGTGTTTcACTcCCCATTTTTT~GATTATGTTTcGAT 1300 TccGACGTTAATTACGTTTcTCAGTGTATcAATCTGTGGATCATGCTcGcAGTACTTAATTTTGTTAGTGT~TAATcTGT~ATCATGCGGT~CTTAAT 1400 TTTGTTAAGAGTcCTG~TTTTG~TGCTGATTAGAATGTTAT~A~ATAACTGCAGGcATAGGCAGcATTATCTGTcATTGTTcTGCT~TT~T~TCGT 1500 AG~ATTATTTACGATTGCTcTGATAAAGTcGcATcTG~AAGTCT~AcATGCTTTGAGTGTTGATGTTTAAATGTTGTAAAA~ATATCC~cTATA~cAT 1600 AGAGGGTTGTTATTTTGcTAGTTATCATTTATTTGT~ATTTATTTTGcTAGTTATGTcAcATTCT~ATTGATAGGAGGGTTTGAcCATATATAAGACAA 1700 AAA~AcccGACTAGTGCTTTATTTTGCTGATT~ATTGTTAAAATTGccTCTATGA~cAAAAACAc~cAACcAGTGcTTTACTTTGCTGATTcATTGTTA 1800 AAATTGAccCTATGGAAcAAAAAcA~CcAAcCAGTGCTTTA~TcTGcTGATTCATTGTTA~ATTGcc~TATGAAcTGAGGATTGGTcTGGTTCTcTAT 1900 CcTGCCAAAAATGTGGTAAAT~CCGAAATcTTTGGAGTTGcT~ATTTGGGTAGGGTAGAGATGTccCATTTGGTGTCAAGATTGAGcTGTGAGAATAGA 2000 TGGGGATAG~TGAACTTTTGGTGTGGAAAcATTATT~ATTATTGTcGGATAGTGAATTGGAATTGcTAGCTGCTTTcATAC~AACcGcccACTTATCT 2100 TAT•CTTTTGTGTTCTGcAGG•GTTGGAcAAGATCAGGTT•GAGAGCTTAACGGAcAAGAGCAAGcTGGATG•TcAGCCAGAGCTGTTcATCCATATTGT 2200

A L D K | R F E S L T D K S K L D A Q P E L F I H I V TCCTGACAAGGCCTCCAACA•ACTGTCGATCATTGACAGTGGTATTGGTATGACCAAGTCAGAT•TTGTGAACAACCTGGGTACCATTGCCAGGTCAGGG 2300

P D K A S N T L S I I D S G I G M T K S D L V N N L G T I A R S G ACTAAGGAGTTTATGGAGGCACTGGCTGCTGGTG•TGATGTGTCCATGATTGGTCAGTTCGGTGTTGGATTCTACTCTG•CTAC•TTGTTGCCGAGAGAG 2400 T K E F M E A L A A G A D V S M I G Q F G V G F Y S A Y L V A E R

TAGTTGTGACCACCAAGCACAACGATGACGAGCAGTATGTGTGGGAGTCTCAGGCTGGTGGGTCCTTCACTGTCACACGTGACACATCTGGGGAG•AGCT 2500 V V V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E Q L TGGGAGGGGTACTAAGATCACCCTCTAcCTCAAGGATGATCAGGTACTAACAGAATTTcATTGAATTCTTGTTCAATTTGTGCTGCTTGTATTGCTGAAT 2600

G R G T K I T L Y L K D D Q GCCCTTATAGTT~ATGAATGTGG~TTGCTATTGATAAATGCATCAACTAGTAATTCTGTC~TCGTAGGATTATTACGAATATTGTTGGTACTTGATTTTA 2700 TAGCCACTTTTAGAAAGAA~ATGTTGATGTGCCTCAAGTTTTCACCATAGGCATGCTTGAGCCAGGTGCTTAATCGGTCTGTTTAAGACATAAAAAATGT 2800 CT~TATGTAGGGGCATTTGGTGAATTGATTGAACAGAATGAA~ATATTTCA~ACACTGTTGACATTGTGATAATCTAGATAGATGCCTTAACATTTCGAA 2900 •TC•TGCTGTGAAAATTATGTTAGTATGTTTCTGCCTTGGATGTTATGAGTAATCATGTTACACATGTACTTAGGATCTATAAGCTTATTGTTGAGCTCA 3000 TAAACTGATTGTGTATGCATTTTTTTTTCTTTGGGTCTGGTTTTGTGTCAGTTTGATTG~GTGTGGTTGTCTAATACTTCCTCCGTTTCACAATGTAAGT 3100 CATTCTAGCATTTCCCATATT•ATAGTGATATTAATGAGTTTAGACATAT•TCTCTATCTAGATTCATTAACATTAATATGAATATAGGAAATGTTAAAA 3200 TGACTTACATTGTGAAA•GGAGGGAGTACTATGAATTAATTAGTAACAAGTATTACTGTTCTACTGTTGTGAATTTTTAGAATCACCTGAACT•CTAAGT 3300 TGAACTGTGAAATGTG~TATAAGGTGTCCTGGCTCTAATGT~TTCATTTGTTATTGACAACTTG~TCATG~TTCCTGCAGTTGGAATACCTTGAAGAGCG 3400

L E Y L E E R CCGCCTCAAGGACCTGATcAAGAAGCACTCTGAGTTTATCAG•TA••CTATCTCTCTATGGA•TGAGAAGACCA•TGAGAAGGAAATTTCTGATGATGAA 3500

R L K D L I K K H S E F I S Y P I S L W T E K T T E K E I S D D E GATGAGGAAGAGAAGAAGGATGCTGAGGAGGGGAAGGTTGAGGATGTTGATGAAGAGAAGGAAGAAAAGGAGAAGAAAAAGAAGAAGATCAAGGAGGTTT 3600 D E E E K K D A E E G K V E D V D E E K E E K E K K K K K I K E V

