Plant Enolase: Gene Structure, Expression, and Evolution · The Plant Cell, Vol. 3, 719-735, July 1991 O 1991 American Society of Plant Physiologists Plant Enolase: Gene Structure,

The Plant Cell, Vol. 3, 719-735, July 1991 O 1991 American Society of Plant Physiologists

Plant Enolase: Gene Structure, Expression, and Evolution

Dominique Van Der Straeten,a Renato A. Rodrigues-Pousada,a Howard M. Goodman,b and Marc Van Montagua,’

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

Hospital, Boston, Massachusetts 021 14 Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General

Enolase genes were cloned from tomato and Arabidopsis. Comparison of their primary structures with other enolases revealed a remarkable degree of conservation, except for the presence of an insertion of 5 amino acids unique to plant enolases. Expression of the enolase genes was studied under various conditions. Under normal growth conditions, steady-state messenger and enzyme activity levels were significantly higher in roots than in green tissue. Large inductions of mRNA, accompanied by a moderate increase in enzyme activity, were obtained by an artificial ripening treatment in tomato fruits. However, there was little effect of anaerobiosis on the abundance of enolase messenger. In heat shock conditions, no induction of enolase mRNA was observed. We also present evidence that, at least in Arabidopsis, the hypothesis that there exists a complete set of glycolytic enzymes in the chloroplast is not valid, and we propose instead the occurrence of a substrate shuttle in Arabidopsis chloroplasts for termination of the glycolytic cycle.

INTRODUCTION

Enolase (2-phospho-~-glycerate hydrolase, EC 4.2.1.1 1) is a ubiquitous enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate, the only de- hydration step in the glycolytic pathway. The enzyme has been purified and/or the genes have been cloned from diverse sources: Escherichia coli (Weng et al., 1986), Bacillus subtilis (D. Vereecke, unpublished data; Verma, 1989), yeast (Brewer, 1981; Chin et al., 1981; Holland et al., 1981), Drosophila (Bishop and Corces, 1990), Xenopus (Segil et al., 1988), chicken (Russell et al., 1986), Peking duck (Wistow et al., 1988), and severa1 mammals (Saki- mura et al., 1985b; Forss-Petter et al., 1986; Lazar et al., 1986; Giallongo et al., 1990). The yeast enzyme is by far the best characterized and its crystal structure has been determined at high resolution (Lebioda et al., 1989; Stec and Lebioda, 1990). Both cis elements and trans-acting factors governing metabolic regulation and coordination of the two structural genes ENOl and €NO2 have been analyzed (Cohen et al., 1986, 1987; Holland et al., 1987; Machida et al., 1989; Brindle et al., 1990). Moreover, enolase was found to be identical to the heat shock protein HSP48 and, hence, important in thermal tolerance and growth control of yeast (lida and Yahara, 1985). In higher vertebrates, three different isozymes of enolase have been

’ To whom correspondence should be addressed.

described (a , p, and y), and their cell type and developmental specificity have been demonstrated (Forss-Petter et al., 1986; Giallongo et al., 1986). Interestingly, the lens structural protein 7-crystallin has been identified as a-enolase in different vertebrates (Wistow et al., 1988).

Very little information is available on plant enolases. The enzyme has been purified and partially characterized from potato (Boser, 1959) and spinach (Sinha and Brewer, 1984), but our knowledge on enolase gene and protein structure and its biological relevance in plants remains extremely poor. It is generally accepted that fermentative or anaerobic metabolism is not prevalent throughout the normal life cycle of photosynthetically active cells, but in certain nongreen and anaerobically treated tissues, which are dependent on glycolysis and oxidative phosphorylation for their energy supply (Goodwin and Mercer, 1983), this enzyme becomes important. Moreover, photosynthetic cells have been shown to break down carbohydrates in the dark, mainly by the glycolytic pathway (Goodwin and Mercer, 1983).

In this paper, we present insights on the role of enolase in plant life. Enolase genes and cDNAs were cloned from tomato and Arabidopsis. The primary and secondary structures of plant enolases were compared with their bacterial, yeast, and animal counterparts. We also present evidence that the occurrence of two forms of each glycolytic enzyme, a cytosolic and a plastid isoform (Gottlieb, 1982),

720 The Plant Cell

cannot be generalized to include enolase. In addition, we describe the expression of enolases (mRNA and protein) in normal and in stressed plant tissues. Based on these data, we present some general conclusions concerning glycolysis in anaerobic and heat shock-treated plant tissue.

RESULTS

lsolation and Characterization of Tomato and Arabidop- sis Enolase Genes

The amino acid sequence of the N terminus and of several tryptic peptides from a 45-kD protein originating from a highly purified tomato 1 -aminocyclopropane-1 -carboxylate (ACC) synthase preparation revealed about 50% similarity with yeast enolase (Van Der Straeten et al., 1990). Mixed oligonucleotides were synthesized based on the main sequences of peptides P26 and P46 (Figure 1 in Van Der Straeten et al., 1990). A tomato (cv Orlando) cDNA library was constructed in Xgt l l , and from 25,000 recombinant clones, we obtained seven hybridizing phages, five of which had an insert size above 1.2 kb. The complete sequence of the largest clone (TENOl) is shown in Figure 1 A. The cDNA of 1554 bp contains an open reading frame encoding a polypeptide of 47.7 kD (444 amino acids) with a calculated pl of 5.6, a 5‘-untranslated leader of 60 bp, and a trailing sequence of 162 bp. In contrast to what was observed for the yeast enolase genes (Holland et al., 1981), codon usage is unbiased in the tomato gene, with the exception of Lys, where AAG is used in 30139 cases.

One member of the tomato enolase gene family was isolated from a VNTF Cherry X Charon 35 genomic library screened with the TENOl cDNA as a probe. The 3-kb Hindlll fragment, which was sequenced (Figure 1 A) and designated gTEN02, covers 75% of the coding region (3261444 residues). The intronlexon structure is presented in Figure 18. The 12 introns invariably contain the GT and AG dinucleotide consensus at their respective 5‘ and 3‘ ends (Csank et al., 1990), with the exception of the 5‘ end of the third intron, where GC occurs. This could inhibit splicing of the intron, implying that gTEN02 might be a pseudogene. The coding regions of gTEN02 and the TENO1 cDNA are 89% identical and thus TENOl most probably is derived from a different gene because this divergence is not readily accounted for by cultivar differences. Howevet, the amino acid similarity is 96%, as only 14 base substitutions lead to translational changes.

Figure 2 presents a genomic DNA gel blot that confirms the existence of an enolase multigene family in tomato. Hybridization of the TENOI Bglll-EcoRI fragment to total genomic tomato DNA revealed three Bglll and three EcoRl restriction fragments, some of which had an intensity higher than one copy equivalent (Figure 2, lanes 4). It would seem, therefore, that the tomato genome contains

at least three, and probably more, genes encoding enolase. Under high-stringency conditions (65OC, 250 mM phosphate buffer), strong hybridization was observed to Ara- bidopsis (Figure 2, lanes 1 and 2), rice (Figure 2, lanes 3), and tobacco DNA (Figure 2, lanes 5), indicating a high degree of interspecies similarity. Three hybridizing bands were present in the rice lanes, whereas multiple bands were observed with tobacco DNA. Thus, the existence of multiple enolase isoforms appears to be fairly common in the plant kingdom.

In Arabidopsis, however, hybridization occurred to a single Bglll and EcoRl restriction fragment, both of which were approximately 7 kb in length and had the intensity of one copy equivalent. The unique Arabidopsis enolase gene was cloned by hybridization with mixed oligonucleotide probes to about 30,000 colony-forming units from a genomic cosmid library with the mixed oligonucleotide probes (see above). Figure 3A presents the complete sequence of the Arabidopsis enolase gene (4553 bp). The sequence covers 1060 bp of the 5’-untranslated region and 786 bp at the 3‘ end. Putative TATA and CAAT boxes (Joshi, 1987), as well as potential polyadenylation signals (Dean et al., 1986), are underlined. The gene contains 11 introns, all displaying the consensus dinucleotides at their boundaries (Csank et al., 1990) (Figure 38). The positions of exon boundaries are conserved between tomato and Ar- abidopsis, with the exception of exons 6, 7, and 1 O of the Arabidopsis gene, which are joining two or three corresponding exons of the tomato gene. Although the plant enolases that we have cloned are most similar to the mammalian (Y isoform, there is no conservation of intron organization between the human (Y gene (Giallongo et al., 1990) and the Arabidopsis or tomato enolase genes. Sim- ilarities between the entire tomato and Arabidopsis coding regions are 80% at the DNA and 90% at the amino acid level.

Because several glycolytic enzymes are anaerobically inducible and carry the anaerobic responsive element (ARE) core consensus sequence in their 5’ end or first intron (for a review, see Dennis et al., 1987), we have looked for its presence in the Arabidopsis enolase gene. The essential or core anaerobic region TGGTTT was found in the first intron, 228 bp downstream from the translational start. The surrounding sequence is 62% similar to the 13-bp sequence of the maize alcohol dehydrogenase 1 (ADH1) ARE core consensus, which is also present in the first intron of the maize sucrose synthase gene (Springer et al., 1986).

