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,
aLaboratorium voor Genetica, Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General
Hospital, Boston, Massachusetts 021 14

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-~-glyceratehydrolase, 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Bacillus subtilis (D. Vereecke, unpublished data; Verma,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 develop-
mental 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 struc-
tures 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 en-
zyme, 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 se-
quences of peptides P26 and P46 (Figure 1 in Van Der
Straeten et al., 1990). A tomato (cv Orlando) cDNA library
was constructed in X g t 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
1A. 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 (Hollandet 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 1A) 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 differ-
ences. 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 phos-
phate 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 gen-
omic 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 bound-
aries (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 1O of the
Arabidopsis gene, which are joining two or three corre-
sponding 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 downstreamfrom 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 induc-
ible, 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
144
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
288
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 390
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

1 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 ~ ~ ~CTTccTGCAAATCCCATCCTTGCTGTGTCACTTGCAGTCTGTAAAGCT~GCTGCTGTCAAGAAG
tgggttgg~~ttttttg~~~g
CTTGGTGCAAATGCCATCCTTGCTGTGTCACTTGCTGTGTGTAAAGCTGGCGCTGCTGTCAGGAAT 456
L G A N A I L A V S L A V C K A G A A V K r K n
144
471

287
h31
573
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
575 g c s t g t a t t t t c t c = t g t t a a a a g a t g t a g t t a a a t g a t g c t t a g ~ a a a t t a c t a a t t a g t t g t t a t t t g ~GAATTTATGATTCTTCCTGTTGGAGCTTCTAG~AA~GGCCATGAAGATGGGCTGTGAAGTGTAT
~g
GAATTTATGATCCTTCCTGTTGGAGCTGCTAA~CAAGGAGGCCATGAAGATGGGCTGTGAAGTCTAT 6/12
E F U I L P V G A SaSnF K E A U K U G C E V Y
717 C A C C A ~ G A A Gg 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
CACCAlTTCAAG 654
H H L K
860 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
1004 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
1148 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 gGCTGTGATTAAGAAGAAATATGGCCAG ~tgg=agttt~=t=t~~tt=t~g
GCTGTGATTAAGAAGAAATATGGTCAG 681
A V I K K K Y G Q
1289 GATCCAACAAATGTTGGTGATGAGGGTGGTTTCGCTCCTAATATCCAG gcaaagtaeaaarttatgctgttgaatggcatactgtegaaeggata~t~ttttacatcatgtgactatggettttgaaaattcagGAGAACAAG
GATGCCACAAATGTCGGTGATGAGGGTGGTTTTGCTCCTAATATCCAG GAGAACAAG 738
D A T N V G D E G G F A P A I Q E N K
1432 G A G G G T C T T G A A T T G C T C A A G A C T G C C A T C G ~ G C A G G T T A T A ~ G G ~ g t a ~ g = t ~ t t g ~ = t g ~ a t t t a ~ a t ~ ~ g a ~ ~ t t t c t ~ ~ ~ a ~ c g ~ t c t c a ~ c ~ a t g t ~ g a ~ g ~ t ~ a g a = t ~ t t t t = = t t t t ~ g G T T G T
GAAGGTCTTGAATTGCTCAAGACAGCCATTGAGAAAGCAGGTTACACTGGAAAG GTGGTCATT 801
E G L E L L K T A I E K A G Y T G K V V I
1576 GGAATGGATGTTGCAGCATCTGAATTCTATGGAAAGGAC~CTTATGATTTGAACTTCAAAGAAGAGg t a t a t c c t t t c t g a c t a t g ~ ~ t a g t ~ g t g c a t t c a s g t ~ g t ~ t ~ c ~ ~ g t ~ ~ ~ g ~ a t ~ t t g ~ a g t g g ~ t t t t ~ t
GGAATGGATGTTCCAGCATCTGAA~ACGGAAAGGACAAATCTTATGACCTGAACTTCAAAGAAGAG 870
G M D V A A S E F Y G K D K T s Y D L N F K E E
1719 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
1863 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 tAACMCAATGGTTCCCAAAAGATCTCAGGTGACCAGCTCAAAGATTTGTAC
gttccat~tttg~~g
ACCAATGACGGCTCACAAAAGATATCAGGTGACCAACTCAAGGACTTATAC 921
NsN NdG S Q K I S G D Q L K D L Y
2006 AAGTCATTTGTGTCTGAGTACCCTATTGTTTCGATAGAAGATCCATTTGACCAAGATGA~GGGAGACCTATGCTAAGCT~CTGCTGAAATTGGGCAGAAAGTACAAATTGTAGGAGATGACCTTCTCGTCACCAATCCTAAG
AAGTCATTTCTGTCCGAGTACCCTATTGTTTCAATTGAAGATCCATTTGACCAAGATGACTGGGAGACCTATGCTAAGCTCACTGCTGAGATTGGGGAGCAAGTACAGATTGTCGGTGATGACCTTCTTGTCACCAACCCTAAG 1065
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
2150 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
2294 tatcagttgratctgtgcaccggtgcag AGAGTGGCCAAGGCAATCAGTGMAAGACTTGCAATGCCCTTCTTCTTAAG g t a c t t a c c a t a c c a t c t a a g c ~ ~ c t g ~ c ~ t g t ~ ~ t ~ t t ~ a t t t g g a g a t ~ ~ ~ t ~ t g t t g ~ a ~
AGGGTCGCCMGGCAATTGCAGAGAAGACTTGCAATGCTCTTCTTCTCAAG 1116
R V A K A I SaE K T C N A L L L K
2436 rercrgcrtctaa~trcgcgcg=agGTTAACCAAATTGGCAGCGTGACTGAGAGTATCGAAGCTGTG~TGTCTAAGCAAGCTGGTTGGGGTGTAATGA~AGCCACCGCAGg r a a g g a g a ~ t g t g a t g c t c t g = ~ ~ t ~ g
GTTAACCAAATCGGTAGTGTGACCGAGAGTATTGAAGCTGTGAAGATGTCCAAGAAGGCAGGTTGGGGTGTAATGACCAGTCACCGCAG 1205
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
2578 g c t ~ g c a g a s a g a g g a a a r t c a ~ t t a t g c t c ~ g t c g t t t a t g ~ c ~ ~ t t g t t ~ ~ ~ ~ ~ t ~ t t tTGGAGAGACGGAGGATACCTTTCATTGCTGATCTTGCT~GGCG
gggt~~t~ggtg~~g gtaattttgtaeat
TGGAGAAACAGAAGATACCTTCATTGCTGATCTTGCTGTCGGTTTGTCAACG 1257
G E T E D T F I A D L A V G L S T
2720 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 ~ gGGACAAATCAAGACCGGAGCTCCCTGCAGATCAGAACGTCTTGCAAAGTACAACCAG
tgg~tt~tttt~g gtact
GGACAAATCAAGACTGGAGCTCCTTGCAGGTCAGAGCGTCTCGCCAAGTACAACCAG 1314
G Q I K T G A P C R S E R L A K Y N Q
2862 c a c c c c a a ~ a g ~ a c a g ~ ~ t a c t ~ ~ g ~ t g g t a t t ~ c ~ a t t = ~ = t g g ~ g c t t c t c t c t g c a g c c a g ~ t c t g ~ t t t ~ t ~ g t t ~ ~ t t ~ ~ ~ t ~ a g C T C T T G A G A A T T G A A G A G G A A C T T G G T T C A G A T G C T G T A T A T G C C G G A G C A A G C
CTGTTGAGGATCGAAGAGGAACTCGGATCAGAGGCTGTTTATGCAGGAGCAAGC 1368
L L R I E E E L G S DeA 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 1512 ~t~tga~~tt~tg~a~t~a~
f r k p v e p y .
ctcaaaaacaagttttgtttt~tg~ggtttt~tt~gt~tttt 1554

