The Plant Journal (1998) 16(4), 453–464
The plant PTS1 receptor: similarities and differences to its
human and yeast counterparts
Christine Wimmer1, Markus Schmid1, Marten Veenhuis2
and Christine Gietl1,*
1Institute of Botany, Technische Universitaet Muenchen,
Arcisstr. 16, D-80333 Munich, Germany, and
2Department of Microbiology, GBB, Biological Centre,
University of Groningen, Kerklaan 30, NL-9751 NN Haren,
The Netherlands
Summary
Two targeting signals, PTS1 and PTS2, mediate import of
proteins into the peroxisomal matrix. We have cloned
and sequenced the watermelon (Citrullus vulgaris) cDNA
homologue to the PTS1 receptor gene (PEX5). Its gene
product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human
and yeast counterparts, and exhibits 11 repeats of the
sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located
mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and
some yeast peroxisomes. Transformation of the Hansenula
polymorpha peroxisome deficient pex5 mutant with
watermelon PEX5 resulted in restoration of peroxisome
formation and the synthesis of additional membranes
surrounding the peroxisomes. These structures are labeled
in immunogold experiments using antibodies against the
Hansenula polymorpha integral membrane protein Pex3p,
confirming their peroxisomal nature. The plant Pex5p was
localized by immunogold labelling mainly in the cytosol
of the yeast, but also inside the newly formed peroxisomes.
However, import of the PTS1 protein alcohol oxidase is
only partially restored by CvPex5p.
Introduction
Microbodies belong to the basic set of membrane-bounded
organelles in cells of higher and lower eucaryotes. As
multipurpose organelles they exhibit a broad spectrum of
enzymatic endowments as required by the organism, by
its cells and tissues at different developmental stages as
Received 14 July 1998; revised 28 September 1998; accepted 6 October 1998.
*For correspondence (fax 149 89 28922167;
e-mail [email protected]).
Abbreviations: AO, alcohol oxidase; PCR, polymerase chain reaction; PTS1,
peroxisomal targeting signal 1; PTS2, peroxisomal targeting signal 2;
CvPex5p, Citrullus vulgaris PTS1 receptor protein; TPR, tetratricopeptide
repeat (34 amino acid repeat).
© 1998 Blackwell Science Ltd
well as by prevailing environmental conditions. In plants,
glyoxysomes play a key role in the conversion of fat to
sucrose during germination in fatty seedling tissue; leaftype peroxisomes in photosynthetically active tissues of
green cotyledons and leaves are required for photorespiration (for reviews see Gietl, 1996; Olsen, 1998); peroxisomes
in root nodules of nitrogen-fixing plants are involved in
urate metabolism (for review see Schubert, 1986). In mammals, peroxisomes are involved in the β-oxidation of very
long chain fatty acids and in the biosynthesis of cholesterin
and ether lipids (Wiemer and Subramani, 1994). In yeasts,
the number, volume and protein composition of peroxisomes adapts to the available carbon and nitrogen sources.
During growth on glucose, only a few small peroxisomes
are present; a strong proliferation of peroxisomes is
induced when Saccharomyces cerevisiae is grown on oleic
acid or Hansenula polymorpha on methanol or alcylated
primary amines (Veenhuis and Harder, 1987).
Despite the diverse functions of peroxisomes in different
species, their protein targeting signals and import
machineries are highly conserved (Faber et al., 1994a; Gietl
et al., 1994; Trelease et al., 1994). Two different topogenic
signals have been identified as necessary and sufficient to
direct precursors of matrix enzymes from the cytosol into
peroxisomes. One of these, designated PTS1, includes the
C-terminal tripeptide Ser-Lys-Leu (SKL) or conservative
variants thereof. The range of permissible deviations within
the PTS1 has shown an unexpected degree of species
and enzyme specificity. The general consensus sequence
demands a small uncharged amino acid in the first position,
a positively charged amino acid in the second position,
and a hydrophobic amino acid in the third. For higher
plants, Met is often present in the third position: the
multifunctional β-oxidation protein from cucumber (MFP II)
ends with a -PRM sequence (Preisig-Mueller et al., 1994);
isocitrate lyase exhibits -SRM or -ARM in oilseed rape,
cottonseed, cucumber, tomato, castor bean and loblolly
pine (Beeching and Northcote, 1987; Comai et al., 1989;
Janssen, 1995; Lee et al., 1997; Mullen and Gifford, 1997;
Olsen et al., 1993; Reynolds and Smith, 1995). Malate
synthase is targeted by -SKL or -SRL in cottonseed, rape,
cucumber, pumpkin and castor bean (Hayashi et al., 1996;
Olsen et al., 1993; Trelease et al., 1996; Turley et al.,
1990), whereas glycolate oxidase from spinach uses -ARL
(Volokita, 1991). In H. polymorpha alcohol oxidase is targeted by the PTS1 variant -ARF, whereas dihydroxyacetone
synthase and catalase use -NKL or -SKI (Didion and
Roggenkamp, 1992; Hansen et al., 1992). From these
species- and enzyme-specific variations in the PTS1
453
454 Christine Wimmer et al.
sequence one might expect that a PTS1 receptor recognizes
PTS1 signals of its own species more efficiently than
signals of distantly related species. The second targeting
signal, designated PTS2, is located in the N-terminus of
the protein and has been found in thiolase, malate dehydrogenase, citrate synthase, amino oxidase (for review see
Gietl, 1996) and in a molecular chaperone (hsp70; Wimmer
et al., 1997) from microbodies. Again, considerable speciesspecific variations from the consensus sequence RL/I-X5H/QL are observed.
Yeasts have proven to be very useful model systems to
study peroxisome biogenesis. Genetic approaches resulted
in various mutants impaired in peroxisome biogenesis
leading in turn to the identification of, at present, 19 PEX
genes which are required for peroxisome formation. Two
pathways are involved in the import process of peroxisomal
matrix proteins, corresponding to the two peroxisomal
targeting signals PTS1 and PTS2 (Rachubinski and
Subramani, 1995). The recognition of the two targeting
signals is mediated by PTS receptors. Yeast and human
cells selectively deficient in the PTS1 or PTS2 import
pathways have been instrumental in identification of the
PTS receptors. The PTS1 receptor Pex5p has been identified
in four yeast species and in humans (for review see Distel
et al., 1996), whilst the PTS2 receptor Pex7p has been
characterized in S. cerevisiae and in humans (for review
see Subramani, 1997).