Because enolase was reported to be heat shock inducible, as are several other glycolytic enzymes in yeast (lida and Yahara, 1985; Piper et al., 1988), we searched for sequence similarity to the plant consensus heat shock element (HSE) 5’-T-TTC--GAA-A-3’ (Schoffl et al., 1989) in the Arabidopsis enolase promoter. Maximal similarity was in the region 833-844, where the sequence 5’-TATTCTGCAAAA-3’ occurs. Although 75% similar to

Plant Enolases 721

A

1

144

287 h31

575

717

860 1004 1148

1289

1432

1576

1719 1863

2006

2150 2294

2436

2578

2720

2862

B

c8~aaactataaacttttttctcttcactcttttct~ctatttct~tgttcacagatct~TGG~CTATCAAATCCATTAAAGCTCGTCAGATCTTTGACAGCCGTGCTAACCCCACAGTCGAGGTT~TGTACATATCTCA m a t i k s i k a r q i f d s r g n p t v e v d v h i s

AATGGTGTGTTTGCCAGAGCTGCTGTTCCAAGTGGTGCATCCACCGGAATTTACGAAGCCCTTGAATTGAGGGATGGAGGATCTGACTACCTCGGAAAGGGTGTTTCGAAGGCTGTTAA~TGTCAA~CAATTATTGGCCCT n g v f a r s a v p s g a s t g i y e a l e l r d g g s d y l g t g v s L s v n n v ~ ~ l l g p GCTCTTGTTGGCAAGGACCCAACTGATCAGACTGGTCTTGATAACTTCATGGTTCATCAGTTAGATGGAACTCAAAATGAGTGGGGTTGGTGCAAAG~G a l v g k d p t d q t g l d n f m v h q l d g t q n e w g w c k e k

a a g c t t e a c a t t e g g r ~ a c a t g t ~ a c t a t c a g a ~ g t g t ~ ~ ~ ~ ~ t t ~ ~ ~ ~ ~ t t t ~ ~ ~ t g g g t t g g ~ ~ t t t t t t g ~ ~ ~ g CTTccTGCAAATCCCATCCTTGCTGTGTCACTTGCAGTCTGTAAAGCT~GCTGCTGTCAAGAAG CTTGGTGCAAATGCCATCCTTGCTGTGTCACTTGCTGTGTGTAAAGCTGGCGCTGCTGTCAGGAAT L G A N A I L A V S L A V C K A G A A V K r K n

H I A N d L A G N K K L V L P V P A F N V I N G G S H A G N K L A U Q gcstgtat t t tc tc=tgt taaaagatgtagt taaatgatgct tag~aaat tactaat tagt tgt ta t t tg~~g GAATTTATGATTCTTCCTGTTGGAGCTTCTAG~AA~GGCCATGAAGATGGGCTGTGAAGTGTAT

GAATTTATGATCCTTCCTGTTGGAGCTGCTAA~CAAGGAGGCCATGAAGATGGGCTGTGAAGTCTAT E F U I L P V G A S a S n F K E A U K U G C E V Y

C A C C A ~ G A A G g t t t c a e s c a g t t g t t c t a g ~ t t ~ t ~ t t ~ ~ ~ t t ~ g t ~ t t t ~ t t t g t t t ~ ~ ~ ~ g t ~ ~ ~ ~ g ~ ~ t ~ g ~ g g ~ t ~ ~ g t t g t t ~ t t t t ~ t t t t ~ ~ t t t t ~ ~ t t t ~ t t t g ~ g g t ~ ~ a t t a g g c t a g a a a a t a c t g t g c CACCAlTTCAAG H H L K t c c e t e t g c c a ~ a ~ t ~ t a t g t g a r g g t e r t t c c t t t e a g t t ~ ~ t t t ~ ~ ~ ~ ~ t t a ~ t g a a a t a ~ c t t t t g g ~ t t c c a t t t g ~ t g g a a a c a t t c t a c c ~ t t t c t c a g c t g a g a g a g a ~ t a g t a a a a a ~ t a t g t a c a a c t ~ t c g t g a c c t c t c a c t t a a t t g t t g g ~ g c t g a ~ g a c a a ~ t t t ~ t a ~ t t t g c a t a t t c ~ ~ g c ~ t a t g t g t c ~ g ~ a g c c t t t ~ t t t c t t g a a c t c c g r g c a c a t c c a a a c t a c g c t a c a r a a a a r g g g ~ ~ = ~ a ~ ~ g t a t t ~ ~ ~ t t t ~ t t g ~ t g t ~ a = ~ t ~ ~ = ~ t t t t t ~ t t t t ~ = t t ~ t = t ~ g = t g ~ t g g = a g t t t ~ = t = t ~ ~ t t = t ~ g GCTGTGATTAAGAAGAAATATGGCCAG

GCTGTGATTAAGAAGAAATATGGTCAG A V I K K K Y G Q

GATCCAACAAATGTTGGTGATGAGGGTGGTTTCGCTCCTAATATCCAG gcaaagtaeaaarttatgctgttgaatggcatactgtegaaeggata~t~ttttacatcatgtgactatggettttgaaaattcagGAGAACAAG GATGCCACAAATGTCGGTGATGAGGGTGGTTTTGCTCCTAATATCCAG GAGAACAAG D A T N V G D E G G F A P A I Q E N K G A G G G T C T TGAATTGCTCAAGACTGCCATCG~GCAGGTTATA~GG~gta~g=t~ttg~=tg~attta~at~~ga~~tttct~~~a~cg~tctca~c~atgt~ga~g~t~aga=t~tttt==tttt~gGTTGTCATC GAAGGTCTTGAATTGCTCAAGACAGCCATTGAGAAAGCAGGTTACACTGGAAAG GTGGTCATT E G L E L L K T A I E K A G Y T G K V V I GGAATGGATGTTGCAGCATCTGAATTCTATGGAAAGGAC~CTTATGATTTGAACTTCAAAGAAGAG g t a t a t c ctttctgactatg~~tagt~gtgcattcasgt~gt~t~c~~gt~~~g~at~ttg~agtgg~tttt~t GGAATGGATGTTCCAGCATCTGAA~ACGGAAAGGACAAATCTTATGACCTGAACTTCAAAGAAGAG G M D V A A S E F Y G K D K T s Y D L N F K E E c t t t c a t c t t g a t g g t t c t g a a g c c c a ~ ~ t a a c ~ a ~ a ~ c ~ t ~ a ~ ~ t g ~ ~ ~ a ~ t t a ~ ~ ~ t t t t g g ~ ~ ~ t ~ t ~ t t c c g g t g a t ~ g a c a t c ~ a t ~ ~ g t ~ ~ t g ~ ~ t a ~ t ~ a ~ ~ ~ ~ t ~ t ~ ~ t ~ ~ ~ t ~ ~ t t g ~ a ~ a t a g ~ t a t t c ~ a g t g a g c t g g a a a t t a t a g g a c r c r t t t ~ c t g ~ g g t a g t t t ~ ~ t g t ~ t g a t t t ~ t t ~ t = t ~ ~ t = = g ~ t t t g = t t = a a t t g t t c c a t ~ t t t g ~ ~ g AACMCAATGGTTCCCAAAAGATCTCAGGTGACCAGCTCAAAGATTTGTAC

ACCAATGACGGCTCACAAAAGATATCAGGTGACCAACTCAAGGACTTATAC

AAGTCATTTGTGTCTGAGTACCCTATTGTTTCGATAGAAGATCCATTTGACCAAGATGA~GGGAGACCTATGCTAAGCT~CTGCTGAAATTGGGCAGAAAGTACAAATTGTAGGAGATGACCTTCTCGTCACCAATCCTAAG AAGTCATTTCTGTCCGAGTACCCTATTGTTTCAATTGAAGATCCATTTGACCAAGATGACTGGGAGACCTATGCTAAGCTCACTGCTGAGATTGGGGAGCAAGTACAGATTGTCGGTGATGACCTTCTTGTCACCAACCCTAAG K S F V S E Y P I V S I E D P F D Q D D W E T Y A K L T A E I G Q e K q V Q I V G D D L L V T N P K g t a e a a c t g a a g c g t t a c a c a a c r a g t t g t c a t t t t c t t g c t t ~ g ~ a t t g c a t g c t t a g a t g t t c c ~ t ~ a t t ~ ~ ~ g g ~ a a t g ~ t ~ g ~ g ~ ~ ~ ~ t ~ t t ~ t t g t g t ~ t ~ ~ g ~ g t t ~ ~ t t t ~ t ~ a a t c c c t t g g a a a t t ~ t a t a a t tatcagttgratctgtgcaccggtgcag AGAGTGGCCAAGGCAATCAGTGMAAGACTTGCAATGCCCTTCTTCTTAAG g t a c t t a ccataccatctaagc~~ctg~c~tgt~~t~tt~atttggagat~~~t~tgttg~a~

N s N N d G S Q K I S G D Q L K D L Y

AGGGTCGCCMGGCAATTGCAGAGAAGACTTGCAATGCTCTTCTTCTCAAG R V A K A I S a E K T C N A L L L K

rercrgcrtctaa~trcgcgcg=ag GTTAACCAAATTGGCAGCGTGACTGAGAGTATCGAAGCTGTG~TGTCTAAGCAAGCTGGTTGGGGTGTAATGA~AGCCACCGCAG graaggaga~tgtgatgctctg=~~t~g GTTAACCAAATCGGTAGTGTGACCGAGAGTATTGAAGCTGTGAAGATGTCCAAGAAGGCAGGTTGGGGTGTAATGACCAGTCACCGCAG V N Q I G S V T E S I E A V K U S K Q k A G W G V M T S H R S

gct~gcagasagaggaaartca~ttatgctc~gtcgtttatg~c~~ttgtt~~~~~t~tttgggt~~t~ggtg~~g TGGAGAGACGGAGGATACCTTTCATTGCTGATCTTGCT~GGCG gtaattttgtaeat TGGAGAAACAGAAGATACCTTCATTGCTGATCTTGCTGTCGGTTTGTCAACG G E T E D T F I A D L A V G L S T

g c a c t t t t g t c t t a t c t a a t t t a t a g c g t g t = ~ ~ = t = g g a a t a g t c t t t g g t a c ~ ~ ~ t t ~ t g t ~ t ~ t ~ g t g g ~ t t ~ t t t t ~ g GGACAAATCAAGACCGGAGCTCCCTGCAGATCAGAACGTCTTGCAAAGTACAACCAG gtact GGACAAATCAAGACTGGAGCTCCTTGCAGGTCAGAGCGTCTCGCCAAGTACAACCAG G Q I K T G A P C R S E R L A K Y N Q

caccccaa~ag~acag~~tact~~g~tggtatt~c~att=~=tgg~gcttctctctgcagccag~tctg~ttt~t~gtt~~tt~~~t~agCTCTTGAGAATTGAAGAGGAACTTGGTTCAGATGCTGTATATGCCGGAGCAAGC CTGTTGAGGATCGAAGAGGAACTCGGATCAGAGGCTGTTTATGCAGGAGCAAGC L L R I E E E L G S D e A V Y A G A S

TTCCGCAAGCCCGTTGAGCCCTACTAA a t t ~ c a g e a g t t c c a a g t g t t g a g a t t g t t g a g t t g t t g ~ t ~ t g c c a ~ ~ ~ a t a ~ ~ t t ~ ~ t ~ t t a g ~ t t g g t ~ ~ t t t t a g g ~ t t a t a t t ~ t ~ t g a ~ ~ t t ~ t g ~ a ~ t ~ a ~ a ~ g ~ a g ~ f r k p v e p y . c t c a a a a a c a a g t t t t g t t t t ~ t g ~ g g t t t t ~ t t ~ g t ~ t t t t

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Figure 1. Nucleotide and Deduced Amino Acid Sequences of the Tomato Enolase cDNA TENO1 (EMBL Accession No. X58108) and the Tomato Enolase Gene gTENO2 (EMBL Accession No. X58109).