Figure 1. Nucleotideand Deduced Amino Acid Sequences of the Tomato EnolasecDNA 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 numberedon the right, the genomic sequence (gTEN02)
on the left. DNA sequences have been aligned, noncoding regions are in lower-casetype. 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-casetype.
(B) Intron/exon structure of the tomato enolase gene gTENO2. The boxes represent expressed regions.
722 The Plant Cell

1 2 3 Arabidopsis, tomato, chicken (0 form; Russell et al., 1986),

B E B E B E B E B E
kb
1 of the amino acid alignment. The derived consensus se-
-6.0
-4.0
-3.0
-2.0
-1.5
--1.0
-0.5
Figure 2. Genomic DNA Gel Blot Analysis of A. thaliana (ecotype
C24), Oryza saliva (cv Taipei), L. esculentum (cv Orlando), and
Nicotiana tabacum.
Lanes 1, A. thaliana, 1 ^g of total DNA; lanes 2, A. thaliana, 1 P.Q
of nuclear DNA; lanes 3, rice, 3 /jg of nuclear DNA; lanes 4,
tomato, 5 ng of total DNA; lanes 5, tobacco, 20 ^9 of nuclear
DNA. 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. Exposure
was for 6.5 hr on flash-sensitized film.
soybean HSE2, this sequence does not correspond en-
tirely to the required hyphenated dyad, and, moreover, it
is not interlocked with a second heat shocklike element,
another essential requirement for heat shock inducibility
(Schoffl et al., 1989).
Primary and Secondary Structure Comparisons of
Enolases
Figure 4 shows an amino acid sequence alignment and
secondary structure predictions for prokaryotic (B. subtilis;
D. Vereecke, unpublished data; Verma, 1989) and different
eukaryotic enolases. The eukaryotic sequences are from
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 inspection
quence covers all amino acids found in the active site
region of yeast enolase (Lebioda et al., 1989), with the
exception of Lys345 and Lys396, which are replaced by Gly
and Glu, respectively, in the chicken @ form. The high
degree of similarity between enolases is also shown in
Table 1, where pain/vise comparisons are presented. Plant
enolases have a lower similarity when compared with yeast
(either ENO1 or ENO2) than do higher animals compared
with yeast. When compared with animal sequences, plant
enolases have the highest similarity to the form. The
sequence around Tyr46 in the form, which was shown to
be phosphorylated in Rous sarcoma virus-infected chicken
embryo fibroblasts (Cooper et al., 1984), is identical to the
same region in plants. Whether this is of any significance
in plant cells remains to be elucidated. Finally, higher
eukaryotic enolases have an increased number of cys-
teines (>5), all of which appear at positions different from
the single cysteine in Bacillus or yeast enolase.
Another striking feature is the occurrence of a 5-amino-
acid insertion unique to plant enolases (QNEWG, residues
101 to 105). The influence of this insertion on the second-
ary structure was investigated. Predicted structures are
presented in Figure 4, "averaging" four different methods.
The pentapeptide unique to plants is part of a predicted
coil region, which is also predicted in coil for the other
enolases and corresponds to a coil region in a model
based on high-resolution x-ray crystallography of yeast
enolase (Lebioda et al., 1989). Therefore, the pentapeptide
is 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 fact
that the secondary structures of different enolases are in
good agreement over aligned positions. It was further
tested by "averaging" the secondary structures of all spe-
cies ("join pred." in Figure 4) and comparing them with the
model of Lebioda et al. (1989). The alignment in Figure 4
shows a reasonable agreement between both models,
although the position and/or the extent of a secondary
structure is often missed by a few residues. Helices are
more faithfully predicted than sheets (e.g., the last 0 sheet
is not seen in our averaged secondary structure). The
predictions 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 accurate
than previous predictions of enolase secondary structure
(Sawyer etal., 1986).
 Plant Enolases 723

