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Plant Physiol. (1998) 116: 733-742
Cloning of a Tobacco Apoplasmic Invertase Inhibitor1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.
Suc is the predominant sugar in higher plants. It serves several
important functions, including acting as the major carbohydrate transport form, as a storage compound, and as an osmoprotectant (Eschrich, 1989 Although the regulation of CWI activity by changes in gene expression
has been well documented, little is known about possible posttranslational regulation. Since CWI is localized outside the symplasm, a posttranslational modification of its structure leading to
enzyme activation or inactivation would have to be operative in the
specific ionic environment of the apoplasmic space. Clearly, by
lowering the apoplasmic pH, cells may activate CWI because of its low
pH optimum (pH 4.5). At lower pH the uptake of the resulting hexoses
via H+/monosaccharide co-transporters is also
stimulated (Sauer and Stadler, 1993 Recently, we characterized an entirely different regulatory mechanism
based on interaction of CWI with an inhibitory polypeptide co-localized
with CWI in the apoplasmic space (Weil and Rausch, 1994 A developmental study of the expression of CWI and INH in tobacco
suspension-cultured cells has shown that both proteins are expressed
during the entire culture period, although CWI inhibition has been
observed only after the medium was Suc depleted (Krausgrill et al.,
1998 To further characterize the structure of the CWI-INH complex and the
physiological role of INH for CWI regulation during plant development,
we have now cloned the tobacco inhibitor. We present proof of function
for the Nt-inh1-encoded protein, after heterologous expression in Escherichia coli, and demonstrate the
specificity of INH binding to CWI. To our knowledge, the
Nt-inh1 clone represents the first fully characterized plant
INH, a member of a novel protein group with moderate but significant
sequence conservation. A comparison of transcript levels for
CWI and Nt-inh1 in different tissues reveals that during
plant development their expression is not always coordinate.
The origin and growth conditions of Agrobacterium
tumefaciens-transformed tobacco (Nicotiana tabacum cv
Petit Havana) cells were previously described (Weil and Rausch, 1990 Purification and Tryptic Digest of INH Protein
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Frommer and Sonnewald, 1995; Ruan and Patrick, 1995;
Stitt and Sonnewald, 1995). Higher plants metabolize Suc either by Suc
synthase or via invertases (Stitt and Sonnewald, 1995; Sturm et al.,
1995; Zrenner et al., 1995). Suc synthase is localized in the cytosol
or bound to the inner side of the plasma membrane (Delmer and Amor,
1995) and catalyzes an equilibrium reaction, whereas invertases, which
catalyze an irreversible hydrolysis, exist in isoforms located in the
apoplasmic space (CWI), in the vacuole (VI), and in the cytosol
(Frommer and Sonnewald, 1995; Sturm et al., 1995). Recently, several
CWI and VI isoforms were cloned and their expression was studied with
respect to developmental regulation and tissue- or cell-specific
expression (Sturm et al., 1995; Weber et al., 1995; Cheng et al., 1996)
and in response to wounding and pathogen attack (Sturm and Chrispeels,
1990; Zhang et al., 1996). In particular, CWI may be involved in phloem
unloading (Miller and Chourey, 1992; Ruan and Patrick, 1995; Weber et
al., 1995), regulation of sink strength (Weil and Rausch, 1990; Roitsch et al., 1995; Ehness and Roitsch, 1997), and hexose production for
wound- or pathogen-induced respiration (Sturm and Chrispeels, 1990;
Zhang et al., 1996). Recently, hexoses formed in the apoplasmic space
were proposed to act, after cellular uptake (Truernit et al., 1996), as
metabolic signals strongly affecting the expression of other genes
(Herbers et al., 1996; Jang et al., 1997).
; Truernit et al., 1996). Thus, it
is noteworthy that for Chenopodium rubrum
suspension-cultured cells a coordinate induction of transcripts for CWI
and a specific Glc transporter isoform has been demonstrated (Ehness
and Roitsch, 1997).
; Weil et al.,
1994; Krausgrill et al., 1996). The inhibitory protein shares some
characteristics with several INHs previously described (Schwimmer et
al., 1961; Pressey, 1968, 1994; Jaynes and Nelson, 1971; Bracho and
Whitaker, 1990; Ovalle et al., 1995). The tobacco (Nicotiana
tabacum) INH is a heat-stable nonglycosylated protein (Weil et
al., 1994). Inhibition of CWI by INH is modulated by the Suc
concentration, the substrate apparently protecting the enzyme by
binding to CWI and/or INH. Whereas the Km
value for Suc hydrolysis was 0.6 mm (Weil and Rausch,
1990), the half-maximum substrate protection was observed with 1.2 mm Suc (Weil et al., 1994). The N-terminal sequence
obtained from the purified tobacco INH shows similarity with the N
terminus of an INH expressed in tomato (Lycopersicon
esculentum) fruit (Pressey, 1994; Weil et al., 1994). Partially
purified inhibitors isolated from tobacco suspension-cultured cells and
from tomato fruit both inhibit tobacco CWI and tomato VI; however, no
substrate protection was found for tomato VI (Sander et al., 1996).