(A) DNA and amino acid sequences of tomato enolases. The cDNA (TENOl) is numbered on the right, the genomic sequence (gTEN02) on the left. DNA sequences have been aligned, noncoding regions are in lower-case type. Deduced amino acid sequences are shown in the one-letter code, in upper-case type. Nonidentical amino acids of the TENO1 clone are in lower-case type. (B) Intron/exon structure of the tomato enolase gene gTENO2. The boxes represent expressed regions.

722 The Plant Cell

1 2 3

B E B E B E B E B Ekb

1-6.0-4.0

-3.0

-2.0

-1.5

--1.0

-0.5

Figure 2. Genomic DNA Gel Blot Analysis of A. thaliana (ecotypeC24), Oryza saliva (cv Taipei), L. esculentum (cv Orlando), andNicotiana tabacum.

Lanes 1, A. thaliana, 1 ̂ g of total DNA; lanes 2, A. thaliana, 1 P.Qof nuclear DNA; lanes 3, rice, 3 /jg of nuclear DNA; lanes 4,tomato, 5 ng of total DNA; lanes 5, tobacco, 20 ^9 of nuclearDNA. B, Bglll; E, EcoRI. One-copy reconstruction on the right.Filters were hybridized with a 32P-labeled 1.5-kb Bglll-EcoRI frag-ment of the TENO1 cDNA clone described in Methods. Exposurewas for 6.5 hr on flash-sensitized film.

soybean HSE2, this sequence does not correspond en-tirely to the required hyphenated dyad, and, moreover, itis not interlocked with a second heat shocklike element,another essential requirement for heat shock inducibility(Schoffl et al., 1989).

Primary and Secondary Structure Comparisons ofEnolases

Figure 4 shows an amino acid sequence alignment andsecondary structure predictions for prokaryotic (B. subtilis;D. Vereecke, unpublished data; Verma, 1989) and differenteukaryotic enolases. The eukaryotic sequences are from

Arabidopsis, tomato, chicken (0 form; Russell et al., 1986),X. laevis (Segil et al., 1988), rat (7 form; Sakimura et al.,1985a), human (« form; Giallongo et al., 1986), and yeast(ENO1 gene product; Holland et al., 1981).

A number of deductions can be made from inspectionof the amino acid alignment. The derived consensus se-quence covers all amino acids found in the active siteregion of yeast enolase (Lebioda et al., 1989), with theexception of Lys345 and Lys396, which are replaced by Glyand Glu, respectively, in the chicken @ form. The highdegree of similarity between enolases is also shown inTable 1, where pain/vise comparisons are presented. Plantenolases have a lower similarity when compared with yeast(either ENO1 or ENO2) than do higher animals comparedwith yeast. When compared with animal sequences, plantenolases have the highest similarity to the « form. Thesequence around Tyr46 in the «form, which was shown tobe phosphorylated in Rous sarcoma virus-infected chickenembryo fibroblasts (Cooper et al., 1984), is identical to thesame region in plants. Whether this is of any significancein plant cells remains to be elucidated. Finally, highereukaryotic enolases have an increased number of cys-teines (>5), all of which appear at positions different fromthe single cysteine in Bacillus or yeast enolase.

Another striking feature is the occurrence of a 5-amino-acid insertion unique to plant enolases (QNEWG, residues101 to 105). The influence of this insertion on the second-ary structure was investigated. Predicted structures arepresented in Figure 4, "averaging" four different methods.The pentapeptide unique to plants is part of a predictedcoil region, which is also predicted in coil for the otherenolases and corresponds to a coil region in a modelbased on high-resolution x-ray crystallography of yeastenolase (Lebioda et al., 1989). Therefore, the pentapeptideis expected not to influence the tertiary structure signifi-cantly. However, it is possible that the 4 amino acids,adjacent to the insertion, extend the neighboring a helix.

The validity of the predictions was supported by the factthat the secondary structures of different enolases are ingood agreement over aligned positions. It was furthertested by "averaging" the secondary structures of all spe-cies ("join pred." in Figure 4) and comparing them with themodel of Lebioda et al. (1989). The alignment in Figure 4shows a reasonable agreement between both models,although the position and/or the extent of a secondarystructure is often missed by a few residues. Helices aremore faithfully predicted than sheets (e.g., the last 0 sheetis not seen in our averaged secondary structure). Thepredictions are in any case more reliable than those gen-erated using the Chou-Fasman method (Chou and Fas-man, 1978; data not shown) and seem to be more accuratethan previous predictions of enolase secondary structure(Sawyer etal., 1986).

Plant Enolases 723

A 1 atcaaaagaaatagcactaaaggctcggaggaagcctgatgaaacatggaagattgtgctctattttcttctgacaatttttacataa~aaaacgcatttgtttactatttttttcata

1 2 1 taaaacatgaaaaacttatatttgaattaatcgaaattaaaattattaacagaaatatctaagtttatatgaaccttttaacaaaaaaaaaagtttataagaacataaaaatcataatag 241 tttagcaacatttaaattattttc~aaaattagtaacttagattaaaataaatattagatcacctcataatcttgagtttgaaactccaaa~caaagagcatccaaaaatcCg~gC~ 361 aaCacCacattttgataagaattatagaactagtgatttgcattttaaatgttgatacatatagaataagcataatcaaacaatgattactgaaaaatatg~ccattaatatcgt~~~~ 481 aaatggt~atggacattgaaaccctagtggagaatttgtcacataagtaaggcccaaagtttttgacccacaaacatatccattaagttatagtttagcgaaacccctttaacaaaaaa 601 gaaaattttc~actagtgaattgtttct~gaga~tctgtacaaccatccaaatttcaaacatggtataaaagatgttattgacaaaataaaaatggaaacagtgaaacgtata~Cgg~ 7 2 1 aaatqgaataaaatctagatgcc~tat~ttattcttacttgttctaaagtctttaataaaaatagtcggtattacttggacaaggagcaaaacaatatggaaaaaactcttctattctgc E41 aaaaggcgtgcagcgcatc~tttggcttcttgc~tcagagctgactgttctcatccaacggctgttattaaaacaatccaacggttttggc~tccgtgac~ctttatatatC~~~ 9 6 1 ccagaccaccaacccatttcctcagctactactgttgaagcgattctCactaaaaccctCgaacacatcgcctttatCtctttCtctaga~tactcgct ATGGCTACTAXACCGTT ~ A T X T V

1079 GTTM~CTAGACAGATCTCGACA~GTGGTMTCCCACCGTTGAG gttagtttctccgatcacttttgt~tttcccagtcactttccggctttgtacagtattc~gacggatctg

1198 tttgtttgatgactatccgatgct~a~accactattcaatcgtttttttgtaaacctgaattgatctagtagtcgtacgtgaatgagat tt tgtgaacgatgatcggtgatttg 1318 ~tctcggcgatttggatcgtgagttgtcgatgatggagttgattttgtttatatgatttttgcgacggatctatttatttccatctggt~tttcgattgtttgctaatgacga

V K A R Q I F D S R G N P T V E

1438 ~ t t t g ~ ~ ~ ~ g ~ ~ ~ 9 C t a ~ t g a t t t c t C t g ~ t g ~ t g t t g g t t t a g G 1 T G A T A ~ C A C A C G T C * M T T A T T M ~ T T A C A ~ ~ ~ ~ G ~ ~ C A C T G G T A T C T A T G A G V D I H T S N G I K V T A A V P S G A S T G I Y E ~~ _ _ . . . _

1557 ~ A ~ T G A G G A ~ C T A C C T T gtgagagactgcgatctgtttgtgtgtttttttagtgttagtagagttatttcaattgatcatg

1676 ttgactgattcgttatttgtttatatcactag G A T C ~ G C C A ~ A C T T A - ~ G gtgagtgactcactttaagctactCtcagaaCatCaaa

1794 t t t g t g t c t a a t t c t a t g c g c t a g g c t c a c t c a t t t t a t t t t t g t t g c t t t t a t t a t t t c t t g c a g G A C C ~ ~ G C A G A C T G C T A ~ ~ C ~ ~ T C C A T G M C ~ A C G G A

1914 ACCCAAMCGAGTGGGGGTGGTGCMGCAMAG gtacttaaaagggaaaattcattttttgtgtattc~cttaagaatagaaggcaagtctgaccatcttgcgccttctatctgtgttt

2033 ag C T T G G A G C C M T G C G A 1 T C T T G C ~ T G ~ T C ~ ~ T G T T G T C A ~ ~ A T T C C T C T A T A C M G gtagaaCgatCtggCaCtattaagttggttCCCtC