CTCATGAGTGGTCCCTGGTCAACAAACAGAAGCCTATCTGGATGAGGAAGCCTGAGGAGATCACTAAGGAGGAGTATGCTGCTTTCTACAAGTC~CTGA~ 3700 S H E W S L V N K Q K P I W M R K P E E I T K E E Y A A F Y K S L T AAACGACTGGGAGGAGCATCTTGCTGTCAAGCACTTCTCTGTGGAGGGTCAGCTGGAATTCAAAGCTGTTCTCTTTGTTCCCAAGAGGGCACCATTCGAC 3800 Q T T G R S I L L S S T S L W R V S W N S K L F S L F P R G H H S T CTCTTTGATACGAGGAAGAAGCTCAACAA~ATCAAGCTCTACGT~CGCCGAGTCTTCATCATGGACAACTGTGAGGAGCTGATc~TGAGTGGCTGAGCT 3900 L F D T R K K L N N I K L Y V R R V F I M D N C E E L I P E W L S

TTGTcAAGGGCATTGTTGACT••GAGGATCTTCC•CTCAACATCTCCCGTGAGATGCTGCAGCAGAACAAGATT•TCAAGGTCATCAGGAAGAACCTTGT 4000 F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I R K N L V CAAGAAGTGCGTTGAGCTCTTCTTCGAGATTGCTGAGAACAAGGAAGACTACAACAAGTTCTATGAAGCCTTCTCCAAGAACCT•AAGCTCGGTATCCAC 4100

K K C V E L F F E I A E N K E D Y N K F Y E A F S K N L K L G I H GAGGA~TCCACCAAcAGAAACAAGATTGCCGAGCTcCTGAGGTACCA~TCCACcAAGAGCGGCGATGAGCTGACCAGC~TCAAGGATTACGTGACGAGGA 4200 E D S T N R N K I A E L L R Y H S T K S G D E L T S L K D Y V T R

TGAAGGAAGGCCAGAACGACATATACTACATCA•CGGCGAGAG•AAGAAGG•CGTGGAGAACTCC•CCTTCTTGGAGAAGCTGAAGAAGAAGGGTTA•GA 4300 M K E G Q N D I Y Y I T G E S K K A V E N S P F L E K L K K K G Y E GGTCCT~TACATGGTTGATGCCATCGACGAGTATGCTGTTGG~CAG~TCAAGGAGTTTGAGGGCAAGAAGCTCGTCT~TGCCA~CAAGGAGGGACTCAAG 4400

V L Y M V D A I D E Y A V G Q L K E F E G K K L V S A T K E G L K CTTGATGAGAGCGAGGACGAGAA•AAG•GGAAGGAGGAGCTCAAGGAGAAATTCGAGGGTCTCTGCAAGGTCAT•AAG•AGGTGCTCGGAGACAAGGTCG 4500 L D E S E D E K K R K E E L K E K F E G L C K V I K E V L G D K V

AGAAGGTGGTGGT•TCGGACCGTGTGGTGGACTCCCCGTGCTG•CTCGTCACCGG•GAGTATGGCTGGACGG••AACATGGAGAGGATCATGAAGGCCCA 4600 E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A Q GGCGCTGAGGGACTCGAGCATGGCCG•GTACATGTCGAGCAAGAAGACGATGGAGATCAACCCGGAGAACGCCATCATGGAGGAGCTCCGCAAGCGCG•C 4700

A L R D S S M A G Y M S S K K T M E I N P E N A I M E E L R K R A GACGCCGACAAGAA~GACAAGTCCGTCAAGGACCTCGTCTTGCTCCTCTTCGAGACGGCCCTCCTCACCTCCGGCTTCAGCCTCGACGACCCCAACACCT 4800 0 A D K N D K S V K D L V L L L F E T A L L T S G F S L D D p N T

TCGGCAGCAGGAT~CACCGCATGCTCAAGCTCGGCCTGAGCATCGACGAGGACGAGACGGCGGAGGCCGACACCGACATGCCGCCGCTCGAGGACGACGC 4900 F G S R I H R M L K L G L S I D E D E T A E A D T D M P P L E D D A CGGCGAGAGCAAGATGGAGGAGGTCGACTAAGCGGGTTTCCTTTTTTTT•CC•T•TCGTCAAATCCTTCGCAGCGAAAGTAcAC•ATGGAGCATTGGAGC 5000

G E S K N E E V D . TAGCTTATCTGTTTTACCTTTTGAACATCGCGTTGTTGGGTCGGTTCGGTTTATTTTGTGCGATTTG~TTTACCTTTA~CTAGCCTGCAATGCATGGGGC 5100 TAGATTGTGCTATGGGTTCTCGAA•TGCGTACGTTAATTATATTGCTAAATATGAGTATTATATTAGTGTC•TCATGAGTTGTCTATTTTTTCCTCTGCA 5200 AGTTAGAGGTGTATCAATTAATTTTGCGAATATGCTTCTGCCTCAATTCCCACCTTCGAGCACCAGCTTTGCAATTTACAGTTACAGAAA~ATATGATTG 5300 CGTAGTGTGAAT 5312

Fig. 2. Nucleotide sequence of the rice hsp82B gene and deduced amino-acid sequence of the polypep- tide

Isolation ofgenomic sequences. A genomic library was constructed from Oryza sativa var. indica, cv. Taichung native 1 nuclear DNA. This D N A was partially digested with Sau3A and cloned into the BamHI site of L Charon 35 (Loenen and Blattner 1983). The 3, Charon 35 library was screened by plaque hybridization with the partial cDNA hsp82 clone, 21G3, which contains 510 bp of the coding region and 54 bp of T-untranslated region, at 65 ~ C, in 3 • ( I • 150mM NaCI, 1 5 m M Na3-citrate, pH 7.0), 0.5% SDS0 5 • Denhardt ' s solution (Denhardt 1966) Washing was done at 68 ~ C, 0.5 • SSC. Different overlapping restriction frag- ments containing the gene were subcloned from L Charon 35 into pUC 18 (Yanisch-Perron et al. 1985) or the T3T7 vector (Boehrin-

ger, Mannheim, Germany). Identification of HindIII, EcoRI, and XbaI subclones, containing the 3' part of the hsp82 genes was done by hybridization with the partial cDNA clone 21G3. Parts further upstream of the gene were identified by successive screens with overlapping portions of the different subclones (localized chro- mosome walking).

Sequence analysis. Sequence analyses were performed by the meth- od of Maxam and Gilbert (1980).