A
1 atcaaaagaaatagcactaaaggctcggaggaagcctgatgaaacatggaagattgtgctctattttcttctgacaatttttacataa~aaaacgcatttgtttactatttttttcata
121 taaaacatgaaaaacttatatttgaattaatcgaaattaaaattattaacagaaatatctaagtttatatgaaccttttaacaaaaaaaaaagtttataagaacataaaaatcataatag
241 tttagcaacatttaaattattttc~aaaattagtaacttagattaaaataaatattagatcacctcataatcttgagtttgaaactccaaa~caaagagcatccaaaaatcCg~gC~
361 aaCacCacattttgataagaattatagaactagtgatttgcattttaaatgttgatacatatagaataagcataatcaaacaatgattactgaaaaatatg~ccattaatatcgt~~~~
481 aaatggt~atggacattgaaaccctagtggagaatttgtcacataagtaaggcccaaagtttttgacccacaaacatatccattaagttatagtttagcgaaacccctttaacaaaaaa
601 gaaaattttc~actagtgaattgtttct~gaga~tctgtacaaccatccaaatttcaaacatggtataaaagatgttattgacaaaataaaaatggaaacagtgaaacgtata~Cgg~
721 aaatqgaataaaatctagatgcc~tat~ttattcttacttgttctaaagtctttaataaaaatagtcggtattacttggacaaggagcaaaacaatatggaaaaaactcttctattctgc
E41 aaaaggcgtgcagcgcatc~tttggcttcttgc~tcagagctgactgttctcatccaacggctgttattaaaacaatccaacggttttggc~tccgtgac~ctttatatatC~~~
961 ccagaccaccaacccatttcctcagctactactgttgaagcgattctCactaaaaccctCgaacacatcgcctttatCtctttCtctaga~tactcgct ATGGCTACTAXACCGTT
~ A T X T V
1079 G T T M ~ C T A G A C A G A T C T C G A C A ~ G T G G T M T C C C A C C G T T G Agttagtttctccgatcacttttgt~tttcccagtcactttccggctttgtacagtattc~gacggatctg
G
V K A R Q I F D S R G N P T V E
1198 tttgtttgatgactatccgatgct~a~accactattcaatcgtttttttgtaaacctgaattgatctagtagtcgtacgtgaatgagat tt tgtgaacgatgatcggtgatttg
1318 ~tctcggcgatttggatcgtgagttgtcgatgatggagttgattttgtttatatgatttttgcgacggatctatttatttccatctggt~tttcgattgtttgctaatgacga
1438 ~ t t t g ~ ~ ~ ~ g ~ ~ ~ 9 C t a ~ t g a t t t c t C t g ~ t g ~ tGg1t Tt g G gAtTt At a
~ gC 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 gtgagagactgcgatctgtttgtgtgtttttttagtgttagtagagttatttcaattgatcatg
C T T
A L E L R D G G S D Y L G K G V S K
1676 ttgactgattcgttatttgtttatatcactagG A T C ~ G C C A ~ A C T T A - ~gtgagtgactcactttaagctactCtcagaaCatCaaa
G
A V G N V N N I I G P A L I G K
1794 tttgtgtctaattctatgcgctaggctcactcattttatttttgttgcttttattatttcttgcagGACC~~GCAGACTGCTA~~C~~TCCATGMC~ACGGA
D P T Q Q T A I D N F M V H E L D G
1914 ACCCAAMCGAGTGGGGGTGGTGCMGCAMAG gtacttaaaagggaaaattcattttttgtgtattc~cttaagaatagaaggcaagtctgaccatcttgcgccttctatctgtgttt
T Q N E W G W C K Q K
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 gtagaaCgatCtggCaCtattaagttggttCCCtC
ATACMG
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
H I A N L A G N
2271 CCCMGATTGTGCTACCAGTTCCTGC~MCGTCATCMTGGTGGATCCCATGCCGGAMCMGCTTGCTATGCAGGAG~A~ATC~CCTGTTGGAG~~MGGM
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
2391 G C C A T U L A G A T G G G T G T G G M G ~ A C C A C C A ~ M Ggtaaatctccaatattttttggtctttgagatgctcttcaatgaatattccatatacatgtattactttttcgcagatgg
A M K M G V E V Y H H L K
2510 ttctgtgaactaacat~~ttacatgttta~gcttaaccatcttgt~tcatatgtttttcca~TGTGATTMGMGMGTAC~CA~A~CA~TG~TGATGM~T
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 ~gtctgtctcttatctgatgta~tttggctgtcctt
G
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
2749 ttacccgaaacataattcatgaaactaatggatcccattcaattttttccag GTTGTCATTGGMTGGATGTTGCCGC-GAGTTCTAC~MGACMGACCTACGACTTGMC
V V I G ~ D V A A S E F Y S E D K T Y D L N
2867 TTCAMGAAGAG g t g a a a t t t c t t t t t c c t t a ~ a a c a g c t t g t c a g g a t a g a g t g t g a g a a a c c t g t t c t t a a c t t ~ a g c t g t t g c g c t c t t t g t t t c t c t gNc aNg MNC MGC M ~ G C
F K E E
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
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
3106 ACCACTGAGn;TGOMCCGAGG~GATTGTCGGTGA~mG-TCACTMCCCCMG gtttgtttttatttgccagtgttcttaccagtttttttttgttgtctgcCCtCtCt
T T E C G T E V Q I V G D D L L V T N P K
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
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
3344 AGTATCGAGGCAGTTMGATGTCGMG~~G~GGGAG~~CCAGCCACAGMGTGG~CCGAGGACACA~TTG~~AGCCG gtaag
~GC~TCCACT
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
3463 ttgaaaccattgcaagcatttaactagcaatctcatgctatatcttttgtcggactctcatttggatctttctgtctatacagGGACAMTCAAMCCGGTGCTCClTGCAGATCCGAG
G Q I K T G A P C R S E
3582 CGTCTTGCCMGTACMCCAG gtttaacatctcagaccacta~aaagactttatgtttgacaaccagagtacattgtctaatatgtgtcttgtttctcaacag~CGTATTGAG
R L A K Y N Q L L R I E
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 MatggagcttttagaagcaaagtggtCttCtttgtgaCg~gg~g~~g~tgRCCt
C C C T A ~ M
E E L G S E A I Y A G V N F R K P V E P Y .
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