The intracellular location of both targeting signal receptors is still a matter of debate. In a species- and experimentdependent manner these proteins have been predominantly or exclusively located in either the cytoplasm, the
peroxisomal membrane, or the peroxisomal matrix (for
reviews see Erdmann et al., 1997; McNew and Goodman,
1996; Subramani, 1996; Van der Klei and Veenhuis, 1996).
As a consequence, three different models are proposed
for the functional roles of Pex5p and Pex7p: a ‘shuttle
mechanism’ in the course of which the PTS receptors bind
the newly formed cargo proteins in the cytosol and direct
them to the membrane-bound peroxisomal translocation
machinery, an ‘extended shuttle mechanism’ in which the
cargo-loaded receptors cross the peroxisomal membrane
together with their cargo proteins and a ‘pulling mechanism’ in which the receptors inside the peroxisomes promote
import by pulling the proteins to be imported into the
organelle.
Components of the plant peroxisomal import machinery
have so far not been described. In the present paper
we report the cDNA sequence for the watermelon PTS1
receptor (CvPex5p). Similar to HsPex5p and HpPex5p it is
found, in fractionation experiments, primarily in the cytosol
fraction with a small amount associated with the microbodies. Transformation of the H. polymorpha pex5 mutant
strain with the watermelon PTS1 receptor resulted in forma-
Figure 1. cDNA and derived amino acid sequence of the peroxisomal
targeting signal-1 receptor (PEX5) from Citrullus vulgaris.
Primers used for PCR are marked with arrows (accession no. AF068690).
tion of peroxisomal membranes and in partial restoration
of the PTS1 import defect.
Results
Isolation of a full length cDNA clone coding for the
watermelon PTS1 receptor (CvPex5p)
A full-length cDNA clone coding for the receptor of the
peroxisomal targeting signal 1 (PTS1 receptor) was isolated
by PCR taking advantage of the expected sequence homology to the published PTS1 receptors of humans and four
different yeasts. PCR primers were chosen according to
the highest sequence similarities within the conserved
tetratricopeptide repeats (TPR) of the five organisms. The
isolated plant cDNA clone comprises 2505 bp with a 59
untranslated region of 63 bp, an open reading frame of
1941 bp, a 39 untranslated region of 483 bp and a poly(A)tail. The cDNA clone codes for a protein of 647 amino acids
with a calculated molecular mass of 72.7 kDa (Figure 1). A
line-up of the amino acid sequences with the putative plant
Pex5p confirmed homology to all five published PTS1receptor sequences. The highest homology was found with
the two versions of the human Pex5p, which differ in
length (639 and 602 amino acids, respectively) because of
alternative splicing (Dodt et al., 1995). The yeast Pex5
polypeptide chains with an overall length of only 612 aa
(S. cerevisiae), 598 aa (Y. lipolytica), 576 aa (P. pastoris)
and 569 aa (H. polymorpha) have a shorter N-terminal
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Plant PTS1 receptor 455
Figure 2. Amino acid sequence alignment of watermelon, human and
H. polymorpha Pex5p.
Identical residues are indicated by an asterisk, similar residues by a dot
below the sequence. TPR repeats are numbered and overlined; the amino
acids characteristic for TPR motifs in positions 8, 24 and 32 of each motif
are boxed. The 5-amino acid long repeating motifs in the N-terminal half
of the proteins are boxed and shadowed.
Leu or Met, position 24 of the repeat) serves as a knob.
These residues interact between adjacent helices of the
correct orientation. Furthermore, a Trp in position 4 and
an Ala in positions 20 and 27 of the repeat are highly
conserved. The helix-breaking Pro residue is present at the
C-terminus of the repeat (position 32). Pex5 proteins exhibit
seven TPR motifs in their C-terminal half (Figure 2). In
plants, motifs 1–3 and 5–7 fit well to the consensus,
whereas motif 4 is shortened to 31 amino acids and seems
to be less conserved. However, motif 4 also exhibits the
weakest homology in other organisms. A comparison
among the watermelon TPR motifs reveals that the homology is restricted primarily to the TPR consensus (position
8s, 24 and 32 within the motifs, Figure 2). A comparison
among the individual TPR motifs of watermelon and
humans, however, exhibits a stronger homology: for
example, 21 identical and 28 similar amino acids out of 34
are present in motif 6 (Figure 2). The plant Pex5p also has
11 repeats of the sequence W-(A/V/N)-(E/D/N/Q/S)-(E/S)-(Y/
F/Q) in its N-terminal half at aa positions 5, 140, 157, 174,
187, 267, 281, 293, 309, 321 and 340; seven similar motifs
are observed in the human Pex5p (Dodt et al., 1995)
(Figure 2). This motif is less redundant in yeasts: the
Y. lipolytica Pex5p has five copies, the P. pastoris Pex5p
four, the H. polymorpha Pex5p three and the S. cerevisiae
Pex5p only two similar motifs in the N-terminal half of the
protein. The function of this motif remains to be elucidated.
The watermelon Pex5p has a dual location
region than the plant and human Pex5 proteins. An alignment of the plant Pex5p with the 639 amino acids of the
human Pex5p is shown in Figure 2. Within the TPR region
the identity at the amino acid level is 43% and the similarity
63%, whereas the total proteins show 29% identity and
45% similarity. The H. polymorpha Pex5p is also included
in Figure 2, since we used H. polymorpha as a heterologous
host for functional studies. A comparison of the plant
Pex5p with the yeast proteins revealed 35–38% identity
and 59% similarity within the TPR region but only 23%
identity and 40% similarity for the total protein.
The plant Pex5p homologue contains seven tetratricopeptide repeats (TPR) like the human and yeast Pex5p’s.