A L E L R D G G S D Y L G K G V S K

A V G N V N N I I G P A L I G K

D P T Q Q T A I D N F M V H E L D G

T Q N E W G W C K Q K

L G A N A I L A V S L A V C K A G A V V S G I P L Y K 2151 tgtccatggaagataagattgttactgctttggctacttggttttttcggtgataaatgttctaagcttttccctttgtgtggtatttgggtgcag~CA1TGC~CC1TGC~GTMC

2271 CCCMGATTGTGCTACCAGTTCCTGC~MCGTCATCMTGGTGGATCCCATGCCGGAMCMGCTTGCTATGCAGGAG~A~ATC~CCTGTTGGAG~~MGGM

2391 GCCATULAGATGGGTGTGGMG~ACCACCA~MG gtaaatctccaatattttttggtctttgagatgctcttcaatgaatattccatatacatgtattactttttcgcagatgg

2510 t t c t g t g a a c t a a c a t ~ ~ t t a c a t g t t t a ~ g c t t a a c c a t c t t g t ~ t c a t a t g t t t t t c c a ~ T G T G A T T M G M G M G T A C ~ C A ~ A ~ C A ~ T G ~ T G A T G M ~ T

H I A N L A G N

P R I V L P V P A F N V I N G G S H A G N K L A M Q E F H I L P V G A A S F K E

A M K M G V E V Y H H L K

S V I K K K Y G Q D A T N V G D E G 2630 G G G m G C A C C A M C A T T C A G G A G M C M G G A G ~ T C ~ 1 T G C T C M G A C T G C T A T C G A G M G G C T G G A T A C A C T G G ~ G gtctgtctcttatctgatgta~tttggctgtcctt

2749 ttacccgaaacataattcatgaaactaatggatcccattcaattttttccag GTTGTCATTGGMTGGATGTTGCCGC-GAGTTCTAC~MGACMGACCTACGACTTGMC

2867 TTCAMGAAGAG gtgaaat t tc t t t t tcct ta~aacagct tgtcaggatagagtgtgagaaacctgt tct taact t~agctgt tgcgctct t tgt t tc tctgcagMCMCM~GC N N N G

2986 T C T C A G M G A ~ C T G G T G A T G C T C T A A A G G A C C P G T A C M G

3106 ACCACTGAGn;TGOMCCGAGG~GATTGTCGGTGA~mG-TCACTMCCCCMG gtttgtttttatttgccagtgttcttaccagtttttttttgttgtctgcCCtCtCt

3225 taaaatgctcacatCtctcttatactcttttcttattccag A G A G ~ C T M G G C M T C G C f f i A G M G ~ C M T G ~ ~ M G G T T M C C A M T C G G A T C T G T M C C G A G

3344 A G T A T C G A G G C A G T T M G A T G T C G M G ~ ~ G ~ G G G A G ~ ~ C C A G C C A C A G M G T G G ~ C C G A G G A C A C A ~ T T G ~ ~ A G C C G ~ G C ~ T C C A C T gtaag

3463 ttgaaaccattgcaagcatttaactagcaatctcatgctatatcttttgtcggactctcatttggatctttctgtctatacag GGACAMTCAAMCCGGTGCTCClTGCAGATCCGAG

3582 CGTCTTGCCMGTACMCCAG gtttaacatctcagaccacta~aaagactttatgtttgacaaccagagtacattgtctaatatgtgtcttgtttctcaacag~CGTATTGAG

3701 G A G G A G T T G G G A T C A G A O G ~ ~ A C G ~ G ~ ~ C G C ~ C C T G ~ G M C C C T A ~ M atggagcttttagaagcaaagtggtCttCtttgtgaCg~gg~g~~g~tgRCCt

3820 gagtttgatcatttgctttaattaaata~aacgttctgtttttgtttcttctttgtttggtttcttacgtcttttgttgaaccctttttgggaaaa~tactcatttttgtaagggaaac 3940 atgagaatgcttgcctttgtcgaggacggtagccccttatttcaatctaatttc~ctttactCtttaatcttc~atgtgat~ttatgatctctggtgacatacgatggagttt~a 4060 caaaaataaaataagttcttcattattatatta~aattgcaactctagtaaaggttttaaaatagacctgcttgtgttcatgactactattattcaacaagagttcacacgtaaaaaagg 4180 tgtgaacadthtctgaagatgactagaaaaacattcatacaaaat~gtagataaatcattttct~tcggtttatcacttttatcgttcactttctgttgaaataagcttaa~aagt 4300 gatggtgaaggacaaaaatttcttg~atttgg~tgtagaataataacaagtacttggtttattaacagatattcaataagcaggtttgcccgagtggttaagggggaagacttaagttc 4420 t~tgcacataagtgcgcgtgggttcgaaccccacagcctgcatctttctCtttattttttgaaattttcaatgcatgaagtttttaggaaaaaagattttaggaaaaatatgcaggCtg 4540 tggggttcgaaccc

G F A P N I Q E N K E G L E L L K T A I E K A G Y T G K

V V I G ~ D V A A S E F Y S E D K T Y D L N

F K E E

S Q K I S G D A L K D L Y K S F V A E Y P I V S I E D P F D Q D D W E H Y A K M

T T E C G T E V Q I V G D D L L V T N P K

R V A K A I A E K S C N A L L L K V N Q I G S V T E

S I E A V K M S K K A G W G V M T S H R S G E T E D T F I A D L A V G L S T

G Q I K T G A P C R S E

R L A K Y N Q L L R I E

E E L G S E A I Y A G V N F R K P V E P Y .

B

H

Figure 3. Nucleotide and Deduced Amino Acid Sequences of the Arabidopsis Enolase Gene (EMBL Accession No. X58107).

(A) DNA and amino acid sequences of the gAENO clone. Noncoding regions are in lower-case type. Deduced amino acids are in the one- letter code. Putative CAAT and TATA boxes and polyadenylation signals are underlined. The ARE core sequence (TGGTTT) in intron 1 is boxed . (B) Intron/exon structure of the gAENO clone. The boxes represent expressed regions.

724 The Plant Cell

A R D S R G PTVOJ VF'SGASTG EA E RD GKGV AV N I P DGT K K M P Y I V D W B B E L Z D S R G N P l E T G A F G R A L V P S G A S T G E Y ~ V ~ R D G D K D R X L G K G V L T A V N N V N E I ~ GFDVTEQN-DGT ENKGKL MATI~RQIFDSRGNPTIHTSNGIWTAAVF'SGASTGIYE&ELRDG GSDYLGKGVSKAVGNVNNIIGPALI GKDPTQQTAIPNWtlELDGTNEWGWBm M A T ~ Q I R o S R G N P T I S N G V F A R A A V P S G A S T G I Y E U & R D G CSDYLGKGVSKAVNNVNSIIGPALV GKDPTWTGLDNLYYHQLDGTNEWGKKEKL

S I ~ D S R G D P T V B V R L U A K G H F R A A V F ' S G A S T G L H E & E U D G D & K U I . G ~ T I G P & L € X X I S W ~ K V ~ M D G T E N K S K F M S I K N I M E I F D S R G N P T V W ~ G L F R A A V P S G A S T G I Y E Q L E L . R D N D K T R X L G K G V G R A W Y J % % ' F L G P A L C T Q N L N V ~ D G T E N K S E MSI-DSRGNPW- R A A V F ' S G A S T G I Y E U & R D G D K Q B U G ~ N S T I A P A L I S S G L ~ ~ E T E N K S K F M S I W E L F D S R G N P T V W L U X S & G L F R A A v P S G A S T G I Y E U & R D N D K T U W G K G V ~ L N f l E ~ E T E N K S K F

AVSKVYARBWDSRGNP'l'VEVELTTEKGW R B I V P S G A S T G V H & A L D R D G D K S ~ K G D & ! & N ~ I D V K W K A V D D F L I S L E T A N K S U C C C ~ C C C C C C C E ~ C C H E E C E E C C C C C C C C ~ C C C C C ~ C ~ C C C ~ C C C ~ C C C C C C C C C C C C ~

BBEEEEEEE EEEBBEEEB EE EEEEE P H H H H H H H H H H H

conserve6 B A C I L L U S A R A B I D O P S I S TOMATO C H I C K E N ( B E T A 1 F R C G R A T (GAMMAI HUMAN ( A L P H A I Y E A S T (1 I j o i n pred. L E B I O D A et a1

conserve6 B A C I L L U S A R A B I D O P S I S TOMATO C H I C K E N ( B E T A 1 F R C G R A T ( GAMMA I HUMANIALPHAI Y E A S T (1 I j o i n pred. L E B I O D A et a1

conserved B A C I L L U S A R A B I D O P S IS TOMATO C H I C K E N ( B E T A 1 FROG R A T (GAHMAI HUMANIALPHAI Y E A S T (1 I jo in pred. L E B I O D A et a1

conserved B A C I L L U S A R A B I D O P S I S TOMATO C H I C K E N ( B E T A I FRCG R A T (GAMMA 1 HUMAN(ALPHA1 Y E A S T (1 I 3 o i n pred. L E E I O D A et a1

. l . I . l . I . I . I . I . I . I . I . l m 1100]

G A N A I L V S A A A P L Y L . d G G HA QEFM P GA F A G H LK G VGDEGG A P N I GANALLG-IPLIQYL GGFNSKTLPVPMMlIVNGGEHADNNV M P V G A P N F P G L N TAVGDEGGFAPNL GAN-GAWSGIPLY-L A G N P K I V L W P A F N V I N G G S H A G L P V G P m Y G Q D A T N V G D E G G F A P N I G - G A A U N V P D &"VLWPAFWINGGSHAG L P V G B Y G Q D A T N V G D E G G F A P N I GANAILGVSL&!SHAGAAEKGVPLYBtLIBPL AGNTELILPVPAFNVINGGSHAGN L P V G A Y G K D A T N V G D E G G F A P N L G - G A A € X G V P m D L A G N P E V I L W P A F N V I N G G S H A G L P V G A D S P F K Y G K D A T N V G D E G G F A P N L GANm-GAAmD- A G N S D L I L P V P A F W I N G G S H A G L P V G m m - Y G K D A T N V G D E G G F A P N I G A N & L G m K A G A V E K G V P m D L A G N S E V I L P V P A F W I N G G S H A G L P V G P M Y G K D A T N V G D E G G F A P N L G A N P DLSKSKTSPWLPVPFLNWNGGSHAG A F T G ~ G S ~ T K K R Y G A S A G N V G D E G G V A P N I C-CC- C C C C C E E E C C C C C E E E C C C C C C C C ~ E E C C C P Hccccccccccccccccccc -- EEEE EEEEE