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 polyadenylationsignals 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

conserve6 AR D S R G PTVOJ VF'SGASTG EA E RD GKGV AV N I P DGT K K

BACILLUS 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 EGFDVTEQN-DGT
I~ ENKGKL
ARABIDOPSIS MATI~RQIFDSRGNPTIHTSNGIWTAAVF'SGASTGIYE&ELRDG GSDYLGKGVSKAVGNVNNIIGPALI G K D P T Q Q T A I P N W t l E L D G T N E W G W B m
TOMATO 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
CHICKEN(BETA1 S I ~ D S R G D P T V B V R L U A K G H FR 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 ENKSKF
FRCG M S I K N I M E I F D S R G N P T V W ~ G L FR 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 ENKSE
R A T (GAMMAI 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 ENKSKF
HUMAN ( A L P H A I MSIWELFDSRGNPTVWLUXS&GLF 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 ENKSKF
Y E A S T (1I 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 ANKSU
j o i n pred. 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 ~
L E B I O D A et a1 BBEEEEEEE EEEBBEEEB EE EEEEE P H H H H H H H H H H H
. l . I . l . I . I
m . I . I . I . I . I
1100]
. l

conserve6 GANAIL V S A A A PLY L . d G G HA QEFM P GA F A G H LK G VGDEGG A P N I

BACILLUS GANALLG-IPLIQYL GGFNSKTLPVPMMlIVNGGEHADNNV M P V G A P N F P G L N TAVGDEGGFAPNL
ARABIDOPSIS GAN-GAWSGIPLY-L AGNPKIVLWPAFNVINGGSHAG L P V G P m Y G Q D A T N V G D E G G F A P N I
TOMATO G-GAAUNVPD &"VLWPAFWINGGSHAG L P V G B Y G Q D A T N V G D E G G F A P N I
CHICKEN(BETA1 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
FRCG G-GAAXGVPmDL AGNPEVILWPAFNVINGGSHAG 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

--
R A T ( GAMMA I GANm-GAAmD- AGNSDLILPVPAFWINGGSHAG LPVGmm-YGKDATNVGDEGGFAPNI
HUMANIALPHAI G A N & L G m K A G A V E K G V P m D L AGNSEVILPVPAFWINGGSHAG L P V G PM Y G K D A T N V G D E G G F A P N L
Y E A S T ( 1I G A N PDLSKSKTSPWLPVPFLNWNGGSHAG 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
j o i n pred. - C C -C 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 Hccccccccccccccccccc
P
L E B I O D A et a1 EEEE EEEEE
I . l . I . I . I . I . I . I . l . l . I .
(1501