These characteristic 34 amino acid repeats of the TPR
protein family are defined by a degenerate consensus
sequence. Only three amino acids with a well-defined
distance are conserved within the TPR motif. The TPR
sequence can be folded into an α-helix with two subdomains, A and B, that form a ‘knob’ and a ‘hole’. The
‘knob’ of one helix fits to the ‘hole’ of an adjacent helix
(Goebl and Yanagida, 1991). The most conserved amino
acids in the TPR motifs determine the characteristic of the
helix: Gly (or Ala, position 8 of the repeat) has a small side
chain and forms the hole, while the bulky Tyr (or Phe or
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Antibodies against the watermelon Pex5p were raised in
rabbits by injecting the overexpressed protein, which
started at the 4. Met in the coding region (Figure 1, amino
acids 208–647). The quality of the new antibodies was
demonstrated as follows (Figure 3). The pre-immune serum
did not recognize watermelon proteins (Figure 3, lane 1).
The α-CvPex5p antibodies immunoreacted with the His
tagged protein used in immunisation, which has a calculated molecular mass of 49.6 kDa (Figure 3, lane 2). In a
crude protein extract from 3-days-old watermelon cotyledons a protein with an apparent molecular mass of 85 kDa
was recognized by the α-CvPex5p antibodies (Figure 3,
lane 3). This discrepancy between the observed molecular
mass by SDS-PAGE and the calculated molecular mass of
72.7 kDa for the plant Pex5p was also found for the human
Pex5p: it migrated with an apparent molecular mass of
80 kDa instead of its predicted molecular mass of 67 kDa
(Dodt et al., 1995). A significant difference between the
theoretical and empirical relative molecular mass has also
been described for the P. pastoris Pex5p (McCollum et al.,
1993). Furthermore, truncated proteins were detected
which have a similar migration behaviour as the CvPex5p
used for immunisation. Since only the full-length CvPex5p
had a slower migration behaviour than expected, we
456 Christine Wimmer et al.
Figure 4. Western blot analysis of CvPex5p in watermelon cotyledons
grown for 1 d, 2 d, 3 d and 5 d in the dark.
The CvPex5p is already present at day 1 and is strongly induced at days 2
and 3 when truncated products also become visible. It is found in the
cytosolic supernatant (S) and in the organellar pellet (P).
Figure 3. Western blot analysis of a crude protein extract from 3-day-old
watermelon cotyledons (lane 1, 3 and 4) and purified CvPex5p (4 ng) used
for immunization (starting with Met 208, see Figure 1). Lane 1, decorated
with pre-immune serum; lane 2s and 3, decorated with specific antiPex5p-antibodies; lane 4, decorated with serum that was depleted for
specific antibodies.
assume this to be due to the highly hydrophobic N-terminal
amino acids of the PTS1 receptor in plants and humans.
No watermelon proteins were detected when the Western
blot was decorated with the antiserum pre-incubated with
the His tagged protein used in immunisations (Figure 3,
lane 4). The immunoreactivity with the 85 kDa protein and
its truncated form was abolished, thus confirming that this
is the Pex5 protein.
Watermelon cotyledons grown for 1 d, 2 d, 3 d and 5 d
in the dark were fractionated by differential centrifugation
into a soluble fraction corresponding to the cytosol, and
an organellar fraction. The presence of the Pex5p in these
fractions was examined by immunoblotting (Figure 4). In
all fractions a protein with an apparent molecular mass of
85 kDa was recognized by the α-Pex5p antibodies. The
watermelon Pex5p is already detectable in 1-day-old cotyledons. It is strongly induced at days 2 and 3, when truncated
products down to 49 kDa also become visible, and is
still present in 5-days-old cotyledons. The double band
recognized at 33 kDa seems to result from a cross-reacting
protein already present in dry seeds. This 33 kDa protein
is mainly present at day 1 in the organellar pellet and
decreases with time.
The watermelon Pex5p was detectable in the cytosolic
supernatant as well as in the organellar pellet (Figure 4).
Regarding its presence in the organellar pellet, the protein
is assumed to be localized in glyoxysomes. Glyoxysomes
are fragile organelles, surrounded only by a single membrane; matrix proteins could partially leak out during isola-
tion of organelles and would then be found in the ‘cytosolic
fraction’. We therefore compared the distribution of the
CvPex5p with that of the glyoxysomal matrix enzyme
malate dehydrogenase (gMDH) in the cytosolic and
organellar fractions from 2-days-old watermelon cotyledons by immunoblotting (Figure 5). This allows for the
calculation of the in vivo ratio of CvPex5p between cytosol
and glyoxysomes. Since only a small amount of Pex5p
was expected to be localized in glyoxysomes, 10 times
more organellar pellet than cytosol (calculated from the
plant fresh weight) were subjected to SDS-PAGE and the
proteins transferred to nitrocellulose. Antisera against
CvPex5p and gMDH were used to visualize the distribution
of the proteins. The Western blot analysis assigned approximately 40% of the loaded Pex5p to the supernatant and
approximately 60% to the organellar pellet (Figure 5, lane
1 and 2). Given the fact that 10 times more organelles were
loaded than cytosolic fraction, the portion of the CvPex5p
within the organelle is less than 10%, with more than 90%
of it in the cytosolic fraction. In comparison to that, gMDH
shows a very different distribution and is found only in
very low amounts in the cytosolic fraction due to leakage
from glyoxysomes during fractionation (Figure 6, lane 3
and 4). We conclude that the presence of the CvPex5p in
the cytosolic fraction could reflect an in vivo localization
in the cytosol and not just leakage from glyoxysomes
during fractionation. Definitive proof for its localization in
the cytosol will have to be corroborated by alternative
methods.
The organellar fraction shown in Figure 5 was further
fractionated on a discontinuous sucrose gradient; the
samples on the gels were loaded on an equivalent fraction
basis (Figure 6). At the interface between 57 and 50%
sucrose only glyoxysomes are found (fraction 4), at the
interface between 50 and 43% sucrose the plastids (fraction
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Plant PTS1 receptor 457
Figure 5. Distribution of the CvPex5p (peroxisomal targeting signal 1
receptor; PTS1-R) and the glyoxysomal matrix enzyme malate dehydrogenase (gMDH) between cytosolic supernatant (S) and organellar pellet (P)
in 2-day-old watermelon cotyledons as determined by Western blot analysis.
Ten times more organelles were loaded than cytosolic fraction. The PTS1R is located primarily in the cytosol, whereas gMDH is found in low
amounts in the cytosolic fraction due to leakage from glyoxysomes during
fractionation.