Southern analysis. Genomic DNA from Oryza sativa was prepared according to Dellaporta et al. (1983). The D N A was separated on

60 F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes

a 0.8% agarose gel and transferred to nylon filters (Hybond, Amer- sham, UK). Hybridization was performed in 3 • SSC, 5 • Den- hardt's solution, 0.5% SDS at 65 ~ C; washing conditions were 0.5 x SSC, 0.5% SDS at 65 ~ C.

Amplification using the polymerase chain reaction (PCR). The posi- tion of the introns was confirmed by sequencing a 322-bp and 648-bp PCR product, obtained as done by Saiki et al�9 (1985), from cDNA samples using described oligonucleotides, homologous to regions in exon 1 (sense 001), exon 2 (antisense 002, sense 003), and exon 3 (antisense 004). The sequences of the oligonucleotide primers used for amplification were 5 ' -GCAAGCTTCCGGCCT- CCGCCTCCGC-3 ' (sense 001), 5 -ATGGTACCCAGGTTGTTC- ACAAGAT-3 ' (antisense 002), 5 ' -TGGGTACCATTGCCAGGT- CAGGGAC-3 ' (sense 003), 5 ' -TTGAATTCCAGCTGACCC- TCCACAG-3 ' (sense 004).

Measurements of Na/K. Plant material was dehydrated at 180 ~ C for 10 h. Ions were extracted with 10 mM acetic acid at 90 ~ C for 2 h. Debris was removed by centrifugation. Ion measurements were done by use of a Beckmann flame spectrophotometer.

Northern analysis. Total R N A was prepared according to Jones et al. (1985) and transferred to Hybond-N membranes. Hybridization to randomly primed probes was carried out using standard proto- cols (Maniatis et al. 1982).

Results

Characterization of a member of the rice hsp82 gene fami- ly. A screening of a root cDNA library led to the isola- tion of an incomplete cDNA clone, E3, which contains an open reading frame with 62% homology to HSP83 of Drosophila (Blackman and Meselson 1986) and 61% homology to HSP90 of yeast (Farrelly and Finkelstein 1984). As later experiments show hsp82 is not osmotically regulated�9 It seems likely it was isolated initially because the salt-stressed plants were recovering from an un- controllable increase in temperature that had occurred approx. 10 d prior to the planned treatment�9 This clone was used to isolate another partial cDNA, 21G3, from a different member of the same gene family and to the isolation of three overlapping clones of genomic DNA, encompassing approx�9 15 kb and encoding three copies of the hsp82 gene. We have designated these three copies as hsp82A through C. The gene corresponding to cDNA clone E3 was designated hsp82D.

These genes may represent only some members of the hsp82 family�9 Hybridization of genomic DNA to the partial cDNA clone 21G3 showed at least three distinct BamHI and EcoRI restriction fragments, and possibly more (Fig. 1). Based on analysis of these and other gels, we conclude there may be four or five copies of hsp82- related genes in rice.

The nucleotide sequence and the expected translation product of hsp82B are shown in Fig. 2 and 3. This gene contains two introns. The exon boundaries were predict- ed roughly from homology between possible translation products and other HSP82 proteins (Fig. 5), comparisons between intron consensus sequences (Hanley and Schuler 1988) and, more specifically, by sequencing a PCR- derived cDNA extending from nucleotide 832 to 2220 and from 2220 to 3694.

1

63

125

187

249

311

3 7 3

435

4 9 7

559

621

MASETETFAFflAEINQLLSLI INTFYSNKEIFLRELISNSSDALDKIRFESLTDKSKLDAQP

ELFIHIVPDKASNTLSI IDSGIGMTKSDLVNNLGT IARSGTKEFMEALAAGADVSMI~flF~V

~FY~AYLVAERVVVTTKHNDDEflYVWESOAGGSFTVTRDTSGEQLGRGTKITLYLKDDflLEY

LEERRLKDLIKKHSEF ISYPISLW~EKTTEKEISDDEDEEEKKDAEEGKVEDVDEEKEEKEK

KKKK--KKKfK~SHEWSLVNKQKPIWMRSPEEITKEEYAAFYKSLTNDWEEHLAVKHFSVEGOLEF

KAVLFVPKRAPFDLFDRKKLNNIKLVVRRVFIMDNCEEL IPEWLSFVKGIVDSEDLPLNISR

EMLQQNK ILKVIRKNLVKKCVELFFEI AENKEDYNKFYEAFSKNLKL GIHEDSTNRNKIAEL

LRYHSTKSGEDELTSLKDYVTRMKEGQNDIYY ITGESKKAVENSPFLEKLKKKGYEVLYMVD

AIDEYAVGQL~EFEGKKLVSATKEGLKLDESEDEKKREELKEKFEGLCKVIKEVL]GDKEKVV

VSDRVVDSPCCLVTGEYGWTANMERIMKAOLARDSSMAGYMSSKKTMEINPENAIMEELRKR

ADADKNDKSVKDLVLLLFETALLTSGFSLDDPNTFGSRIHRMLKLGLSIDEDETAEADTMPL

683 EDDAGESKMEEVD*

Fig. 3. Expected translation product of the rice hsp82B gene. Trian- gles indicate potential ATP-binding motifs; asterisks indicate puta- tive leucine zipper. Conserved charged domains are boxed. Consen- sus carboxy-terminal peptide is double-underlined

hsp82A hsp82B hsp82D

4666

GAATTCCATCATGGACGAGCTCCGCAAGCGT GCCGATGCTGACAA

�9 �9 'CG . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . C" "C . . . . .

hsp82A hsp82B hsp82D

GAACGACAAGTCT GTGAAGGACCT GGTGATGCTGCTCTTCGAGAC

. . . . . . . . . . . . C �9 �9 . . . . . . . . C. .CT . . . . C . . . . . . . . . . .

�9 ' ' T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

h s p 8 2 A

hspS2B hsp82D

T G C C C T C C T G A C C T C C G G C T T C A G C C T T G A G G A C C C C A A C A C C T T

G . . . . . . . . C . . . . . . . . . . . . . . . . . C �9 � 9 . . . . . . . . . . . . . .