conserved E L A I AG D A SEF Y Y YP SIED W Q V DDL V

BACILLUS G S N E F A L O T I V E A I E K AG F K P G E KEDGKY HLSGEGVVKT-KYPIIS
A R A B I D O P S IS 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
TOMATO QENKEG
-Y TG GKDKSYDLNFKEESNDGSQKISGDQUQLUSFVSEYPIVS
C H I C K E N ( B E T A 1A
O
G
Y
K
A
It . IEA
LIN
IL TD RDGRYDLDFK SPPDPKRLITGEQLG.8IYRGFIKDYPVVS
FROG K-AGY PD RDGKYDLDFK S P D D P S R Y I S P D K ~ N Y P W S
R A T (GAHMAI LEG
NY
- TEKMYI RDGKYDLDFK S P A D P S R C I T G @ L w F V R N Y P V V S
HUMANIALPHAI &NKEGLEI.LKTAIGKAGY TDINVI RSGKYDLDFK SPDDPSRYISPD-IKDYPVVS
Y E A S T (1I HG- DGKVIU KDGKYDLDFKNPNSDKSKWLTGPQUUUSUJXRYPIVS
j o i n pred. 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 EEC E E C C C ~
L E B I O D A et a1 HHHHHHHHHHHHHHH EEEEEEE EEEE HHHHHHHHHH EEEEE
I . I . I . I . I . / . l . l . I . I . I .
1250/ 1300(
conserved N K L VNQIG E A G SHRSGETED I A D V GQI GAP R R AK NQL R I E L A G
BACILLUS QIGTLT~GYTAVISHRSGETEDSTIADIAVATNAGQIKTGAPS
ARABIDOPSIS QIGWESLZA.U(@KKAGWVMTSHRSGETEDTFI"fGLSTGQIKTGAPC
TOMATO QlGWES~KKAGWGVMTSHRSGETEDTFIAQUVGLSTGQIKTGAPC
CHICKEN (BETAI QIGWESI~CK~QSHGWOVYVSHRSGETEDTPIBPLWQLCTGQIEQGAPC
FRCG QIGTVT~QSNGWSHRSGETEDTPIBPLWQLCXQIKTGAPC
R A T (GAMMA 1 QIGSV~AQENGWGWSHRSGETEDTPIBPLWQLCTGC?IKTGAPC
HUMAN(ALPHA1 QIGSVT~QANGWQVHVSHRSGETEDTPIBPLWQLCTGQIKTGAPC
Y E A S T ( 1I QIGT~~GWOVYVBHRSGETEDTPIBPLWQLRXQIKTGAPA
3 o i n pred. CCHHHHHHHHHWHHHHHHHEECCCCC-CCCCEEEEECCCCCCCCCEELfUEEECCCCCCCCCCCCCP Hccccccccccc
L E E I O D A et a1 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

the pT7-7 expression vector. The hybrid polypeptide lacks

Table 1. Percent Amino Acid Similarity between Different
Prokaryotic and Eukaryotic Enolases 11 N-terminal amino acids from enolase, which are re-
placed by 9 amino acids encoded by the polylinker of the
1 2 3 4 5 6 7 8 9 pT7-7 vector. Figure 5A shows that the recombinant pro-
1 43.0 42.6 43.2 41.6 42.6 39.5 43.0 43.0 tein, induced by temperature shift of a K38(pGP1-2/pT7-
2 95.4 51.1 53.5 60.8 59.6 59.4 61.3 7-TENO1) culture, migrates at 56 kD in denaturing poly-
3 50.4 53.0 59.7 57.5 58.0 60.1 acrylamide gels (SDS-PAGE; Laemmli, 1970). The dena-
4 89.9 64.5 58.7 63.6 65.4 tured recombinant product was recovered from prepara-
5 66.8 59.8 64.0 66.6 tive Laemmli gels and used to generate a polyclonal anti-
6 78.3 82.0 87.8
serum in rabbits.
7 80.8 81.5
83.2 Furthermore, the complete coding region of TEN01 was
8
g expressed under control of the Tac promoter in E. coli.
The isopropyl-0-o-thiogalactopyranoside (IPTG)-induced
1, B, subtilis; 2, yeast ENO1 gene product; 3, yeast ENO2 gene polypeptide migrated as a 56-kD protein on SDS-PAGE
product; 4, A. thaliana; 5, tomato; 6, X. /aew's; 7, chicken /3 form; (Figure 5B). This molecular mass discrepancy (the calcu-
8, rat 7 form; 9, human form. Percent identity was calculated
lated value is 48 kD) is difficult to explain because it cannot
by the PC/Gene program, Release 6.01 (A. Bairoch, University of
Geneva, Switzerland). be due to glycosylation. We found, however, that the
enolase protein from a partially purified tomato extract also
migrates at an anomalously high molecular mass. The
enolase antibody recognizes both a 56-kD and a 45-kD
Absence of Plastid Enolase in Arabidopsis product on a protein gel blot of this tomato extract (Figure
5C). The 45-kD polypeptide also corresponds to enolase
There is a single nuclear-encoded enolase gene in because its N-terminal sequence is 45% homologous to
Arabidopsis because nuclear DMA gel blots (Figure 2, lanes
2) showed one band and total genomic DMA also yielded
one hybridizing band (the weak signal above is from non-
digested DMA remaining in the wells; Figure 2, lanes 1). A B
1 2 1 2 3 4 5 1 2
Any chloroplast-encoded enolase must have a highly di-
kD
verged sequence. Moreover, sequence comparison be-
tween TENO1 and both the tobacco and liverwort chloro-
plast genome sequences (IntelliGenetics Suite, Release
5.37, IntelliGenetics, Mountain View, CA) did not reveal *. -92 -
any significant similarity. Indeed, in those cases that have
been studied, cytosolic and plastid glycolytic enzymes are
all encoded by the nuclear genome (Weeden and Gottlieb,
1980; Cerff and Kloppstech, 1982; Gottlieb, 1982; Shih et
-45 -
al., 1986).
In addition, no homology was found between the
enolase 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 fact
that the Arabidopsis enolase N terminus is very similar to -30-
the Bacillus and yeast N-terminal sequences (Figure 4),
supports the idea that the Arabidopsis enolase polypeptide Figure 5. Expression of Recombinant Tomato Enolase in E. coli.
is not imported into the chloroplast. Further evidence in
support of this conclusion is based on our inability to detect (A) Expression of the translational fusion T7-7-TENO1 in
enolase in purified chloroplasts by either activity or immu- K38(pGP1-2) cells. Lane 1, negative control (insert in reverse
noassays (data not shown). Both assays were positive on orientation); lane 2, temperature-induced recombinant enolase.
total leaf extracts. The arguments presented above The induced 56-kD protein is indicated by the arrowhead.
(B) Expression of intact recombinant enolase in JM109 cells. Lane
strongly indicate that Arabidopsis chloroplasts lack at least
1, cells transformed with pBTac2 without IPTG; lane 2, after IPTG
part of the glycolytic pathway. induction; lane 3, cells transformed with pBTac2-TENO1 without
IPTG; lane 4, after IPTG induction; lane 5, molecular mass mark-
Expression and Activity of Tomato Enolase in E. coli ers. The induced 56-kD protein is indicated by the arrowhead.
(C) Immunoblot analysis of tomato enolase. Lane 1, partially
Tomato enolase was expressed in E. coli in two different purified tomato enolase extract; lane 2, extract from induced
ways. A translational fusion was made to the 010 gene in K38(pGP1-2/pT7-7-TENO1).
726 The Plant Cell