3), at the interface between 43 and 33% sucrose the
mitochondria (fraction 2), and at the interface between 33
and 20% sucrose mainly endoplasmic reticulum (fraction
1) (Hock, 1973; Sautter and Hock, 1982). In fraction 3, we
also found intact glyoxysomes associated with the plastids
which cannot be separated without destroying the organellar envelope membranes. Fraction 4 contains pure glyoxysomes, whereas the organelles in the upper fractions of
the gradient are contaminated by glyoxysomal marker
enzymes released from glyoxysomes during fractionation.
The same holds true for mitochondrial marker enzymes,
which are found in fraction 2 (intact mitochondria), but
also in fraction 1 and on top of the gradient due to leakage
during fractionation. Therefore, we divided the organelles
after harvesting from the sucrose gradient in two halves
and treated one half with trypsin to remove proteins
not protected by the organellar membrane. The gradient
fractions were characterized by Western blot analysis using
antisera against CvPex5p, glyoxysomal and mitochondrial
malate dehydrogenase (Figure 6) and by measuring the
marker enzyme activities isocitrate lyase and fumarase for
glyoxysomes and mitochondria (Figure 7). The Pex5p and
glyoxysomal malate dehydrogenase were distributed over
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Figure 6. The peroxisomal targeting signal 1 receptor (PTS1-R) is in part
associated with glyoxysomes.
An organellar pellet was subfractionated on a discontinuous sucrose
gradient: 1, endoplasmic reticulum; 2, mitochondria, 3, plastids with
glyoxysomes; 4, glyoxysomes. The mitochondrial matrix enzyme malate
dehydrogenase (mMDH; a and b), the glyoxysomal matrix enzyme malate
dehydrogenase (gMDH; c and d) and the CvPex5p (peroxisomal targeting
signal 1 receptor, PTS1-R; e and f) were analyzed by Western blot analysis
before (a, c and e) and after tryptic digestion of the isolated fractions (b, d
and f).
the whole gradient (Figure 6c,e), but were protease-protected only in fractions 3 and 4 (Figure 6d,f) indicating their
localization within intact glyoxysomes. On the other hand,
mitochondrial malate dehydrogenase (mMDH) is found in
fractions 1 and 2 (Figure 6a) but is protease protected
mainly in fraction 2 (Figure 6b). These findings are supported by the distribution and protease protection of the
glyoxysomal marker enzyme isocotrate lyase (Figure 7a)
and the mitochondrial marker enzyme fumarase
(Figure 7b). Isocitrate lyase activity is found in all four
gradient fractions. However, the protease protection is 45%
in fraction 4, 66% in fraction 3, and only 11% in fraction 2
and 7% in fraction 1, indicating the presence of intact
glyoxysomes in fractions 3 and 4. Fumarase activity on the
other hand is present and protease-protected predominantly in fraction 2 (74% protease-protected) and fraction
1 (51% protease-protected) with very low amounts of
activity in fractions 3 and 4.
These data support that the PTS1-receptor is localized in
the glyoxysomal compartment. Its location in the cytosol
is expected but not proven for plants in vivo. This would
458 Christine Wimmer et al.
Figure 7. Characterisation of the organellar fractions separated on a
discontinuous sucrose gradient by measuring the glyoxysomal matrix
enzyme isocitrate lyase (a) and the mitochondrial matrix enzyme fumarase
(b) before and after tryptic digestion of the isolated fractions.
1, endoplasmic reticulum; 2, mitochondria; 3, plastids and glyoxysomes;
4, glyoxysomes.
be compatible with the ‘shuttle mechanism’ in the course
of which the PTS1 receptor transports the cargo protein
only from the cytosol to the membrane-bound translocation machinery, and also with the ‘extended shuttle mechanism’ in which the cargo-loaded receptors cross the
peroxisomal membrane together with their cargo proteins
similar to the import of proteins into the nucleus (Goerlich
and Mattaj, 1996). Its localization within glyoxysomes,
however, supports the model of the ‘extended shuttle
mechanism’, whereby the cargo-loaded receptors cross
the peroxisomal membrane and can be found within the
organelle (Erdmann et al., 1997).
The peroxisome formation and import deficiency of the
H. polymorpha pex5–1 mutant is partially restored by the
watermelon Pex5p (CvPex5p)
We tested whether the plant Pex5p could substitute for its
homologue in H. polymorpha. Earlier complementation
experiments with interspecific hybrid cDNA clones from
yeasts and humans demonstrated that the C-terminal half
of the protein, which comprises the TPR domain, can be
exchanged between different organisms (Dodt et al., 1995;
Wiemer et al., 1995). The less conserved N-terminal half
of the PTS1 receptor complemented so far only in a speciesspecific manner. The following constructs were used for
transformation of the Hppex5 mutant: the full length plant
Figure 8. Western blot analysis of a crude protein extract from Hansenula
polymorpha pex5–1 mutant transformed with plant full-length PEX5 (lane 1),
with the hybrid clone coding for the H. polymorpha N-terminal half and
the plant C-terminal half (lane 2) or with the H. polymorpha PEX5 (lane 3).
Upper panel, decorated with antibodies against the CvPex5p; lower panel,
decorated with antibodies against the HpPex5p.
cDNA clone and a hybrid clone coding for the H. polymorpha N-terminal half (aa 1–271) and the plant C-terminal
half (aa 377–647) of the Pex5p were inserted into the
H. polymorpha shuttle vector pHIPX4 m under the control
of the alcohol oxidase promoter. The coding sequence for
the H. polymorpha Pex5p was used as a positive control.
The hybrid protein of the Hansenula N-terminal half and
the plant C-terminal half is shorter (542 aa) than both the
plant (647 aa) and the Hansenula (569 aa) protein.
The H. polymorpha transformants expressing either the
plant Pex5p or the hybrid Pex5p were unable to grow on
methanol as a sole carbon source. However, they could be
cultivated on glycerol/methanol as carbon source. Peroxisome-deficient strains can grow on glycerol: the advantage
of a glycerol/methanol mixture is that it gives a good
induction of the alcohol oxidase promoter.