. . . . . . G . . . . . . . . . . . . . . . . . . T ' G . . . . . . . . . . . . . . . . .

hsp82A hspS2B h~p82D

C G G C A C C A G G A T C C A C C G G A T G C T C A A G C T C G G C C T G A G C A T C G A

. . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . .

hsp82A hsp82B hsp82D

CGAGGACGAGTCTGCTGAGGCTGACGCCGACATGCCGCCGCTGGA

. . . . . . . . . . A ' G ' ' G . . . . . C ' ' ' A . . . . . . . . . . . . . . . . C ' "

. . . . . . . . . . . . C ' ' C . . . . . . . . . . . . . . . . . . . . T . . . . . . . .

hsp82A GGACGACGCCGGCGAGAGCAAGATGGAGGAGGT CGACTAA

hsp82B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hsp82D . . . . . . . . . . . . . . . . . CG . . . . . . . . . . . . . . . . . . . . .

493

Fig. 4. Comparison of nucleotide sequence of the 3'-coding region of hsp82A, hsp82B, and hsp82D in rice. Identical nucleotides in all three sequences are indicated by dots in hsp82B and hsp82D. Only those bases of the hsp82B or hsp82D sequences which differ from that of hsp82A are shown. The non-coding region is not presented because there is no significant homology. Complete sequences are present in the EMBL Data Bank, with accession numbers Z11920, Z15018, and Z15024

F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes

Os M . . . . A••TETFAFQAEINQLL•LIINTFY•NKEIFLRELI•N••DALDK•RFE•LTDK•KLDAQPELFIHI•PDKA•NTLSIID•GIG•TK•DL 8 6

At . A D V Q M A D A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . R L . . . . . N K . . . . . . . . . . . . . A ' '

Hs ' P E E V . . . . . . . . . . . . . A - - M . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . Y . . . . . P . . . . S G K . . K . D . I . N P Q E R . . T L V ' T . . . . . . A . .

Dm ' P E E A . . . . . . . . . . . . . A - ' N . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . Y . . . . . P . . . . S G K . . Y . K L I ' N ' T A G ' ' T ' ' ' T . . . . . . . . .

Sc . . . . . . . . . . . . . . . . . T " N . . . . . . V . . . . . . . . . . . . . . A . . . . . . . . Y K . . S . P K Q ' E T E ' D . . . . R . T . K P E Q K V ' E ' R . . . . . . . . A E -

Ec .KGQ . . . . . . R G . . S , V K . . . H - M - H S L . . . . . . . . . . . . . . A . . . A . . L . . R A . S N P D L Y E G D G . . R V R V S F . . D K R ' ' T ' S ' N ' V ' ' ' R D E V

Os •NNLGT•AR•GTKEFMEAL•-AAGADVSM•GQFGVGFYSAYL•AERVV•TTKHNDDE•YV•••WESQAGG•FTvTRDTSGEQLGRGTKITLYLKD 171

At . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . V D " P . . . . . . . S ' F ' "

H s I . . . . . . . K . . . . A . . . . . . . Q . . . . I . . . . . . . . . . . . . . . . . . K ' ' ' I R . . . . . . . . A . . . . . . S . . . . . . . . . A D H . . P I ' M ' ' ' V I ' H ' ' E

Dm . . . . . . . . K . . . . A . . . . . . . Q . . . . I . . . . . . . . . . . . . . . . . D K . T - - S . N . . . . . . . . . . . . . S . . . . . . . . . A D N S ' P . . . . . . . V ' ' I ' E

Sc I . . . . . . . K . . . . A . . . . . . . S . . . . . . . . . . . . . . . . . L F . . . D . . Q ' I S ' S . . . . . . I . . . . . . N . . . . . . . . L ' E V N - R I . . . . I L R ' F ' ' '

Ec I D H . . . . . K - F L E S ' L ' S ' G S D R A K ' S Q L . . . . . . . . . . . F I . . D K , T . R . R A A G E K P E N G V F ' ' ' A G E ' E Y " - A ' I T K ' - - D " ' E ' " H ' R E

Os D Q L E Y L E E R R L K D L I K K - H S E F I S Y P I S L W T E K T T E K E I - - S D D E D E E E K K D . . . . . . . . A E E G - K V E D V - - D E E K E E K E K K K K K - - - I K E V S H E 2 4 4

A t . . . . . . . . . . . . . . V . . . . . . . . . . . . Y . . . . . . . . . . . . . . . . . . . D ' P ' K . . . . . . . . E N ' ' ' E ' ' E . . . . . . . . KDG . . . . . . . . . . . . . . .

Hs ..T . . . . . . . V - E V V . . . . . Q , . G . , . T . Y L . . E R . . . . . . . . . . A . . . . G E . K E E E D K D D - . K P . I . . . G S . ' ' D D S G K D . . . . TKK . . . . K Y I

Dm ' ' T D . . . . S K I - E I V N . . . . Q . . G , , . K . L V - ' E R ' ' ' V . . . . . . A D D . , . E . G D E K K E M E T D E P ' I ' ' ' G E ' ' - D A D K ' D ' D A ' ' K K T ' " K Y T

Sc . . . . . . . . K - I . E - V I - R . . . . V A . . . Q . V V T . E Y . . . V P I P E E . K K D . E - K D E E K K D E E K K D E D D K K P K L E E V D E " E K ' P ' T ' K V . . . . . EVQ

Ec G E D ' F ' D D W V R S I . S . . Y . D H . A L . V E ' ' I ' ' R E ' ' D G T V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

Os W • • - L • N K Q K P I W M R S P E E I T K E E Y A A F Y K S L T N D W E E H L A v K H F s V E G Q L E F K A V L F V P K R A P F D L F D T R K K L N N • K L v v R R V F I M D N c E E L • P 3 3 2

At " E ' " I . . . . . . . L ' K . . . . . . . . S . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . . . . . . . .

Hs D Q E E . . . . T . . . . T . N . D D - - Q - - - G E . . . . . . . . . . D . . . . . . . . . . . . . . . R . L . . I - R . . . . . . . E N K . : K . . . . . Y . . . . . . . . S . D . . . .