yeast enolase and identical to amino acids 50 to 59 in the
tomato polypeptide predicted from the TEN01 clone. The
45-kD product is probably the result of artificial processing
during purification.
Interestingly, the intact recombinant protein was active
in . co// extracts. Enolase activities were on average
fivefold higher than in the noninduced controls (specific
activities 4690 units/^g of protein and 950 units//jg of
protein in controls). In contrast, K38(pGP1-2) cells trans-
formed with the construct with the modified N terminus
did 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 growth
temperature with IPTG to avoid thermal denaturation. We
conclude that an intact N-terminal region is essential for
plant enolase activity.
Expression of Enolases in Plants: Tissue and
Developmental Specificity
Expression of enolase genes was examined in different
plant tissues. First we investigated differences in mRNA
levels in photosynthetic and nongreen tissue. Figure 6A
presents an RNA gel blot of tomato leaf and root tissue
probed with the TENO1 cDNA. Because the leaf sample
contains 10 n<3 of poly(A)+ RNA and the root lane 100 ng
of total RNA, which is approximately equivalent to 1 ^g of
poly(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 independent
hybridization experiments with different samples). Similar
results were obtained in Arabidopsis (data not shown).
In addition, we investigated whether this difference in
steady-state mRNA levels is also observed for enolase
protein. Figure 6B presents a protein gel blot of protein
from different tissues of Arabidopsis. The antibody recog-
nized a 56-kD protein in Arabidopsis. The level of enolase
seemed reasonably constant in all tissues. Because gly-
colytic activity is expected to be higher in nonphotosyn-
thetic tissue (Goodwin and Mercer, 1983), we compared
enolase activities in Arabidopsis roots and leaves. Specific
activities in roots were as high as 107,000 units/mg,
whereas leaf activities were at least fivefold lower. It is
concluded that tissue-specific regulation of enolases is
complex and, although occurring at the transcriptional or
post-transcriptional level, apparently also involves post-
translational controls.
A second aspect of interest is the developmental control
of enolase expression. It has been proposed that the
carbon flux through glycolysis increases during fruit rip-
ening (Stitt et al., 1986). The expression pattern of enolase
mRNA at different stages of tomato fruit ripening was
analyzed (Figure 6C). Surprisingly, the 1.6-kb messenger
was present in green fruit but undetectable in pink fruit.
However, when pink fruits were artificially ripened by a
combined treatment with LiCI, hormones, and wounding
(Van Der Straeten et al., 1990), the enolase mRNA was

ENOLASE AND ACC-SYNTHASE KINETICS

100 R

90 7
c/i
80 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 ^g
of 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, pink
tissue; lane 3, artificially ripened pink tissue. A random primed probe of the TENO1 Bglll-EcoRI fragment has been used. Exposure was
for 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.