The introduced genes resulted in the production of the
relevant proteins as could be demonstrated by Western
blot analysis (Figure 8). The plant Pex5p (647 aa) reacted
with the anti-CvPex5p antibodies (Figure 8, upper part,
lane 1), the yeast Pex5p (569 aa) reacted with the antiHpPex5p antibodies (Figure 8, lower part, lane 3), whereas
the hybrid protein (542 aa) was recognized by both of the
antibodies (Figure 8, upper and lower part, lanes 2). No
degradation products or truncated proteins could be
detected.
Morphological analysis showed that methanol-induced
cells of Hppex5 did not contain peroxisomes (Figure 9a). In
contrast, cells producing the watermelon Pex5p contained
peroxisomes associated with additional membranous
layers (Figure 9c). However, import of AO protein was not
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Plant PTS1 receptor 459
Figure 9. The peroxisome and import deficiency of the H. polymorpha pex5
mutant is partially restored by the watermelon Pex5p, but not by the
Hansenula-watermelon hybrid Pex5p.
Electron micrographs prepared from ultrathin sections of KMnO4-fixed cells
(a, c and e) and aldehyde-fixed cells (b, d and f) of H. polymorpha
transformants. P, peroxisome; V, vacuole; M, mitochondrion; N, nucleus.
Bars 5 0.5 µm. (a) Untransformed pex5 mutant after induction in methanol/
ammonium sulphate containing medium. The cells do not contain
peroxisomes. A large alcohol oxidase crystalloid is found in the cytosol (*).
(c) Cells expressing the watermelon Pex5p contain peroxisomes; additional
membrane profiles are often found surrounding the peroxisomes. (e)
Cells expressing the Pex5 fusion protein; peroxisomal structures are not
detectable, an alcohol oxidase cristalloid is found in the cytosol (*). (b, d
and f) Immunocytochemistry of cells expressing the watermelon PEX5
gene with antibodies against the PTS1 proteins alcohol oxidase (b), the
plant Pex5p (d) and against the integral peroxisomal membrane protein
Pex3p (f).
complete since AO crystalloids were also found in the
cytoplasm. In cells synthesizing the Pex5 fusion protein
with the N-terminal half of H. polymorpha linked to the plant
TPR domain, peroxisomal structures were not detectable
(Figure 9e); the cells contain large cytosolic AO crystalloids
and some multi-layered membrane structures. The control
gave the expected results in that HpPEX5 normally restored
peroxisome formation (data not shown).
Immunocytochemistry with antibodies against the PTS1
protein AO was carried out in order to investigate the
extent of restoration of a PTS1 directed import. In Hppex5
cells expressing the watermelon Pex5p, AO labeling was
observed in the peroxisomal matrix, but also in the cytosol
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
including on the crystalloids and in the nucleus (Figure 9b).
At high expression rates AO cristalloids are formed which
are occasionally found inside the nucleus when import of
PTS1 proteins is not fully functional (Van der Klei et al.,
1991a). In cells expressing the Pex5 hybrid protein, AO
was found only in the cytosol (not shown). Immunogoldlabelling with α-CvPex5p antibodies revealed that the plant
PTS1 receptor was found in the cytosol and inside the
peroxisomes and – due to overexpression – also in the
nucleus (Figure 9d). Both the peroxisomal membranes
and the additional membrane profiles surrounding the
peroxisomes in the plant Pex5p producing cells (Figure 9c)
were labeled with antibodies against the integral membrane protein HpPex3p (Baerends et al., 1996), indicating
that they were of a peroxisomal nature (Figure 9f). We
conclude that synthesis of the hybrid PTS1 receptor of
H. polymorpha and watermelon in a Hansenula mutant
deficient of the peroxisomal PTS1 receptor does not restore
the formation of peroxisomes and AO import. In contrast
to this, synthesis of the watermelon PTS1 receptor in the
same mutant led to the formation of peroxisomes and to
a limited import of AO protein. These data basically show
that the watermelon PTS1 receptor can function in Hansenula. However, AO was also found in the cytosol, indicating
that the expression of the watermeoln PEX5 gene only
partially restores the AO import defect in the Hppex5. In
H. polymorpha, complementation of the growth defect on
methanol can only be achieved if import of the AO into
peroxisomes is complete, as already small amounts of
AO in the cytosol prevent normal growth (Van der Klei
et al., 1991b).
Discussion
We have identified the watermelon PEX5 gene, a plant
homologue of the PTS1 receptor described for humans
and yeasts. Its protein product, CvPex5p, exhibits typical
sequence features of this receptor: the C-terminal half of
the protein containing seven TPR motifs is responsible for
binding the C-terminal tripeptide peroxisomal targeting
signal (PTS1) in humans (Dodt et al., 1995; Wiemer et al.,
1995) and yeasts (Brocard et al., 1994; Szilard et al., 1995;
Terlecky et al., 1995; Van der Klei et al., 1995). Furthermore,
the CvPex5p contains 11 repeats of the sequence W-X2(E/S)-(Y/F/Q) in its N-terminal half, which are also characteristic for the human Pex5p, but less frequent and distinct in
the yeast homologues. This repeated motif might have
functions specific for higher eucaryotes. The high homology among plant, human and yeast Pex5 proteins demonstrates that peroxisome assembly in higher and lower
eukaryotes requires at least some of the same type of
peroxisome assembly proteins.
Similar to the human and H. polymorpha Pex5p, the
plant Pex5p is found in fractionation experiments primarily
460 Christine Wimmer et al.
in the cytosol fraction with a smaller amount sequestered
in the microbodies. If cytosol location of the receptor
will be corroborated by other methods the results are
compatible with a cycling model of PTS1 receptor function
in which the receptor shuttles between the cytosol and the
organellar matrix (Van der Klei and Veenhuis, 1996). The
relative proportion of the protein found in the two locations
may be affected by the relative abundance of the receptor
and newly synthesized PTS1 proteins. Thus, the relative
distribution of the receptor between cytoplasm and peroxisome is difficult to quantify and could vary between cells
and/or organisms, and also reflect specific environmental
conditions.
The restoration of the PTS1 protein import in cells from
patients with peroxisome biogenesis disorders and in yeast
mutants by complementation with the cloned homologous
PEX5 gene provides strong evidence for its function. Plant
mutants disturbed in peroxisome biogenesis (pex mutants)
are not yet available and might not be viable considering
their multiple functions: in germinating seeds glyoxysomes
are necessary for fat mobilisation, while leaf-type peroxisomes are necessary for photorespiration in the light. We
therefore analyzed the function of CvPex5p in yeast by
complementing the PTS1 import defect in the Hppex5
mutant. We introduced the coding sequence for the plant
Pex5p and for a hybrid protein combining the H. polymorpha specific N-terminal half with the plant TPR domain, the
latter being more conserved among different organisms.