Dm E D E E . . . . T . . . . T ' N ' D D ' S Q ' - ' G E . . . . . . . . . . D . . . . . . . . . . . . . . . R ' L ' ' I ' R - T . . . . . E N Q ' ' R . . . . . Y . . . . . . . . . . . D ' ' '

Sc E I E E . . . . T ' ' L ' T ' N ' S D ' ' Q - - - N . . . . . I S . . . . D P ' Y . . . . . . . . . . . . R ' I " I . . . . . . . . . E S K ' ' K . . . . . Y . . . . . . T ' E A ' D ' ' '

Ec • •EK- I • •AQAL•T•NK••• •D. • •KE•• .HIAH•FNDP•TW••NR•••KQ•YT•L.Y•• • • • •W•MW-N•DHKHGL•.Y•Q . . . . . . D A . Q F M .

Os E W L • F v K G I v D • E D L P L N I • R E M L • • N K I L K V I R K N L v K K K c V E L F F E I A • E N K E D Y N K F Y E A F • K N L K L G I H E D • T N R N K • A E L L R Y H • • K • G - 4 2 0

A t .Y . . . . . . V . . . D . . . . . . . . . T . . . . . . . . . . . . . . . . . . . I ' M ' N . . . . . . . . . . T . . . . . . . . . . . . . . . . . . Q . , G . ' ' D . . . . . . . . . . .

Hs - Y . N ' I R ' V . . . . . . . . . . . . . . . . . S . . . . . . . . . I . . . . . L . - - S . L . . , D . . N . K . . . . . . . . . . . . . . . . . . . . . R R L S . . . . . . T S Q ' "

Dm - Y ' N ' M ' ' V . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . Y M . . I E . L T . . D ' ' N ' K ' ' ' D Q . . . . . . . . V . . . . N . . A . L . D F . . F . T S A . . '

Sc . . . . . . . . V ; . . . . . . . . L . . . . . . . . . . M . . . . . . I . . . . L I . A - N . . . . . D S . Q F E . . . S . . . . . I . - . V . . . T Q . . A A L , K . . . . N . . . . V "

Ec N Y - R . . R . L I . . S . . . . . V . . , I . . D S T V T R N L . N A . T . R R V L Q M L E K L . K D D A . K . Q T . W Q Q . G L V - - E - P A ' ' F A ' Q E A ' ' K ' ' ' F A ' ' H T D S

Os DELT•LKDY•TR•KEGQND•YY••GESKKA•EN•PFLEKLKKKGYEVLYM•DAI•EYAVGQLKEFEGKKLV•ATKEGLKLDE•EDEKKRKEELKE 510

At ' ' M ' ' F . . . . . . . . . . . K ' ' F . . . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . YD . . . . . . . . . . . . . . . T E . E K . . . . . . K ' '

Hs - - M - - - S E . . S . . . . T . K S . . . . . . . . . E Q . A . . A . V . R V R . R . F - - V - . T E P . . . . C ' Q . . . . . D ' ' S - - - V . . . . . E . P . D ' E ' ' ' K M ' ' S ' A

Dm . D F C . - A . . - S , . . D N . K H V . F . . . . . . D Q ' S " A ' V ' R V ' A R ' F ' - V - ' T E P . . . . V I Q H , , . Y K ' ' O ' ' ' V . . . . . E . P , D . S . . . K R . ' D ' A

Sc . . . . . . T . . . . . . P , H . K N . . . . . . . . L . . . . K . . . . D A . . A . N F . . . F L T . P . . . . . FT . . . . . . . . T . - D I . - D F E L E E T D . E K A E . E K ' I ' '

Ec $ A Q T V ' E ' ' ' $ . . . . . . EK . . . . . A D . Y A - A K S . . H . . L . R . . . I . . - L L S . R . . . W M M N Y . T . . D . - P F Q - V S - V D E S . E K L A ' ' V D E S A K E A "

Os KFEGLCK••-KE•LGDKVEK••V•DR••D•PCCLV•GEYGWTANMERIMKAQALRD••MAGYM••KKTME•N•ENAIMEE•RKRADADKN•DK•V 598

At S - ' N ' ' ' T . . . . I . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . D - G . . . . . . . . . E . . . . . . . . .

Hs . . - N - - - L M - ' . I . D K . . . . . T I ' N . L ' S . . . . I ' ' S T . . . . . . . . . . . . . . . . . . N . T M . . . M A . , H L . . . . D H P ' V ' T ' ' Q K ' E . . . . . . . A .

Dm ' ' ' S ' ' ' L M ' ' S I ' D N . . . . . . . . N ' L . . . . . . I . . S Q F . . S . . . . . . . . . . . . . . T A T M . . . A G . . Q L . . . . D H P ' V E T ' ' Q K . . . . . . . . . A .

Sc Y E P L T K A L . . . . I ' " Q . . . . . . . Y K L L ' A - A A I R ' ' Q F ' ' S . . . . . . . . . . . . . . . . . $S . . . . . . . F . ' S ' K S P ' I K ' ' K ' ' V ' E G G A Q ' ' T "

Ec . A L T P F I D R V - A L . . E R . K D . R L T H . L T . T - A I V S . D A D E M S T Q . A K L F A - A G Q K V P E V . . . . . . Y I F . L . ' D H V L V . . . . . . . ~ D T E D E A ' F $

Os KDLVLLLFETALLTSGFSLDDPNTFGSRIHRMLKLGLSIDEDE-TAEADTDM ..... P-L-EDDAGESKMEEVD* 659

At . . . . M ' ' Y . . . . . . . . . . . . E . . . . AA . . . . . . . . . . . . . . . . . N V , E ' G . . . . . . . . E ' E ' ' A ' E . . . . . . . . *

Hs . . . . V . . . . . . . . S . . . . . E ' ' Q ' H S N ' ' Y " I . . . . G . . . . . V A ' ' E P N A A V P D E I ' P ' - ' G ' E D A ' R . . . . . *

Dm . . . . I . . . . . S " S . . . . . . S ' Q V H A " ' Y " I . . . . G . . . . . . P M T T - D A Q S A G D A ' S ' V ' ' T E D A ' H . . . . . *

Sc ' ' ' T K ' ' Y . . . . . . . . . . . . E . T S . A . , . N . L I S . . . N . . . . . . . . . T E . A P E A S T A - A P V . E V P A D T E . . . . . *