I . l . I . I . I . I . I . I . l . l . I . (1501

E L A I AG D A S E F Y Y Y P S I E D W Q V DDL V G S N E F A L O T I V E A I E K A G F K P G E KEDGKY HLSGEGVVKT-KYPIIS QENKE-GY TG S E D K T Y D L N ~ P V N N G S Q K I S G ~ E Y P I V S QENKE-GY TG GKDKSYDLNFKEESNDGSQKISGDQUQLUSFVSEYPIVS LLNIIEALEI.tKAAIAOGY T D RDGRYDLDFK SPPDPKRLITGEQLG.8IYRGFIKDYPVVS -KAGY P D RDGKYDLDFK S P D D P S R Y I S P D K ~ N Y P W S LEN-GY T E K M Y I RDGKYDLDFK S P A D P S R C I T G @ L w F V R N Y P V V S & N K E G L E I . L K T A I G K A G Y T D I N V I RSGKYDLDFK SPDDPSRYISPD-IKDYPVVS -GH D G K V I U KDGKYDLDFKNPNSDKSKWLTGPQUUUSUJXRYPIVS t l t l C ~ C C C C C C ~ E E E E ~ C C C C C C C C C C C ~ C C C C C C E C C C C ~ C C C ~ E E C C C C C C C ~ C C C E E E E C C C ~ EC

HHHHHHHHHHHHHHH EEEEEEE EEEE HHHHHHHHHH EEEEE I . I . I . I . I . / . l . l . I . I . I . 1250/ 1300(

N K L VNQIG E A G S H R S G E T E D I A D V G Q I G A P R R AK NQL R I E L A G Q I G T L T ~ G Y T A V I S H R S G E T E D S T I A D I A V A T N A G Q I K T G A P S QIGWESLZA.U(@KKAGWVMTSHRSGETEDTFI"fGLSTGQIKTGAPC Q l G W E S ~ K K A G W G V M T S H R S G E T E D T F I A Q U V G L S T G Q I K T G A P C QIGWESI~CK~QSHGWOVYVSHRSGETEDTPIBPLWQLCTGQIEQGAPC Q I G T V T ~ Q S N G W S H R S G E T E D T P I B P L W Q L C X Q I K T G A P C Q I G S V ~ A Q E N G W G W S H R S G E T E D T P I B P L W Q L C T G C ? I K T G A P C Q I G S V T ~ Q A N G W Q V H V S H R S G E T E D T P I B P L W Q L C T G Q I K T G A P C Q I G T ~ ~ G W O V Y V B H R S G E T E D T P I B P L W Q L R X Q I K T G A P A

C C H H H H H H H H H W H H H H H H H E E C C C C C - C C C C E E E E E C C C C C C C C C E E L f U E E E C C C C C C C C C C C C C P Hccccccccccc EEEEE HHHHHHHHHHHHHH EEEEEE EEEE HHHHHHHHHHHHHHHH

l _ I _ I . I . I . I . I . l . I . I . I . I 14001

Figure 4. Amino Acid Alignment and Secondary Structure Predictions of Prokaryotic and Eukaryotic Enolases.

The consensus sequence is indicated at the top. Active site amino acids are boxed. The predicted secondary structure is presented as follows: residues in an (Y helix are italicized and underlined and residues in a p sheet (extended strand) are in boldface type. All other amino acids are in coil structures. "Join pred." is a predicted secondary structure averaged over all enolases. H, helix; E, extended strand; C, coil. The actual structure of yeast enolase, as reported by Lebioda et al. (1989), is indicated at the bottom. The boxed numbers at the bottom refer to the position in the amino acid sequence.

Plant Enolases 725

Table 1. Percent Amino Acid Similarity between DifferentProkaryotic and Eukaryotic Enolases

12345678g

1 2 3 4

43.0 42.6 43.295.4 51.1

50.4

5

41.653.553.089.9

6

42.660.859.764.566.8

7

39.559.657.558.759.878.3

8

43.059.458.063.664.082.080.8

9

43.061.360.165.466.687.881.583.2

1, B, subtilis; 2, yeast ENO1 gene product; 3, yeast ENO2 geneproduct; 4, A. thaliana; 5, tomato; 6, X. /aew's; 7, chicken /3 form;8, rat 7 form; 9, human « form. Percent identity was calculatedby the PC/Gene program, Release 6.01 (A. Bairoch, University ofGeneva, Switzerland).

Absence of Plastid Enolase in Arabidopsis

There is a single nuclear-encoded enolase gene inArabidopsis because nuclear DMA gel blots (Figure 2, lanes2) showed one band and total genomic DMA also yieldedone hybridizing band (the weak signal above is from non-digested DMA remaining in the wells; Figure 2, lanes 1).Any chloroplast-encoded enolase must have a highly di-verged sequence. Moreover, sequence comparison be-tween TENO1 and both the tobacco and liverwort chloro-plast genome sequences (IntelliGenetics Suite, Release5.37, IntelliGenetics, Mountain View, CA) did not revealany significant similarity. Indeed, in those cases that havebeen studied, cytosolic and plastid glycolytic enzymes areall encoded by the nuclear genome (Weeden and Gottlieb,1980; Cerff and Kloppstech, 1982; Gottlieb, 1982; Shih etal., 1986).

In addition, no homology was found between theenolase N-terminal amino acid sequence and the consen-sus chloroplast target sequence (Schmidt and Mishkind,1986; von Heijne et al., 1989). This, together with the factthat the Arabidopsis enolase N terminus is very similar tothe Bacillus and yeast N-terminal sequences (Figure 4),supports the idea that the Arabidopsis enolase polypeptideis not imported into the chloroplast. Further evidence insupport of this conclusion is based on our inability to detectenolase in purified chloroplasts by either activity or immu-noassays (data not shown). Both assays were positive ontotal leaf extracts. The arguments presented abovestrongly indicate that Arabidopsis chloroplasts lack at leastpart of the glycolytic pathway.

Expression and Activity of Tomato Enolase in E. coli

Tomato enolase was expressed in E. coli in two differentways. A translational fusion was made to the 010 gene in

the pT7-7 expression vector. The hybrid polypeptide lacks11 N-terminal amino acids from enolase, which are re-placed by 9 amino acids encoded by the polylinker of thepT7-7 vector. Figure 5A shows that the recombinant pro-tein, induced by temperature shift of a K38(pGP1-2/pT7-7-TENO1) culture, migrates at 56 kD in denaturing poly-acrylamide gels (SDS-PAGE; Laemmli, 1970). The dena-tured recombinant product was recovered from prepara-tive Laemmli gels and used to generate a polyclonal anti-serum in rabbits.

Furthermore, the complete coding region of TEN01 wasexpressed under control of the Tac promoter in E. coli.The isopropyl-0-o-thiogalactopyranoside (IPTG)-inducedpolypeptide migrated as a 56-kD protein on SDS-PAGE(Figure 5B). This molecular mass discrepancy (the calcu-lated value is 48 kD) is difficult to explain because it cannotbe due to glycosylation. We found, however, that theenolase protein from a partially purified tomato extract alsomigrates at an anomalously high molecular mass. Theenolase antibody recognizes both a 56-kD and a 45-kDproduct on a protein gel blot of this tomato extract (Figure5C). The 45-kD polypeptide also corresponds to enolasebecause its N-terminal sequence is 45% homologous to

A B1 2 1 2 3 4 5 1 2

kD

«*. -92 -

-45 -

•-30-

Figure 5. Expression of Recombinant Tomato Enolase in E. coli.

(A) Expression of the translational fusion T7-7-TENO1 inK38(pGP1-2) cells. Lane 1, negative control (insert in reverseorientation); lane 2, temperature-induced recombinant enolase.The induced 56-kD protein is indicated by the arrowhead.(B) Expression of intact recombinant enolase in JM109 cells. Lane1, cells transformed with pBTac2 without IPTG; lane 2, after IPTGinduction; lane 3, cells transformed with pBTac2-TENO1 withoutIPTG; lane 4, after IPTG induction; lane 5, molecular mass mark-ers. The induced 56-kD protein is indicated by the arrowhead.(C) Immunoblot analysis of tomato enolase. Lane 1, partiallypurified tomato enolase extract; lane 2, extract from inducedK38(pGP1-2/pT7-7-TENO1).

726 The Plant Cell

yeast enolase and identical to amino acids 50 to 59 in thetomato polypeptide predicted from the TEN01 clone. The45-kD product is probably the result of artificial processingduring purification.

Interestingly, the intact recombinant protein was activein £. co// extracts. Enolase activities were on averagefivefold higher than in the noninduced controls (specificactivities 4690 units/^g of protein and 950 units//jg ofprotein in controls). In contrast, K38(pGP1-2) cells trans-formed with the construct with the modified N terminusdid not show activities above basal endogenous levels.This was confirmed when the same plasmid was trans-formed to BL21(DE3) cells and induced at low growthtemperature with IPTG to avoid thermal denaturation. Weconclude that an intact N-terminal region is essential forplant enolase activity.

Expression of Enolases in Plants: Tissue andDevelopmental Specificity

Expression of enolase genes was examined in differentplant tissues. First we investigated differences in mRNAlevels in photosynthetic and nongreen tissue. Figure 6Apresents an RNA gel blot of tomato leaf and root tissueprobed with the TENO1 cDNA. Because the leaf samplecontains 10 n<3 of poly(A)+ RNA and the root lane 100 ngof total RNA, which is approximately equivalent to 1 ̂ g ofpoly(A)+ RNA, and because the signal in root is about

twofold stronger than in leaf, we estimate that the expres-sion in roots is 10-fold to 20-fold higher than in leaves(comparable signals were obtained from two independenthybridization experiments with different samples). Similarresults were obtained in Arabidopsis (data not shown).