Interestingly, the hybrid protein was unable to induce the
formation of peroxisomes and to restore import of PTS1
proteins in the H. polymorpha mutant. The plant TPR
domain thus might be incompatible with the yeast Nterminal half of the protein. On the other hand, the entire
plant Pex5p did induce assembly of peroxisomes, although
the complementation was only partial. The presence of
HpPex3p, an integral peroxisomal membrane protein, identified the genuine nature of the organelles. The limited
import of AO, which is targeted by the PTS1 variant -ARF,
supports the idea that the plant TPRs contain the PTS1binding domain. However, it also hints that specific conformations of the TPR domains determine the efficiency of
recognition of a given tripeptide signal. Alternatively, partial import may be an indication that the plant Pex5p does
not interact efficiently with other components of the yeast
peroxisome import system. Results pointing in the same
direction were obtained with the PTS1 receptor from
another eukaryote, the human Pex5p. It was unable to
complement the P. pastoris pex5 mutant, whereas a fusion
protein combining the N-terminal part of the P. pastoris
Pex5p and the TPR domain of the human Pex5p provided
a differential complementation: expression of this fusion
protein restored the ability of the P. pastoris pex5 mutant
to grow on media containing oleate as sole C-source, but
did not restore the ability to metabolize methanol (Dodt
et al., 1995; Wiemer et al., 1995). It can be envisaged that
the human TPRs cannot recognize the P. pastoris AO with
the PTS1 signal -ARF. The plant Pex5p, on the other hand,
seems capable of an inefficient recognition of the PTS1
motif -ARF and/or a partly successful interaction with the
import machinery. H. polymorpha is especially sensitive to
the presence of AO in the cytosol. Furthermore, the P. pastoris Pex5p failed to functionally complement a H. polymorpha pex5 mutant (van der Klei et al., 1995). An
incomplete import of AO into peroxisomes, however, cannot restore growth on methanol as C-source since already
a small amount of AO in the cytosol prevents normal
growth of H. polymorpha on methanol due to specific
energetic disadvantages (Van der Klei et al., 1991b).
It is remarkable that the plant Pex5p can restore the
formation of peroxisomes in the H. polymorpha pex5–1
mutant. The absence of peroxisomes in the mutant is due
to the requirement of the PTS1 receptor in the microbody
assembly process for targeting PTS1 proteins to the incipient peroxisomal membrane and import apparatus. This in
turn requires that the receptor can recognize and use
the species- and protein-specific C-terminal tripeptides of
H. polymorpha and can interact with the other components
of the import machinery. Apparently, the higher plant PTS1
receptor can recognize a majority of the proteins necessary
for the assembly and import of matrix proteins of H. polymorpha peroxisomes. The Pex5p does not contain targeting information itself but, bound to a PTS1 peptide, it is
recognized and targeted. Thus, binding of the cargo leads
to a modification of Pex5p that allows docking and import
(Van der Klei and Veenhuis, 1996). An inappropriate threedimensional texture of the complex comprising the plant
PTS1 receptor and the Hansenula cargo might prevent full
recognition by the docking protein HpPex14p within the
peroxisomal membrane (Komori et al., 1997). The observed
supernumerary peroxisomal membrane layers are an
indication that the receptor cannot recognize all required
PTS1 proteins or the receptor-cargo complex cannot interact with the docking protein thus leading to unbalanced
targeting and failure of full complementation. The partial
restoration of peroxisome formation and import capability
invites further experimentation in defining the recognition
sites in the plant PTS1 receptors TPR region for individual
tripeptide signals.
Experimental procedures
Plant material
Watermelon seeds (Citrullus vulgaris Schrad., var. Stone mountain, harvest 1994; Landreth Seed Co., Baltimore, MD, USA) were
germinated under sterile conditions in the dark at 30°C for 1–5 d
(Gietl, 1990).
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Plant PTS1 receptor 461
Cloning of the cDNA that encodes the watermelon PTS1
receptor
Poly(A)1RNA was isolated from cotyledons of 2-day-old
watermelon seedlings. A customer cDNA library in the Lambda
ZAP Express vector was made by Stratagene. Isolation of the
cDNA clone for the plant PTS1 receptor (CvPEX5) was carried out
in three steps.
The central part of the cDNA encoding the watermelon PTS1
receptor was amplified by PCR with primers of mixed oligonucleotides. The primers were designed according to highly conserved
regions within the published sequences of the PTS1 receptors
from S. cerevisiae (accession no. L23076, Van der Leij et al.,
1993), P. pastoris (accession no. Z19592, McCollum et al., 1993),
H. polymorpha (accession no. U26678, Van der Klei et al., 1995),
Y. lipolytica (accession no. U28155, Szilard et al., 1995) and humans
(accession no. U19721, Dodt et al., 1995; accession no. X84899,
Fransen et al., 1995; accession no. Z48054, Wiemer et al., 1995).
The Sense1 primer encoded EAGLAFEAAV (59 GAR GCX GGX
YTX GCX TTY GAR GCX GCX GT 39; R 5 G/A, Y 5 C/T, X 5
deoxy Inosine; Figure 1, bases 1180–1208). The Antisense1 primer
corresponds to W N K(R) L G A T(AS) L A N (59 TT XGC XAR XXX
XGC XCC XAR XYX RTT CCA 39; Figure 1, bases 1636–1664). As
template the watermelon cDNA was amplified by PCR from the
library in the Lambda ZAP Express vector using the T3 and T7
promoter primers. The products were purified by a Microspin
S400-HR column (Pharmacia). This step was necessary to exclude
vector sequences and thus unspecific binding of the PCR primers.
A single PCR fragment was generated and cloned into the SrfI
site of pCR-Script (Stratagene). Sequence determination of the
resulting plasmid revealed a 485 bp insert flanked by both PCR
primers (Figure 1, bases 1180–1664). Computer analysis of the
insert sequence revealed high homology to the PTS1 receptors
from humans and yeasts.