Ec E W V E - - - D - Q - . . A E R G T , E . . . L . . . . . R . . N Q - L V . * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61

Fig. 5. Alignment of pro- tein sequences of the HSP90 family. Sequence is given in the one-letter code. Os, amino-acid sequence for the Oryza sativa HSP82B polypep- tide; At, Hs, Dm, Sc, and Ec, published sequences for Arabidopsis (Conner et al. 1990), human (Hickey et al. 1989), Drosophila (Blackman and Meselson 1986), yeast (Farrelly and Finkelstein 1984), and E. coli (Bardwell and Craig 1987) homologues, respec- tively. Capitals indicate where the amino-acid sequences differ, dots where they all match. Hyphens are inserted to maximize alignment

A comparison of the 3' coding region of hsp82A, hsp82B and the partial cDNA clone E3 is shown in Fig. 4. Hsp82A was found to be the corresponding gene of the partial cDNA 21G3. Only a portion of the hsp82C gene containing part of its coding region has been sequenced, and this was 100 % identical to that of hsp82B (data not shown).

The hsp82B gene product is between 67% and 87% identical to the equivalent genes of other eukaryotes and 31% identical to the htpG product of E. coli (Fig. 5). As in all the other eukaryotic homologues, two hydrophilic regions with moderate conservation are present in HSP82B (amino acids 211-255 and 507-552). The largest one in the amino-terminal half, which is totally missing in the E. coli equivalent, is thought to be responsible for the association with, among others, steroid receptors (Catelli et al. 1988). Position 124 (Fig. 3) is the point of

symmetry for two overlapping, but inverted, potential ATP-binding motifs which could contribute to the auto- phosphorylating activity of the protein (Csermely and Kahn 1991). We also note a stretch of evenly spaced leucine, isoleucine, and valine residues that might func- tion as a leucine zipper in some of the intermolecular associations referred to above. The protein HSP82B ends with the peptide MEEVD which is conserved among the HSP82/HSP90 proteins of several species.

Heat shock induces expression of hsp82B. It is possible that some of the copies of HSP82 in rice are involved in normal growth, whereas others are produced principally during stress. Constitutively expressed cognate genes such as hsc82 of yeast lack copies of the heat-shock- regulatory element (GAANNTTC) in their promoter (Borkovich et al. 1989). On the other hand, hsp82B has

62

TCTAGATTGTACTAAATGTTTGGTTCCAAATTAGTTTTGCTGACCAACGA 50

ACCTAGGCCGTGAAATGTTTGGTTCCAAATTAGTTTTGCTGACCAACGAA 100

CCTAGGCCGTGTTCTTTGCAATACTATTCCAACTTCAATAACTCATAACT 150

CATTTGTCACACGCACATTTCTCAAACTATCAAACGTAATTTGTTTCTAT 200

GAAGTTTGATAAAAATATTGTTTTAAAAATCATATTAATATGTATATATA 250

TATATATATATATAAGTTTTTTAATTGATACTTAATTAATCGTGTCAATA 300

GGTTGTTTTGTTTTGCGTGTTACGAGGAAAGGCTTCCAACCTTAACCAAA 350

TAACATAACCCTAAAGATAAAGACCACAAGTCTICATTCGCTTCC/~GAAC 400

CTTC~TGCATCTAATCTGCGATGTGCAGT~AAAATTCATCC 450

TGTAATTAAGTT~TGCATCTAATTTGCGATTTTTTCTATTTT 500

GCAAAGATGCTATTCGAATGACAGTTTTAGTGATATTTTTGTAATTTTTC 550

AGTGGCAGTTTTACAATAACGATTTAAAGCAGTGGTAAATTTATAATTGC 560

CCCTTACAAGAATTATAGTATTCACTATTCCCATACAGTACTAGACCATA 600

GAGATGATGGTTTTCTTAATTTAAACCGACTCCAAGCCTCTCCACTCCTC 650

~~AAAATATAAAAACCATAACCAACCGACTGACATGTGGGC 700

CCGACGACTCGTCAAGTCGTCCATCTCCCAACTCCCCCGCTGGTTCGAGC 750

CCCTGGT AGAACCCCCATCTCCCCCTTATATATCGCCCCC ............... 800

TTTTTCCCCATCTTTCTCCAATCGAATCGCACACGCCACGCGAGCTCCCT 850

CTCCTAAAGCCCTAACCCTAGCCTCCAAGTCGTCGT_C~TCC_(~ 900

CGGCCTCCGCCGCCGCCGCAGCGCC 925

Fig. 6. 5 ' -Untranslated region of the hsp82B genomic clone from rice. Possible heat-shock elements are boxed. Repeats are underlined (dashed and bold); the TATA element is indicated by dots. The (TA) 11-sequence is also underlined

F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes

Fig. 7. A RNA gel blot analysis showing the accumulation ofhsp82 transcripts during heat shock of rice. Analysis of total RNA from rice leaves stressed for various times at 42 ~ C. Heat stress was applied for 0, 10, 20, 30, 45, 60, and 120 min. Each lane contained 20 lag of RNA and was hybridized to the 1.4-kb fragment encom- passing the 3' portion of the hsp82B genomic clone. B Autoradio- gram of proteins labelled with [35S]methionine for 2 h during heat shock. No prominent bands were seen in non-shocked controls (data not shown). C RNA gel blot analysis showing steady-state levels of a rice S-adenosylmethionine synthetase gene during heat shock. Analysis of total RNA from rice leaves in control conditions (26 ~ C) and stressed for 2 h at 42 ~ C. Each lane contained 20 lag of RNA and was hybridized to the 0.85-kb hsp82B specific probe and to a partial clone of a rice S-adenosylmethionine synthetase gene (EMBE Data Bank Accession number 226867)