increased at least 30-fold over green tissue. This significant
rise in mRNA was caused by the chemicals and the wound-
ing treatment because a water control did not lead to any
increase of enolase messenger compared with pink tissue
(data not shown). When the same blot was probed with a
partial tomato ADH cDNA (D. Van Der Straeten, unpub-
lished results), we observed a similar expression pattern
with the exception of pink tissue, where a signal was
detected of equal intensity to green fruit (Figure 6D),
indicating the presence of certain glycolytic mRNA species
in this ripening stage.
The induction of enolase mRNA during the artificial
ripening process was concomitant with an increase in
enzyme activity, as shown in Figure 6E. The kinetics of
enolase 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 enolase
activities were found in green tissue (52,000 units/mg),
despite the fact that it did not have the most elevated
mRNA levels.
Expression of Plant Enolases in Stress Conditions
Anaerobic stress is known to induce the first and last
enzymes 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 were
observed in maize (Bailey-Serres et al., 1988). We have
investigated whether this is preceded by a rise in enolase
mRNA accumulation. Figure 7A presents an RNA gel blot
that shows the effect of anaerobiosis in seedlings and
roots of tomato. As a control for the hypoxic induction, we
analyzed the same samples with a partial tomato ADH
cDNA 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 waterlogged
tomato roots. By contrast, there was a strong induction of
ADH messenger, both in hypoxically treated seedlings (at
least 10-fold) and in roots (at least 30-fold), confirming the
validity of the test conditions. Figure 7C presents an RNA
gel blot of mRNA from submerged Arabidopsis seedlings
probed with the TENO1 cDNA. A low increase in mRNA
level was observed.
In yeast, HSP48 is identical to enolase (lida and Yahara,
1985), although no effect on the ENO1 promoter was
demonstrated (Uemura et al., 1986). Therefore, we tested
whether enolase is heat shock-inducible in plants. The
effect of heat shock on enolase mRNA accumulation was
tested in tomato leaves and roots. Figure 8 shows a
significant decrease in enolase message in both tissues
after 1 -hr exposure to the elevated temperature; the level
Plant Enolases 727
B
1 2 3 4 1 2 3
4.4-
2.4-
1.65-
1.4-
Figure 7. Expression of Enolase and ADH under Anaerobic
Stress.
(A) RNA gel blot of submerged etiolated tomato seedlings and
waterlogged tomato roots. Each lane contains 100 ^g of total
RNA. Lane 1, etiolated seedlings; lane 2, after 20 hr submergence;
lane 3, root tissue; lane 4, root tissue after 40 hr waterlogging. A
32
P-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 on
flash-sensitized film. The arrowhead indicates the enolase mRNA
signal.
mRNA sizes are indicated in kilobases on the left.
was at least partially restored after 2 hr. This result was
confirmed in a separate experiment where samples were
taken 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 and
Key, 1985; Lindquist, 1986).
DISCUSSION
A Most Conserved Polypeptide
In this report we described the cloning of enolases from
Arabidopsis and tomato. To our knowledge, enolase is the
first glycolytic protein from which the primary structure is
elucidated in all kingdoms and for which a detailed tertiary
structure model exists.
728 The Plant Cell
1 2 3 4 5 6
kb
i
-1.65
Figure 8. RNA Gel Blot Analysis of Tomato Enolase under Heat
Shock Conditions.
The TENO1 Bglll-EcoRI fragment was radiolabeled and used as
a probe. Lane 1, 10 Mg of leaf poly(A)" RNA; lane 2, after 1 hr at
40C; lane 3, after 2 hr at 40C; lane 4, 100 Mg of total RNA from
root; lane 5, after 1 hr at 40C; lane 6, after 2 hr at 40C.
Exposure time was 17 hr on flash-sensitized film.
From a primary structure comparison of enolases
throughout evolution, the following can be concluded.
Eukaryotic enolases are, on average, 42% identical to the
prokaryotic Bacillus enzyme. This high degree of conser-
vation is a property of most glycolytic enzymes, which is
also 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 the
active site (Lebioda et al., 1989) remain unchanged. Strik-
ingly, however, yeast enolases have a significantly lower
similarity to their plant counterparts (52%) than to their
animal counterparts (60%). This might imply that the yeast
and plant progenitor genes have diverged earlier than
yeast and animal ancestral genes. This idea is consistent
with the multikingdom tree proposed by Sogin (1989).
Furthermore, the primary structure comparison revealed
the existence of an insertion of 5 amino acids unique to
the plant enolases. A secondary structure prediction, av-
eraging four different methods, allowed us to conclude
that this pentapeptide is residing in a coil region and,
therefore, probably has very little influence on tertiary
folding. This was according to our expectations because
it was demonstrated recently that yeast enolase, as three
other glycolytic enzymes and nine other enzymes, pos-
sesses an a/(i barrel structure, which nonetheless is a
variation on the common theme (Lebioda and Stec, 1988).
All of these enzymes have a core of mostly parallel 13
sheets surrounded by helices. This is a thermodynami-
cally extremely stable structure, perhaps arising from con-
vergent evolution (Fothergill-Gilmore, 1986). Therefore, it
was anticipated that the plant enzyme would not be differ-
ent from other enolases from a secondary and tertiary
structure point of view.
Finally, we evaluated the influence on tertiary structure
of the presence of 5 cysteine residues in plant enolase, as
opposed to a single cysteine in Bacillus and yeast. It seems
highly unlikely that the cysteines in plant enolase would
form intramolecular disulfide bridges, thereby possibly dis-
torting the structure proposed for yeast enolase (Lebioda
et al., 1989). When their positions are superimposed on a
model as presented by these authors, it appears that the
cysteines reside on opposing sides of the barrel, too
distant from each other to allow bridge formation. Inter-
estingly, the cysteine in position 408 in plants, which is
also conserved in all other eukaryotic enolases, is appar-
ently spatially near the position of the unique cysteine in
yeast (position 247), residing close to the active site (Le-
bioda et al., 1989). It has been proven that chemical
modification of this residue in yeast leads to loss of activity
(Oh et al., 1973). However, further investigations will have
to demonstrate that its role in plants and other eukaryotes
might be played by a cysteine located elsewhere in the
chain.
Plant Enolase Usually Appears as Multiple Isoforms
In most organisms, the glycolytic pathway has evolved by
gene duplication, giving rise to different isozymes (Foth-
ergill-Gilmore, 1986). In this way, selection may have pro-
duced enzymes optimal for the chemical differences and
metabolic demands of specific tissues. In most cases
described for plants, these isozymes include at least one
cytosolic and one plastid isoform (Gottlieb, 1982; Shih et
al., 1986). The existence of enolase isozymes has been
demonstrated on the protein level in developing castor oil
seeds (Miernyk and Dennis, 1982). Our data support this
fact on the gene level for several plant species. A genomic
DNA gel blot from four different species revealed the
existence of at least three gene copies for three of them.
Unless these are pseudogenes or sequences split by long
introns, the results indicate the presence of three genes in
rice, three or four in tomato, and an even more numerous
gene family in tobacco. The only exception to the presence