This PCR product was labeled with digoxigenin (DIG, Boehringer) and used as a probe for screening 4 3 105 clones of the
cDNA library in the Lambda ZAP Express vector. One clone
covering bases 619–2445 of the final cDNA clone (Figure 1) was
obtained. Its open reading frame coded for 440 amino acids, a
protein being too short in comparison with the published PTS1
receptors. A probe was generated by PCR covering bases 622–
846 of the final clone and used for screening another 8 3 105
clones. Three different cDNA clones were isolated showing complete sequence identity as far as they overlapped. They contained
the 39 end of the clone but differed in length at the 59 end, the
longest one starting in position 186 (Figure 1). Again a 59 terminal
EcoRI-HindIII restriction fragment covering bases 186–657 of the
final clone was used for screening 1.1 3 106 clones, which resulted
in a cDNA clone covering bases 55–2445 of the final clone.
To isolate the 59 part of the cDNA, first-strand synthesis of cDNA
(ss cDNA) followed by poly(dG)-tailing with terminal transferase
was carried out (Gietl, 1990). The poly(dG)-tailed ss cDNA was
used as a template for PCR with the Antisense2 primer (59 CAC
TCT TTG TGG TGG CCC ATC C 39; Figure 1, bases 129–150) based
on the nucleotide sequence for amino acids DGPPQRV and an
oligo(dC) tail as sense primer. A PCR fragment of about 300 bp
was generated, cloned into pCR-Script (Stratagene) and
sequenced. The insert exhibited the Antisense2 primer and the
necessary adjacent codons for the amino acids (bases 55–128)
known from the longest ds cDNA clone isolated from the library.
In addition, a sequence encoding 18 amino acids including the
start methionine and a 59 untranslated region of 63 bp were found.
Upstream of the start codon a stop codon was found within the
reading frame.
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464
Finally, the complete cDNA clone encoding the watermelon
PTS1 receptor was synthesized by PCR. The N-terminal part was
synthesized using the Sense2 primer, which based on the codons
for MASAWNE (59 GCGGGATCC-ATG GCC TCT GCA TGG AAT
GAG G 39) and the Antisense2 primer (Figure 1, bases 1–22 and
129–150); a BamHI restriction endonuclease cleavage site was
added at the 59 end for cloning purposes. The C-terminal part was
amplified with the Sense 3 primer (comprising the bases 58–76)
and the Antisense 3 primer (59 GCGGGGCCC-TCA TAA TGG GAA
CTC CTT CTG 39; Figure 1, bases 1924–44); an ApaI site was added
for cloning purposes. In a third PCR reaction the full-length cDNA
was produced by mixing the N-terminal and the C-terminal part
of the clone and adding an excess of Sense 2 and antisense 3
primer (Horton et al., 1989). The final PCR product was cut with
BamHI and ApaI, cloned into the appropriate sites of the plasmid
vector pBK-CMV (Stratagene) and controlled by sequence analysis.
Construction of cDNA clones encoding the watermelon
PTS1 receptor or a hybrid gene combining the coding
region for the N-terminal half of the H. polymorpha
Pex5p with the C-terminal half of the watermelon Pex5p
in the H. polymorpha shuttle vector pHIPX4
The E. coli–H. polymorpha shuttle vector pHIPX4 (Gietl et al., 1994)
was modified by introducing additional restriction sites for cloning
purposes: the HindIII–SmaI fragment downstream of the Pmox
promoter (the ‘multiple cloning site’) was removed and replaced
by a double-stranded synthetic oligonucleotide coding for HindIII–
SalI–BamHI–EcoRI–XhoI–ApaI–ClaI and SmaI, resulting in the vector pHIPX4 m.
The full coding region for the watermelon PTS1 receptor was
cut out with BamHI and ApaI from the pBK-CMV clone (see above)
and recloned into a BamHI and ApaI site in pHIPX4 m.
Furthermore, a fusion gene was generated combining the coding
regions for the N-terminal 271 amino acids of the Hansenula
polymorpha Pex5p (Van der Klei et al., 1995) and for the C-terminal
half of the watermelon Pex5p containing the tetratrico peptide
repeat motifs (Figure 1, amino acids 377–647) with the help of the
‘gene splicing by overlap extension’ method (Horton et al., 1989).
For this purpose, the coding region for the N-terminal half of the
H. polymorpha Pex5p was amplified with the primers HpSense
(59 GGGGGATCC-ATG TCA TTT CTG GGA GG 39) and FusionAntisense (39 TA TTA GTC AAG GCA TTG TTA – GGA TTA GGG
AAC TTC CTT CC 59), the latter one covering 20 bases of the
H. plymorpha gene (Van der Klei et al., 1995; Figure 1, bases 795–
914) and 20 bases of the watermelon Pex5 cDNA (Figure 1, bases
1129–1148). The coding region for the C-terminal half of the
watermelon Pex5p was amplified with the primers Fusion-Sense
(complementary to the Fusion-Antisense primer) and the Antisense3 primer (see above). BamHI– and ApaI restriction sites were
added that way at the 59end and 39end, respectively. In a final
PCR, the hybrid gene was produced by mixing the N-terminal
yeast fragment and the C-terminal watermelon fragment and
adding an excess of HpSense and Antisense3 primers. This PCR
fragment was cut with BamHI and ApaI and cloned into the
appropriate sites of vector pHIPX4 m. The H. polymorpha PEX5
gene under the control of the alcohol oxidase promoter was used
as a positive control, its N-terminal half as a negative control. The
constructs were transformed into the H. polymorpha pex5–1
(formerly Hpper3–1) mutant strain (Van der Klei et al., 1995) by
electroporation (Faber et al., 1994b).
462 Christine Wimmer et al.
Overexpression of a cDNA encoding the watermelon
Pex5p for raising antibodies
The cDNA for the watermelon Pex5p starting at the 4. Met in the
coding region (Figure 1, amino acids 208–647) was cloned into the
E. coli-expression vector pQE70 (Quiagen) between the restriction
sites SphI and BamHI; the SphI site also provided the start codon.