hsp82

six of these sequences in its 5' end (Fig. 6). The presence of these elements indicated hsp82B could be responsive to heat. To establish conditions for induction of this gene, three-week-old plants grown at 27 ~ C were trans- ferred to 42~ for 5, 10, 20, 30, 45, 60, and 120 min. Total R N A was then prepared from the leaves and hy- bridized to the 1.4-kb fragment encompassing the 3' port ion of the HSP82B genomic clone. Figure 7A shows that the corresponding m R N A species (estimated to be 2.4 kb in length) that was present in untreated plants began to accumulate to higher levels within 10 rain and reached a maximal relative amount within 120 rain. An autoradiogram of proteins labeled with [aSS]methionine for 2 h showed that several proteins, including ones with the sizes expected for HSP70 and HSP82, were made in large amounts by this time (Fig. 7B). To verify that each lane contained similar amounts of messenger, we re- hybridized filters with a partial clone of a rice S- adenosylmethionine gene (EMBL Data Bank Accession number 226867; OSSAMS1). As seen in Fig. 7C, heat shock did not greatly alter the steady-state levels of this transcript. Filters were also re-hybridized with an hsp82B-specific probe derived from the Y-untranslated region of hsp82B (0.85 kb HindII-HindIII fragment; from nucleotide 4927). The hsp82B-specific probe and the 1.4-kb probe showed similar patterns of hybridization.

The genes hsp70 and hsp82B show pronounced differences in inducibility. Plants shifted f rom 26 ~ C to 42 ~ C trans- pire more quickly and lose the water more quickly

hsp70

c.o+ 03010.10s7 07, 1,3 I [K+] 17.941 18.60 14.35 13.65 1 3 . 9 7 1 1 1 . 7 4

t,o+/K+l o o21oo2 oo, o o.o, lO.ll I

Fig. 8. Correlation between ion-concentration in heat-stressed leaves of rice and hsp70 and hsp82 expression. Upper panel: 20 lag samples of RNA, isolated from leaves of different populations of hydroponically grown rice plants of the same age, after indepen- dently performed heat-shock experiments, were hybridized with hsp82 and hsp70 probes. Lower panel: Na + and K § concentrations (mg.g-1 dry weight) in leaves from rice plants independently subjected to heat shock (2 h, 42 ~ C). Experiments are ordered from lowest Na + accumulation to highest

through evaporation. The result is a build-up of various salts within the plant. After performing independent heat-shock treatments on different populations of hy- droponically grown rice plants of the same age, we mea- sured the internal Na + and K + levels and used this as a measure of the magnitude of the osmotic stress imposed on the plants in each replicate. The leaves of untreated plants contained approx. 0.1 mg Na +- g-1 dry weight and 20 mg K + - g - 1 dry weight. Within 2 h, Na + levels increased between two- and fivefold. When all of the heat-shock experiments were ranked according to inter-

F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes 63

nal Na § accumulations, and then probed with cDNAs for rice HSP70 (Borkird et al. 1991), or HSP82B, the result shown in Fig. 8 was obtained. In general, there was a correlation between the levels of Na + and HSP70 mRNA: preparations with the lowest ion accumulation tended to have lowest levels of HSP70 mRNA. No such correlation was apparent between HSP82B mRNA and either Na + concentrations or HSP70 mRNA. Ion accu- mulations also occur in the absence of heat shock when plants are treated with high concentrations of salt. To verify that osmotic stress did not induce hsp82B, we grew plants for 3 d with 1% NaC1, and investigated whether heat-shock genes were induced. This form of osmotic stress had no effect on hsp82B expression, yet induced hsp70. We presume that the increasing concentrations of NaCI displaced intracellular water or other stabilizing ions causing some proteins to denature, and this in turn provided the primary inducer of HSP70 (Craig and Gross 1991). Proteins can also be denatured by dehydra- tion accompanying growth in ethanol. Plants were treated for 3 d with 0.1% ethanol before the RNA po- pulation was examined. Again, hsp70 was induced whe- reas hsp82B was not (Fig. 9). Drying plants for 3.5 h, by removing them from hydroponic medium, had similar effects on the two genes (data not shown).

A number of other hormone or chemical treatments was investigated, including growing plants with 5-20 gM abscisic acid, 100 gM salicylic acid, 10 gg- m1-1 chitosan, and 1 gM stigmasterol. None induced hsp82B (data not shown). Neither darkness nor 100 laM para- quat had any effect on hsp82. However, transferring

Fig. 9A, B. RNA gel blot analysis of the expression of hsp82 and hsp70 genes from rice in different conditions. A Total RNA was analyzed from rice leaves grown under normal conditions (28 ~ C) heat-stressed for 2 h (42 ~ C) or grown for 3 d with 0.1% ethanol (0.1% EOH). B Total RNA was analyzed from rice leaves grown under a normal light regime (with and without 100 gM paraquat) or grown in continuous dark for 3 d (with and without paraquat). Each lane contained 20 gg of RNA and was hybridized with hsp82, hsp70 and sam probes as described in Fig. 7

light-grown plants to continuous darkness for 3 d led to a marked increase in HSP70 RNA levels, without any accompanying effect on HSP82B RNA (Fig. 9).

We should note that due to high conservation between members of the hsp70 gene family, cross-hybridization is not excluded. The signals obtained in Northern analysis may thus be the summation of the expression of several hsp70 genes. No differences in hybridization patterns were obtained whether hybridizations were done with an hsp82B-specific probe (Y-untranslated region) or with a probe which can hybridize to other members of the rice hsp82 gene family.

Discussion

We have isolated and partially characterized several genes belonging to the hsp82/90 family. Three of these genes lie within a cloned 18-kb region of the genome. Southern analysis of total DNA, and sequence analysis of several partial cDNA and genomic clones indicates that there must be four or five copies of hsp82-related genes in rice. Most of the work in this paper has focused only on the middle member of the isolated gene cluster, designated hsp82B. The hsp82B coding region appeared to be interrupted by two introns. The exon boundaries were confirmed by sequencing PCR-generated cDNA sequences derived from the flanking exons. The position of these introns is identical to the first and third intron of the hsp81-1 gene of Arabidopsis (Takahashi et al. 1992). The position of the first intron is also identical to that of the second intron of the human hsp89a and hsp89~ homologues (Hickey et al. 1989; Rebbe et al. 1989).