In addition, we investigated whether this difference insteady-state mRNA levels is also observed for enolaseprotein. Figure 6B presents a protein gel blot of proteinfrom different tissues of Arabidopsis. The antibody recog-nized a 56-kD protein in Arabidopsis. The level of enolaseseemed reasonably constant in all tissues. Because gly-colytic activity is expected to be higher in nonphotosyn-thetic tissue (Goodwin and Mercer, 1983), we comparedenolase activities in Arabidopsis roots and leaves. Specificactivities in roots were as high as 107,000 units/mg,whereas leaf activities were at least fivefold lower. It isconcluded that tissue-specific regulation of enolases iscomplex and, although occurring at the transcriptional orpost-transcriptional level, apparently also involves post-translational controls.

A second aspect of interest is the developmental controlof enolase expression. It has been proposed that thecarbon flux through glycolysis increases during fruit rip-ening (Stitt et al., 1986). The expression pattern of enolasemRNA at different stages of tomato fruit ripening wasanalyzed (Figure 6C). Surprisingly, the 1.6-kb messengerwas present in green fruit but undetectable in pink fruit.However, when pink fruits were artificially ripened by acombined treatment with LiCI, hormones, and wounding(Van Der Straeten et al., 1990), the enolase mRNA was

ENOLASE AND ACC-SYNTHASE KINETICS100 R

90 7c/i80 5

70 £

60 %50 5

40 5

30 E

20 "2.

10 ;0 2 4 6 8 10 12 14 16 18 20 22 24

INCUBATION TIME (hi

Figure 6. Tissue-Specific and Development-Specific Expression of Enolase in Tomato and Arabidopsis.

(A) RNA gel blot analysis of tomato leaf and root. Lane 1, tomato leaf tissue, 10 ^g of poly(A)+ RNA; lane 2, tomato root tissue, 100 ^gof total RNA. The TENO1 Bglll-EcoRI fragment was radiolabeled and used as a probe. Exposure was overnight on flash-sensitized film.(B) Immunoblot analysis of enolase in different tissues of Arabidopsis. Each lane contains approximately 100 ^g of total protein. Lane 1,flowers; lane 2, stem; lane 3, leaf; lane 4, unripe pods; lane 5, ripe pods; lane 6, roots; lane 7, etiolated seedlings; lane 8, green seedlings.(C) RNA gel blot analysis of tomato fruit at different stages. Each lane contains 10 ^g of poly(A)+ RNA. Lane 1, green tissue; lane 2, pinktissue; lane 3, artificially ripened pink tissue. A random primed probe of the TENO1 Bglll-EcoRI fragment has been used. Exposure wasfor 12 hr on flash-sensitized film.(D) As in (C), but probed with a tomato ADH partial cDNA. Exposure was for 24 hr on flash-sensitized film.(E) Time course of induction of enolase and ACC synthase by artificial ripening.

Plant Enolases 727

increased at least 30-fold over green tissue. This significantrise in mRNA was caused by the chemicals and the wound-ing treatment because a water control did not lead to anyincrease of enolase messenger compared with pink tissue(data not shown). When the same blot was probed with apartial tomato ADH cDNA (D. Van Der Straeten, unpub-lished results), we observed a similar expression patternwith the exception of pink tissue, where a signal wasdetected of equal intensity to green fruit (Figure 6D),indicating the presence of certain glycolytic mRNA speciesin this ripening stage.

The induction of enolase mRNA during the artificialripening process was concomitant with an increase inenzyme activity, as shown in Figure 6E. The kinetics ofenolase induction were very similar to ACC synthase ki-netics (Van Der Straeten and Van Montagu, 1990); never-theless, the activity increase for enolase was much lower(threefold versus 50-fold). However, the highest enolaseactivities were found in green tissue (52,000 units/mg),despite the fact that it did not have the most elevatedmRNA levels.

Expression of Plant Enolases in Stress Conditions

Anaerobic stress is known to induce the first and lastenzymes of the glycolytic pathway at the mRNA level and,in general, also at the protein level (Dennis et al., 1987;Bailey-Serres et al., 1988). For certain intervening glyco-lytic enzymes like enolase, small increases in activity wereobserved in maize (Bailey-Serres et al., 1988). We haveinvestigated whether this is preceded by a rise in enolasemRNA accumulation. Figure 7A presents an RNA gel blotthat shows the effect of anaerobiosis in seedlings androots of tomato. As a control for the hypoxic induction, weanalyzed the same samples with a partial tomato ADHcDNA as a probe (D. Van Der Straeten, unpublished re-sults) (Figure 7B). A fair increase in enolase mRNA oc-curred in submerged tomato seedlings. There was, how-ever, no detectable increase of messenger in waterloggedtomato roots. By contrast, there was a strong induction ofADH messenger, both in hypoxically treated seedlings (atleast 10-fold) and in roots (at least 30-fold), confirming thevalidity of the test conditions. Figure 7C presents an RNAgel blot of mRNA from submerged Arabidopsis seedlingsprobed with the TENO1 cDNA. A low increase in mRNAlevel was observed.

In yeast, HSP48 is identical to enolase (lida and Yahara,1985), although no effect on the ENO1 promoter wasdemonstrated (Uemura et al., 1986). Therefore, we testedwhether enolase is heat shock-inducible in plants. Theeffect of heat shock on enolase mRNA accumulation wastested in tomato leaves and roots. Figure 8 shows asignificant decrease in enolase message in both tissuesafter 1 -hr exposure to the elevated temperature; the level

B1 2 3 4 1 2 3

4.4-

2.4-

1.65-1.4-

Figure 7. Expression of Enolase and ADH under AnaerobicStress.

(A) RNA gel blot of submerged etiolated tomato seedlings andwaterlogged tomato roots. Each lane contains 100 ^g of totalRNA. Lane 1, etiolated seedlings; lane 2, after 20 hr submergence;lane 3, root tissue; lane 4, root tissue after 40 hr waterlogging. A32P-labeled Bglll-EcoRI fragment of TENO1 was used as a probe.Exposure was for 72 hr on flash-sensitized film.(B) As in (A), but probed with a tomato ADH partial cDNA.Exposure was for 24 hr on flash-sensitized film.(C) RNA gel blot of submerged etiolated Arabidopsis seedlings.Both lanes contain 50 ^g of total RNA. Lane 1, control; lane 2,after a 20-hr submergence period. Exposure was overnight onflash-sensitized film. The arrowhead indicates the enolase mRNAsignal.mRNA sizes are indicated in kilobases on the left.

was at least partially restored after 2 hr. This result wasconfirmed in a separate experiment where samples weretaken after 1 hr and 3 hr (data not shown). Consistently,no increase in enolase activity was found (data not shown).This transient response is observed for most cellular pro-teins when a plant is subjected to heat shock (Kimpel andKey, 1985; Lindquist, 1986).

DISCUSSION

A Most Conserved Polypeptide

In this report we described the cloning of enolases fromArabidopsis and tomato. To our knowledge, enolase is thefirst glycolytic protein from which the primary structure iselucidated in all kingdoms and for which a detailed tertiarystructure model exists.

728 The Plant Cell

1 2 3 4 5 6k bi

-1.65

Figure 8. RNA Gel Blot Analysis of Tomato Enolase under HeatShock Conditions.

The TENO1 Bglll-EcoRI fragment was radiolabeled and used asa probe. Lane 1, 10 Mg of leaf poly(A)" RNA; lane 2, after 1 hr at40°C; lane 3, after 2 hr at 40°C; lane 4, 100 Mg of total RNA fromroot; lane 5, after 1 hr at 40°C; lane 6, after 2 hr at 40°C.Exposure time was 17 hr on flash-sensitized film.

that this pentapeptide is residing in a coil region and,therefore, probably has very little influence on tertiaryfolding. This was according to our expectations becauseit was demonstrated recently that yeast enolase, as threeother glycolytic enzymes and nine other enzymes, pos-sesses an a/(i barrel structure, which nonetheless is avariation on the common theme (Lebioda and Stec, 1988).All of these enzymes have a core of mostly parallel 13sheets surrounded by « helices. This is a thermodynami-cally extremely stable structure, perhaps arising from con-vergent evolution (Fothergill-Gilmore, 1986). Therefore, itwas anticipated that the plant enzyme would not be differ-ent from other enolases from a secondary and tertiarystructure point of view.

Finally, we evaluated the influence on tertiary structureof the presence of 5 cysteine residues in plant enolase, asopposed to a single cysteine in Bacillus and yeast. It seemshighly unlikely that the cysteines in plant enolase wouldform intramolecular disulfide bridges, thereby possibly dis-torting the structure proposed for yeast enolase (Lebiodaet al., 1989). When their positions are superimposed on amodel as presented by these authors, it appears that thecysteines reside on opposing sides of the barrel, toodistant from each other to allow bridge formation. Inter-estingly, the cysteine in position 408 in plants, which isalso conserved in all other eukaryotic enolases, is appar-ently spatially near the position of the unique cysteine inyeast (position 247), residing close to the active site (Le-bioda et al., 1989). It has been proven that chemicalmodification of this residue in yeast leads to loss of activity(Oh et al., 1973). However, further investigations will haveto demonstrate that its role in plants and other eukaryotesmight be played by a cysteine located elsewhere in thechain.

From a primary structure comparison of enolasesthroughout evolution, the following can be concluded.Eukaryotic enolases are, on average, 42% identical to theprokaryotic Bacillus enzyme. This high degree of conser-vation is a property of most glycolytic enzymes, which isalso reflected by their exceptionally low rate of evolution,about as low as cytochrome c (Fothergill-Gilmore, 1986).Obviously, most of the residues located in or near theactive site (Lebioda et al., 1989) remain unchanged. Strik-ingly, however, yeast enolases have a significantly lowersimilarity to their plant counterparts (52%) than to theiranimal counterparts (60%). This might imply that the yeastand plant progenitor genes have diverged earlier thanyeast and animal ancestral genes. This idea is consistentwith the multikingdom tree proposed by Sogin (1989).