The necessary restriction sites at the 59-end and 39-end of the
cDNA sequence were added by PCR (primers: Sense-pQE70 59GGGGCATGCTG AAC TTA TCT GCA ATG G 39 and AntisensepQE70 59-CCCGGATCC – TAA TGG GAA CTC CTT CTG 39). The
codon for the 5. Met in the Pex5p (Figure 1, positiom 209) was
changed into the codon for Leu to restore the SphI site. The
pQE70-vector adds an affinity tag consisting of six consecutive
His residues at the C-terminus. Expression was carried out in the
E. coli host M15 (Quiagen) as described by Gietl et al. (1996). The
expressed protein had to be solubilized from inclusion bodies by
6 M guanidinium hydrochloride and was purified under denaturing
conditions on an Ni-NTA resin (Quiagen) according to the manufacturer’s instructions. Aliquots of the main elution peak fraction
were sent to Eurogentec (Seraing, Belgium) for immunization
of rabbits. The antibodies were purified by ammonium sulfate
precipitation and used in a 1: 3000 dilution for Western blot
experiments.
Test for specificity of the antibodies raised against the
watermelon Pex5p
Ni-NTA resin (2 ml of a 50% slurry, Quiagen) was equilibrated
with 10 mMTris–HCl, 150 mM NaCl, pH 8.0 (TBS) and incubated
with 750 µg overexpressed and purified His tagged CvPex5p that
was used for immunization of rabbits (see above), by stirring the
mixture on ice for 1 h. The slurry was washed 5 times with
10 ml TBS to remove unbound His-tagged CvPex5p. The slurry
containing the His-tagged antigen was incubated with 40 µl of
antibodies by stirring the mixture at room temperature for 3 h.
The supernatant was collected from the slurry, adjusted to the
same dilution as the specific anti-CvPex5p antibodies and used
as an antiserum depleted for the specific antibodies.
Preparation of a crude protein extract and isolation of
organelles from watermelon cotyledons
A crude protein extract from 3-day-old watermelon cotyledons
was prepared by grinding 12 pairs of cotyledons in liquid nitrogen
and stirring the powder with 6 ml 50 mM Hepes, 1 mM DTT, 1 mM
EDTA pH 7.4 in the cold room for 1 h. The mixture was centrifuged
in a tabletop centrifuge for 10 min. The supernatant was used
for quality control of the anti-CvPex5p antibody by Western
blot analyis.
Isolation of a crude organellar pellet and separation of organelles
on a discontinuous sucrose density gradient were carried out as
described previously(Gietl et al., 1997): 50 pairs of cotyledons
were homogenized and a 10 500 g organellar pellet was prepared.
The organelles were resuspended in 0.8 ml medium (10 mM
HEPES, 1 mM DTT, 8% sucrose, pH 7.4) and layered onto a 13 ml
discontinuous Suc gradient in 10 mM EDTA, 5 mM MgCl2, pH 7.4
and centrifuged in a swing out rotor (SW 40 Ti) at 80 000 gav for
3 h. The discontinuous gradient was formed by successively
introducing 2.5 ml of 57, 50, 43, and 33% Suc medium and then
1.5 ml of 20 and 10% Suc medium. After centrifugation the visible
bands of microbodies, plastids, mitochondria and endoplasmic
reticulum (from bottom to top) at the interfaces between the
different Suc steps were harvested using a syringe. One hundred µl
of a freshly harvested organelle fraction was treated with 10 µl
trypsin (0.75 mg ml–1 final concentration; 5 min at 4°C) to remove
proteins sticking at the outer surface of organelles; the digestion
was stopped with trypsin inhibitor (3.0 mg ml–1 final concentration). Fumarase and isocitrate lyase as marker enzymes for mitochondria and glyoxysomes were measured. The presence of the
Pex5p within glyoxysomes was demostrated by Western blot
analysis; antibodies against glyoxysomal and mitochondrial malate dehydrogenase (Gietl et al., 1996) were used as a control.
Growth of H. polymorpha transformants for Western blot
analysis and electron microscopy
H. polymorpha pex5 mutant cells transformed with cDNAs coding
for the CvPex5p (culture 1), or for a hybrid protein combining the
N-terminal half of the H. polymorpha Pex5p and the C-terminal
half of the watermelon Pex5p (culture 2), or for the HpPex5p
(culture 3) under the control of the alcohol oxidase promoter
were extensively pre-cultured on YND (0.67% yeast nitrogen base
without amino acids) 1 0.5% glucose. Exponentially growing cells
were shifted to media containing 0.3% glycerol and 0.2% methanol
with an initial optical density (OD) of 0.1 and grown at 37°C.
Peroxisome-deficient strains can grow on glycerol, the advantage
of a glycerol/methanol mixture is that it gives a good induction
of the alcohol oxidase promoter. After 20 h of growth (t 5 20 h)
the cultures had reached an OD of 0.8 (culture 1) and 1.4 (culture
2) and 1.0 (culture 3). Four hours later (t 5 24 h) the ODs were
unchanged suggesting that the cells had reached the stationary
growth phase (i.e. glycerol-consumed, methanol induction). At
this time point (t 5 24 h) samples were taken for Western blot
analysis and electron microscopy.
For preparation of a protein extract, cells were harvested,
resuspended in 12% trichloroacetic acid and left for 15 min on
ice for precipitation and inactivation of all proteins including
proteases. A crude extract using glass beads was prepared as
described previously (Gietl et al., 1994). Finally, proteins, cell walls
and glass beads were centrifuged (4°C, 10 000 g, 15 min) and
washed twice with 80% aceton. The pellet was dissolved in sample
buffer for SDS-PAGE and analyzed after removing undissolved
material by a short spin. Morphology was examined on KMnO4fixed cells. Immunocytochemistry was performed on ultra-thin
sections of Unicryl-embedded cells (Slot and Geuze, 1984) using
polyclonal antibodies against alcohol oxidase, CvPex5p and the
peroxisomal membrane protein Pex3p (Baerends et al., 1996).
Acknowledgements
We thank Ineke Keizer for expert assistance in electron microscopy.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Gi 154/5–1 and 5–2).
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Note added in proof: Recently the sequence of a cDNA encoding Pex5p, a peroxisomal targeting signal type 1 receptor
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Brickner and L.J. Olsen.
GenBank database accession no. AF068690.
© Blackwell Science Ltd, The Plant Journal, (1998), 16, 453–464