The hsp82B gene product is quite similar to the corre- sponding gene products of other eukaryotes. Eukaryotes have been shown to contain a number of different pro- teins that associate with HSP90, including steroid hor- mone receptors belonging to the erb-A superfamily (es- trogen receptor, progesterone receptor, glucocorticoid receptor, mineralocorticoid receptor, androgen receptor; Catelli et al. 1988), arylhydrocarbon receptor (Perdew 1988), actin and tubulin (Sanchez et al. 1985; Koyasu et al. 1986), oncogenic tyrosine kinases (Ziemiecki et al. 1986; Miyata and Yahara 1992), (elF)-2a (Rose et al. 1987), and recently an immunophilin (p59; Peattie et al. 1992). There is also evidence that HSP90 exists in a heteromeric complex with HSP70 in the cytosol. Several motifs could mediate these intermolecular interactions. As in other eukaryotic HSP82/90 proteins, two hydro- philic domains with a high density of charged amino-acid residues are present in HSP82B. We have noticed that there is also a leucine zipper mot i f between the two charged domains that might contribute stability or speci- ficity to heteromeric or homomeric complex formation. The leucine zipper mot i f was first discovered as a con- served sequence pattern in several transcription factors (Landschulz et al. 1988) where it was shown to mediate dimerization (Kouzarides and Ziff 1988). For the HSP90- glucocorticoid receptor interaction, a metal-linked gap- ped zipper model has been proposed (Schwartz et al.

64 F. Van Breusegem et al. : Heat inducible rice hsp82 and hsp70 genes

1993). The presence of these motifs in a rice HSP82 protein may be an indication that the same types of interactions are occurring in plants, perhaps with HSP70, perhaps with structural proteins and enzymes.

Heat-shock proteins are commonly considered to be coordinately regulated. This is only true to a certain extent. Nover et al. (1990) have shown that there are two different types of inducers of these genes: hs-like and non-hs-like inducers. The primary inducer in the hs-like inducing pathway is the accumulation of "abnormal" proteins which regulate transcription through a positive feed-back loop (Ananthan et al. 1986; Craig and Gross 1991). It has been suggested that mistranslated or mis- folded proteins are able to compete with the heat-shock regulatory factor (HSF) as substrates for a ubiquitin- dependent proteolytic pathway. The more this pathway is needed to destroy denatured proteins, the less it is available to inactivate the HSF that in turns induces hsp transcription. Alternatively, the activity of the regu- lator might depend through analogous equilibrium processes on the concentration of free HSP70 versus that bound to denatured protein (Baler et al. 1992). Either mechanism for autoregulated equilibrium would be con- sistent with the expression pattern of hsp70 we have observed in rice. Each condition likely to produce dena- tured proteins had the expected effect on the accumula- tion of HSP70 messenger. Ethanol which can denature proteins and/or stimulate mistranslation induced this gene. Cadmium which is another protein-denaturing agent, has been used by other researchers to induce the gene in other plants (Edelman et al. 1988). We were also able to induce hsp70 with high temperature, or with high salinity, two stresses that have been shown to stimulate protein turnover, either in wheat (Ferguson et al. 1990) or in rice (Kang and Titus 1989). Prolonged darkness was an unanticipated inducer of hsp70 in our experiments, although previous studies of hsp70 in Chlamydomonas (von Gromof f et al. 1989) and of hsp83 in Pharbitis (Felsheim and Das 1992) have shown there is a transient increase in hs gene expression when dark-grown cells are suddenly exposed to light. The growth of plants in dark- ness results in depletion of ATP. Benjamin et al. (1992) have shown that the effects of ATP depletion are suf- ficient to induce the D N A binding of HSF when oxida- tive metabolism is impaired. It is possible that adaptation to either conditions requires the destruction of so many superfluous molecules that the basal activity of the pro- teolytic pathways is taxed.

We expected that at least heat stress would lead to comparable induction of both hsp70 and hsp82. How- ever, rice plants heat-shocked independently and with comparable levels of HSP82 mRNA, showed consider- able variation in HSP70 mRNA levels. The two genes investigated here behaved as if they were controlled through independent sensor systems, each of which re- cognized different denaturing agents or different degrees of damage produced by these agents. The need for differ- ent modes of regulation might indicate that the proteins are being used for different purposes in the cell. Although both have been shown to have chaperonin-like effects in vitro (Beckmann et al. 1990; Wiech et al. 1992), these

activities may be used differentially in vivo. The protein HSP82/90 has been shown, for example, to be associated with inactive steroid receptors and only released when the hormone displaces it (Scherrer et al. 1990). When added in physiological amounts it has also been shown to stimulate eIF-2~ kinase (Rose et al. 1989) and to enhance 15- to 18-fold the in-vitro activity of casein kinase II (Miyata and Yahara 1992). These characteris- tics resemble more a molecular scaffold that must in- teract with other macrornolecules even after they have been synthesized and properly targeted.

If indeed there are different inducers for different types of HSP, there should be demonstrable differences in the regulatory sequences of their promoters. The gene hsp70 may have separate regulatory elements responding to protein denaturation and to other cellular damages caused by heat stress whereas hsp82B would not have the protein-denaturation-dependent control.

Conversely, hsp82B might require the interaction with two different regulatory factors in order to be expressed, one being the conventional heat-shock-element-binding protein which would normally respond to all protein- denaturing conditions, and the other that is uniquely induced or de-repressed during heat stress and not by any other stress. A deletion analysis of the hsp82B promoter should be able to distinguish between these possibilities.

The authors thank R. Villarroel (Laboratorium voor Genetica, Universiteit Gent, Gent, Belgium) for help with the sequencing, both Dr. Van Der Straeten and D. Van den Broeck (Laboratorium voor Genetica, Universiteit Gent, Gent, Belgium) for critical read- ing of the manuscript, Martine De Cock for typing it, and Karel Spruyt and Vera Vermaercke for drawings and photographs. This work was supported by grants from the Belgian Programme on Interuniversity Poles of Attraction (Prime Minister's Office, Science Policy Programming #38), the Commission of the European Com- munities TS2-0053-B (GDF), and the Rockefeller Foundation (FR 86058 #59). A.B.G. is indebted to Conselho Nacional de Desen- volvimento Cientifico e Tecnoldgico (Brasil; RHAE proc. no. 260015188.1) for a predoctoral fellowship.

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