Furthermore, the primary structure comparison revealedthe existence of an insertion of 5 amino acids unique tothe plant enolases. A secondary structure prediction, av-eraging four different methods, allowed us to conclude

Plant Enolase Usually Appears as Multiple Isoforms

In most organisms, the glycolytic pathway has evolved bygene duplication, giving rise to different isozymes (Foth-ergill-Gilmore, 1986). In this way, selection may have pro-duced enzymes optimal for the chemical differences andmetabolic demands of specific tissues. In most casesdescribed for plants, these isozymes include at least onecytosolic and one plastid isoform (Gottlieb, 1982; Shih etal., 1986). The existence of enolase isozymes has beendemonstrated on the protein level in developing castor oilseeds (Miernyk and Dennis, 1982). Our data support thisfact on the gene level for several plant species. A genomicDNA gel blot from four different species revealed theexistence of at least three gene copies for three of them.Unless these are pseudogenes or sequences split by longintrons, the results indicate the presence of three genes inrice, three or four in tomato, and an even more numerousgene family in tobacco. The only exception to the presence

Plant Enolases 729

of multiple genes was Arabidopsis, where a single band was detected on genomic DNA gel blots in both nuclear and total DNA preparations. This demonstrates that there is no requirement for multiple enolase genes to meet the demands of eukaryotic existence.

Arabidopsis Plastids Lack Part of the Glycolysis

As mentioned above, glycolytic enzymes in general pos- sess at least two isoforms, a cytosolic form and a plastid form. In those cases that have been studied, both types are encoded by the nuclear genome (Weeden and Gottlieb, 1980; CerH and Kloppstech, 1982; Gottlieb, 1982; Shih et al., 1986). Shih et al. (1986) provided convincing evidence for the symbiotic theory, which predicts that genes for the chloroplast enzymes have been transferred from the genome of a prokaryotic symbiont to the nucleus. Several reports, however, have mentioned the absence of certain glycolytic activities in chloroplasts (Stitt and apRees, 1979) or nongreen plastids (Macdonald and apRees, 1983; Jour- net and Douce, 1985; Frehner et al., 1990). Our data indicate on the gene level that certain plant species, as demonstrated in Arabidopsis, indeed have an incomplete set of glycolytic enzymes in their plastids. This conclusion was based on severa1 lines of evidence.

First, Arabidopsis possesses a single, nuclear-encoded gene for enolase. Furthermore, this gene encodes a polypeptide whose N-terminal region is highly homologous to the Bacillus and yeast enolase N terminus and, moreover, has no homology to the consensus chloroplast target sequence (von Heijne et al., 1989). Finally, both the enolase protein and its activity are absent in Arabidopsis chloroplasts. Therefore, it seems not only that the Arabidopsis chloroplast enolase gene was lost at some point in evolution, but also that Arabidopsis plastids must bypass this step in glycolysis. The consequence of this fact is different for chloroplasts and nongreen plastids. In chloroplasts, glycolytic enzymes are not involved mainly in glycolysis, but have their function in photosynthetic carbon fixation (dark phase). Enolase is one of the few steps that is not involved in the Calvin-Benson cycle and, therefore, there is no essential need for it in the chloroplast, although enolase activity has been demonstrated in isolated pea chloroplasts, where it plays a role in carbohydrate breakdown in the dark (Stitt and apRees, 1979). The situation is different in nongreen plastids, which have to convert hexose phosphates to pyruvate for fatty acid synthesis and, therefore, need to be able to transform 2-phosphoglycerate in phosphoenolpyruvate. Although we did not check enolase activity in nongreen plastids, we suggest the existence of a substrate-product shuttle to bypass enolase in Arabidopsis nongreen plastids. Such a shuttle system was proposed in some other cases, although, to the best of our knowledge, never for enolase (Stitt and

apRees, 1980; Journet and Douce, 1985; Frehner et al., 1990).

Complex Controlling Mechanisms Regulate Enolase Expression

In nonphotosynthetically active cells, glycolysis plays a role in energy metabolism. In plants, how-ever, the situation is different because plant cells rely primarily on photosyn- thesis for their energy supplies. The question remains: what are the tissues and the circumstances under which glycolysis proceeds in plant cells? Messenger RNA and enzyme activity analyses in tomato and Arabidopsis demonstrated that enolase expression is more prevalent in roots than in green tissue. This is consistent with the higher demand for energy from glycolysis in nongreen tissue. However, a protein gel blot of different tissues of Arabidopsis showed that higher activities in root tissue are not a simple reflection of increased amounts of protein. The fact that there is very little variation on the protein level indicates possible involvement of post-translational modification. Further investigations will be needed to solve this issue.

Several studies have reported an increase of glycolytic intermediates during the ripening process or upon exposure to ethylene or cyanide (Chalmers and Rowan, 1971 ; Solomos and Laties, 1974; Stitt et al., 1986). We have analyzed how this influences mRNA accumulation and enzymatic activities of enolase in tomato fruit.

In green fruits, the prevalence of messenger RNA was lower than in roots. Corresponding enzyme activities were reduced by a factor of two. Surprisingly, enolase mRNA was undetectable in pink fruit, and enzyme activity was approximately fivefold lower than in green fruit. It is con- ceivable that the enolase protein is formed at the mature green stage and subjected to metabolic controls in later stages of ripening. Activation of pre-existing enzymatic activity has been suggested for glycolysis in general (So- lemos and Laties, 1974). Interestingly, when pink fruits were ripened artificially, enolase mRNA accumulated to high levels, at least 20-fold higher than in green fruit. Enzyme activities increased by a factor of three, consistent with a threefold increase in glycolytic carbon flux during ripening, as reported earlier (Solomos and Laties, 1974). There are three possible explanations for the fact that the high prevalence of message was not accompanied by an equal increase in enzymatic activity. First, it may reflect the post-translational controls postulated earlier. Second, it is possible that efficiency of translation is much lower in the artificially ripened tissue than in mature green fruit. This type of regulation has been reported for sucrose synthase in maize (McElfresh and Chourey, 1988; Rowland et al., 1989; Taliercio and Chourey, 1989). The alternative explanation is based on the fact that lithium ions have

730 The Plant Cell

been reported to inhibit enolase (Kornblatt and Musil, 1990), even at concentrations 1 O-fold lower than applied in the artificial ripening process. Due to the inhibition by lithium, the actual active enzyme concentration is dramat- ically lowered and could trigger a feedback mechanism controlling transcription. There are, however, no data available on the irreversibility of this inhibition.

In conclusion, it is clear that both tissue and developmental specificity of enolase in plants exist and that these are regulated by a complex mechanism including controls at the level of transcription, and possibly also at the levels of translation and post-translation.

Enolase, as Other lntervening Glycolytic Enzymes, Shows Low Responsiveness to Anaerobiosis

Under conditions of low oxygen tension, plants have been shown to shift their carbohydrate metabolism from an oxidative to a fermentative pathway. On the cellular level, a specific set of polypeptides is synthesized, the so-called anaerobic polypeptides (Sachs et al., 1980), whereas the synthesis of aerobic proteins is discontinued. Severa1 of the anaerobic polypeptides are associated with glycolysis (for a review, see Dennis et al., 1987). Their induction occurs both at the transcriptional level and at the level of enzymatic activity. However, there is a significant difference in the level of induction for the first and the last enzymes of the glycolytic pathway on the one hand and for the intervening glycolytic enzymes on the other hand. The latter enzymes are generally induced to a fairly low level (twofold) or not at all (Kelley and Freeling, 1984; Bailey-Serres et al., 1988). The strongly hypoxically induced genes have been shown to contain a consensus sequence (ARE), with a core hexanucleotide TGGTTT essential for anaerobic induction (Dennis et al., 1987; Walker et al., 1987; Olive et al., 1990), present in the promoter region and also in the first intron, as for sucrose synthase (Springer et al., 1986). The ARE core was found in the first intron of the Arabidopsis enolase gene. A second region in the maize Adhl ARE containing the hexanucleotide CGGTTT (Walker et al., 1987) was present in the enolase promoter, however, only 27 nucleotides upstream from the putative TATA box, not accompanied by the essential core element and, therefore, probably not functional. It was tested whether the presence of the ARE core in intron 1 is sufficient for anaerobic induction of enolase in Arabidopsis. A very low induction was observed in submerged Arabidopsis seedlings (approximately twofold). It was concluded that the ARE core element necessarily needs to be present in the promoter region to be functional. Earlier studies on maize glyceraldehyde-3-phosphate dehydrogenase (GAPDH), where the core element was found at the required position from the transcriptional start but anaerobic induction could not be demonstrated (Martinez

et al., 1989), implied that the ARE core element is a necessary but not a sufficient condition for high anaerobic induction.

Low anaerobic transcriptional induction of enolase was observed in tomato and compared with tomato ADH induction in the same conditions. In tomato seedlings, a fair increase in steady-state messenger was observed. By contrast, enolase root mRNA levels remained constant. In both tissues, however, ADH mRNA was induced at least 10-fold to 20-fold under the same hypoxic treatment. An expression pattern similar to enolase was reported for GAPDH in rice seedlings and roots under anaerobiosis (Rivoal et al., 1989). The results reported here support the hypothesis that intermediate steps in the glycolytic pathway may be less responsive to anaerobiosis than are the initial and final parts of the pathway.