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 ge-
nome 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 poly-
peptide 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 chloro-
plasts. Therefore, it seems not only that the Arabidopsis
chloroplast enolase gene was lost at some point in evolu-
tion, 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 break-
down 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-phospho-
glycerate 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
Plant Enolases 729
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 dem-
onstrated 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 expo-
sure 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-translationalcontrols 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 1O-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 avail-
able on the irreversibility of this inhibition.
In conclusion, it is clear that both tissue and develop-
mental 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 differ-
ence 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 in-
duced 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 hexanucleo-
tide 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 func-
tional. 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 de-
hydrogenase (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 in-
duction 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 path-
way 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 (1985) proved that yeast enolase is iden-
tical 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 phospho-
glycerate 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 13-day-old dark-grown Arabidopsis and
tomato seedlings was by complete submergence for 20 hr in 1O
mM Tris-HCI, pH 7.0, containing 75 pg/mL chloramphenicol, as

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-litertank 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 40C for 1, 2, or 3 hr.
Assay for Enolase Activity
Activity measurementsfor 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 (1966). Incu-
bations were at 25C 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 25C.
lsolation of Chloroplasts and Protein Extraction
Chloroplasts were isolated from Arabidopsis leaves essentially
according to Mullet and Chua (1983). Mitochondrial contamination
was checked by a cytochrome c oxidase assay (Darley-Usmar et
al., 1987).
Extraction of protein from different plant tissues was as de-
scribed by Bauw et al. (1987). Protein measurements were ac-
cording 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).
Secondary Structure Predictions
Secondary StrUCtUre predictions were based on f o ~ different
r porta et ai. (1g83), followed by a Cscl gradient. The amounts of
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 performedby aligning all enolases and assign-
ing to each position the conformation that was occurring most
frequently.
Plant Enolases 731
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. (1990). Approximately 25,000 plaque-formingunits
were plated and screened on Hybond N (Amersham Corp.) using
mixed oligonucleotide probes (1O0 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)
and 5'-TT IGG IGC (A/G)AA ICC ICC (C/T)TC (A/G)TC ICC IAC-
3' (29-mer, derived from P26). Hybridization was performed at
40C 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 40C, followed by 2 x SSC, 0.1% SDS for 30 min at 40C.
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 con-
ditions 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 65C.
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
uniqueArabidopsis 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
Nuclear DNA was prepared according to Jofuku and Goldberg
(1988). Total DNA preparation was mainly as described by Della-
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 65C 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-per-
cent formaldehyde gels were blotted on Hybond N (Amersham).
732 The Plant Cell
RNA gel blot hybridization was at 42C in a buffer described
previously (Van Der Straeten et al., 1990).
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 produc-
tion. 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 pro-
moter. 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 22C.
The second methodemployed a plasmidconstructed by ligation
of a synthetic linker coding for the first 11 amino acids of tomato
enolase and a TENOl Bglll-Pstlfragment into pBTac2 (Boehringer
Mannheim). JM109 cells were grown at 28C to an Asoovalue 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 condi-
tions, the antiserum was able to reveal 10 ng of the recombinant
protein but did not cross-react with purified yeast or rabbit eno-
lases (Sigma).
ACKNOWLEDGMENTS
We thank Luc Van Wiemeersch for excellent technical assistance,
Jos Van Damme for amino acid sequencing, and Jan Gielen, Jef
Seurinck, and Raimundo Villarroel-Mandiolafor 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 Investigaco Cien-
tfica e Tecnolgica, Portugal, for a fellowship. H.M.G. is sup-
ported by a grant to Massachusetts General Hospital from
Hoechst AG.
Received April 16, 1991; accepted May 21, 1991.
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