Enolase 1s Not a Heat Shock Protein in Plants

lida and Yahara (1 985) proved that yeast enolase is identical to HSP48 and is involved in thermal tolerance and growth control in yeast. Heat shock induction was also reported for other yeast glycolytic proteins, as phosphoglycerate kinase and GAPDH (Piper et ai., 1988). We tested whether enolase might be heat shock inducible in plant cells. When tomato roots and leaves were exposed to elevated temperatures, enolase mRNA accumulation pat- terns were similar to those found for most cellular proteins. An initial decrease in mRNA after the first hour of heat exposure was followed by a restoration of the normal level of expression after 2 hr to 3 hr of heat shock. This kinetic, indicating that growth resumed after a temporary pause (Lindquist, 1986), was found in leaves as well as in roots. As expected, we did not find a significant homology to consensus plant HSE (Schoffl et al., 1989) interlocked with a second dyad element, required for heat shock inducibility. It was concluded that plant enolase is not a heat shock- inducible protein.

METHODS

Plant Material, Growth Conditions, and Stress Treatments

Arabidopsis thaliana (ecotype C24) plants were grown at 22OC and 80% humidity. Tomato plants (Lycopersicon esculentum cv Supersonic) were grown at 24OC and 80% humidity. Tomato fruits (green or pink; cv Orlando) were obtained from a local market.

Artificial ripening conditions were as described elsewhere (Van Der Straeten et al., 1990).

Hypoxic induction of 1 3-day-old dark-grown Arabidopsis and tomato seedlings was by complete submergence for 20 hr in 1 O mM Tris-HCI, pH 7.0, containing 75 pg/mL chloramphenicol, as

Plant Enolases 731

described by Springer et al. (1986). When applied to 1.5-month- old tomato plants, waterlogging conditions were simulated by placing the plants in a 100-liter tank filled with water to 2 to 3 cm above the soil surface for 40 hr at 21 "C.

Heat shock was done by incubation of 2-month-old tomato plants at 40°C for 1, 2, or 3 hr.

Assay for Enolase Activity

Activity measurements for enolase were performed in 50 mM Tris- HCI buffer, pH 7.4, containing 1 mM MgCI2 and 2.5 mM 2- phospho-D-glycerate, as modified from Westhead (1 966). Incu- bations were at 25°C for 10 min. The absorbance at 230 nm was measured against a blank without substrate. A calibration curve was established for phosphoenolpyruvate, the linearity of the reaction was confirmed for each sample, and each value is the average of three independent measurements. One unit is defined as the amount of enzyme that converts 1 nmol of substrate per hour at 25°C.

lsolation of Chloroplasts and Protein Extraction

Chloroplasts were isolated from Arabidopsis leaves essentially according to Mullet and Chua (1 983). Mitochondrial contamination was checked by a cytochrome c oxidase assay (Darley-Usmar et al., 1987).

Extraction of protein from different plant tissues was as described by Bauw et al. (1987). Protein measurements were according to Bradford (1976).

Peptide Sequencing

Proteins separated on 10% Laemmli gels (Laemmli, 1970) were electroblotted onto coated glass fiber sheets, and the N-terminal amino acid sequence of the protein of interest was determined as described previously (Van Der Straeten et al., 1989). Total tryptic digestion, separation, and sequencing of tryptic peptides were as reported earlier (Van Der Straeten et al., 1989, 1990).

RNA Isolation, Construction, and Screening of a Tomato cDNA Library

The construction of a tomato pericarp cDNA library, prepared from RNA induced by artificial ripening, was according to Van Der Straeten et al. (1 990). Approximately 25,000 plaque-forming units were plated and screened on Hybond N (Amersham Corp.) using mixed oligonucleotide probes (1 O0 pmol). The sequences of the oligonucleotides are: 5'-(A/G)TC ICC (A/G)TT IAT IAC (A/G)TT (A/G)AA IGC IGG IAC IGG-3' (33-mer, derived from peptide P46)

3' (29-mer, derived from P26). Hybridization was performed at 40°C in 6 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate), 5 x Denhardt's solution (1 x Denhardt's solution is 0.02% Ficoll, 0.02% PVP, 0.02% BSA), 0.5% SDS, 0.1 mg/mL denatured herring sperm DNA. Filters were washed twice in 2 x SSC for 30 min at 40°C, followed by 2 x SSC, 0.1% SDS for 30 min at 40°C.

and 5'-TT IGG IGC (A/G)AA ICC ICC (C/T)TC (A/G)TC ICC IAC-

Genomic Libraries

A cosmid library of A. thaliana ecotype Columbia was kindly provided by Dr. Neil Olszewski (University of Minnesota). About 30,000 colony-forming units were screened using the same conditions as for the tomato cDNA library. A X Charon 35 genomic library of the tomato cultivar VNTF Cherry was a gift from Dr. Robert Fischer (University of California, Berkeley). Two hundred thousand plaque-forming units were screened with a tomato enolase cDNA (designated TENO1) Bglll-EcoRI random primed probe. Hybridization and washes were at 65°C.

Subcloning and DNA Sequence Analysis

The tomato cDNA inserts were subcloned as EcoRl fragments in pUCl9. A full-length subclone of 1554 bp was named TENOl. A 7.5-kb fragment containing the entire coding sequence of the unique Arabidopsis enolase gene (gAENO) was subcloned in the EcoRl site of pBR325, and a 1.8-kb Hindlll subfragment was inserted into pUCl8. Subclones of the above-mentioned inserts were sequenced according to Maxam and Gilbert (1977). A 3.0-kb Hindlll fragment of a tomato genomic enolase clone (gTEN02) was subcloned in pUCl8 and sequenced with interna1 primers (Sanger et al., 1977).

Nucleic Acid Hybridization Analysis Secondary Structure Predictions

Nuclear DNA was prepared according to Jofuku and Goldberg (1988). Total DNA preparation was mainly as described by Della- porta et ai. (1 g83), followed by a Cscl gradient. The amounts of DNA loaded On gel were correlated with the genome size of the different species used. DNA gels were blotted onto Hybond N (Amersham) and probes labeled by random priming according to the manufacturer's specifications (Amersham). Hybridization was carried out at 65°C as described (Van Der Straeten et al., 1990).

RNA for gel blotting was prepared and purified as reported (Rodrigues-Pousada et al., 1990), and the poly(A)+ fraction was obtained on oligo(dT)-cellulose (Sambrook et al., 1989). Six-percent formaldehyde gels were blotted on Hybond N (Amersham).

Secondary StrUCtUre predictions were based on f o ~ r different methods: the GOR method (Garnier et al., 1978), the homolog method (Levin et al., 1986), the GGBSM method (Gascuel and Goh-", 1988)~ and the method of Fk" and Wodak (1988). The methods were aPPlied for each enOlaSe SeParatelY; a residue was considered likely to adopt a helical, an extended strand, Or a coil conformation if at least three methods were in agreement. A join prediction was performed by aligning all enolases and assign- ing to each position the conformation that was occurring most frequentl y.

732 The Plant Cell

RNA gel blot hybridization was at 42°C in a buffer described previously (Van Der Straeten et al., 1990).

Received April 16, 1991 ; accepted May 21, 1991.

REFERENCES Expression in Escherichia coli, Antibody Production, and lmmunoblotting

Plant enolase cDNA was expressed in E. coli to obtain high amounts of recombinant protein for subsequent antibody production. Two different strategies were used. A 1.5-kb blunted Bglll- EcoRl fragment of TENOl was subcloned in a blunted BamHl site of the expression vector pT7-7 (Tabor and Richardson, 1985) and then transformed into K38(pGP1-2) cells containing the T7 RNA polymerase gene controlled by a temperature-inducible promoter. The recombinant protein, formed after induction at 42"C, lacks 11 amino acids at the N terminus that are replaced by 9 amino acids from the pT7-7 plasmid. The same construct was also transformed into BL21 (DE3) cells (Studier and Moffatt, 1986) to allow induction by IPTG at 22°C.

The second method employed a plasmid constructed by ligation of a synthetic linker coding for the first 11 amino acids of tomato enolase and a TENOl Bglll-Pstl fragment into pBTac2 (Boehringer Mannheim). JM109 cells were grown at 28°C to an Asoo value of 0.5 in LB medium containing triacillin (50 pg/mL); IPTG was added to 1 mM, and the cells were further incubated for 2 hr. Bacteria were harvested and sonicated in a 50 mM potassium phosphate buffer, pH 7.3, supplemented with 1 mM MgCI2. Enolase activities were measured versus noninduced controls.

Two New Zealand White rabbits were immunized with the truncated plant enolase produced in K38(pGP1-2/pT7-7-TENOl) and separated on an SDS-polyacrylamide gel. About 30 pg of protein was injected, first with complete Freund's adjuvant and then three additional times with incomplete adjuvant. Immuno- blotting was according to Towbin et al. (1979) with a 1 :lOOO-fold diluted primary antiserum and an antirabbit alkaline phosphatase conjugate as a secondary antibody (Sigma). Under these conditions, the antiserum was able to reveal 10 ng of the recombinant protein but did not cross-react with purified yeast or rabbit enolases (Sigma).

ACKNOWLEDGMENTS

We thank Luc Van Wiemeersch for excellent technical assistance, José Van Damme for amino acid sequencing, and Jan Gielen, Jef Seurinck, and Raimundo Villarroel-Mandiola for DNA sequencing. We are grateful to Jean Richelle for the prediction of secondary structures and useful discussions, and to Allan Caplan and Tom Gerats for critical reading of the manuscript. We thank Annemie Van Houtven for teaching us the rabbit immunization. We appre- ciate the help of Martine De Cock for layout of the manuscript and our art team for the figures. D.V.D.S. is a senior research assistant of the National Fund for Scientific Research, Belgium. R.A.R.P. is indebted to the Junta Nacional de Investigacão Cien- tífica e Tecnológica, Portugal, for a fellowship. H.M.G. is supported by a grant to Massachusetts General Hospital from Hoechst AG.

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DOI 10.1105/tpc.3.7.719 1991;3;719-735Plant Cell

D Van der Straeten, R A Rodrigues-Pousada, H M Goodman and M Van MontaguPlant enolase: gene structure, expression, and evolution.

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Plant Enolase: Gene Structure, Expression, and Evolution · The Plant Cell, Vol. 3, 719-735, July 1991 O 1991 American Society of Plant Physiologists Plant Enolase: Gene Structure,