). The observed binding of the CWI-INH complex to concanavalin
A-Sepharose (free INH does not bind) suggests that both proteins are in
physical contact throughout the entire culture period, an assumption
confirmed by co-migration of CWI and INH as a stable complex during
native cathodic PAGE. These and other observations have led to the
hypothesis that in suspension-cultured tobacco cells INH operates as a
regulatory switch for CWI, with INH always being bound to CWI but
inducing the inhibitory conformational change only when Suc
concentration decreases below a threshold level (Weil et al., 1994;
Krausgrill et al., 1998).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
,
1994). For the analysis of transcript amounts in different plant
organs, 7-week-old nonflowering and 15-week-old flowering tobacco
(N. tabacum cv SNN) plants from the greenhouse were used.
Tomato (Lycopersicon esculentum) fruits were purchased at
the local market. The photoautotrophic Chenopodium rubrum
cell-suspension culture was a gift from M. Stitt (Heidelberg, Germany).
Fungal invertases (from Saccharomyces cerevisiae and
Candida utilis) were obtained from Sigma.
. After fractionated ammonium sulfate precipitation, the 40 to 85% fraction was desalted and chromatographed twice on sulfopropyl Sephadex using a pH-gradient elution followed by an NaCl-gradient elution. After the
NaCl-gradient peak fraction was separated by SDS-PAGE,
the homogeneous INH protein was electroeluted and subjected
to a second semipreparative SDS-PAGE run. The Coomassie-stained INH
band was excised, and after destaining/shrinking in acetonitrile, it
was subjected to in situ proteolytic digestion with trypsin in 25 mm ammonium bicarbonate for 6 h at room temperature. The released peptides were recovered from the supernatant and separated
by reverse-phase HPLC. Purified peptide fractions were dotted on glass
fiber membranes and subjected to automated Edman degradation on a
sequencer (model 473A, Applied Biosystems).
Cloning of a Nt-inh1 cDNA
Total RNA was prepared from transformed tobacco cells according to the method of Logemann et al. (1987), from which the
poly(A+) fraction was isolated with the Dynabeads
biomagnetic separation system (Dynal, Oslo, Norway). cDNA was
synthesized using the ZAP-Express cDNA-synthesis kit (Stratagene) and
used for reverse-transcriptase PCR. Antisense primers were designed
according to all peptide sequences obtained from the tryptic digest and
used for PCRs in combination with a sense primer derived from the
previously sequenced N terminus of INH (Weil et al., 1994). For
reverse-transcriptase PCR approximately 1 ng of cDNA was used with
Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). With the
following primers a 300-bp large partial INH cDNA was amplified, which
was later used as a probe for library screening and Southern- and
RNA-blot analysis (see below): sense primer
5
-AAGAACACACCIAAC/TTAC/TCA-3 and antisense primer
5-CCAACCATA/TCCATCC/TTCA/TGC-3.
). The library contained
2 × 106 independent clones. Screening and
in vivo excision of the phagemid were performed according to the
manufacturer's instructions (ZAP-Express cDNA-synthesis kit). Plaques
that were 5 × 105 were screened and five
independent clones were isolated. The clones were sequenced by
automatic sequencing (ABI Prism 377 [Applied Biosystems]; TopLab
Laboratories, Munich, Germany).
RNA-Blot and Southern-Blot Analyses
The preparation of total RNA followed the protocol of Logemann et al. (1987). Genomic DNA was isolated from tobacco leaves (N. tabacum cv SNN) according to the method of
Murray and Thompson (1980). Nonradioactive Southern- and RNA-blot
procedures with biotinylated probes and chemiluminescence detection
were performed according to the method of Löw and Rausch (1996),
except that for RNA blots transfer was overnight in 20× SSC. For
Southern-blot analysis the conditions for the high-stringency
wash were based on approximately 85% homology.
Immunoblot Analysis
Immunoblotting (Towbin et al., 1979) was performed as described by
Weil and Rausch (1994), using the semidry procedure. After proteins
were transferred to Immobilon P membranes (Millipore) for 1 h, the
membranes were blocked with 8% BSA and incubated in the different
antisera for 12 h at 4°C. Immunoblots were developed with
anti-rabbit IgG-alkaline phosphatase conjugate (Sigma).
Expression of Nt-inh1 Protein in Escherichia coli
The Nt-inh1 protein was expressed as a fusion protein using the pQE-30 vector from Qiagen (Hilden, Germany), which provides an N-terminal 6× His tag. For this purpose, the Nt-inh1 cDNA was amplified from the cDNA clone (pBK-CMV vector) with Pfu polymerase (Stratagene) using the following primers: sense primer 5-TATATGGATCCAATAATCTAGTAGAAACTA-3 (BamHI
site underlined) and antisense primer
5-ACATAGTCGACTCACAATAAATTTCTGACAATA-3 (SalI site underlined). The amplification product was cloned into the EcoRV-restricted pBluescript SKII vector (Stratagene) from
which it was released with BamHI and SalI and
subsequently ligated with the
BamHI/SalI-restricted pQE-30 vector. The sequence
of the cloned construct was confirmed by automatic sequencing (see
above). Transformation of host cells was performed according to the
manufacturer's instructions (Qiagen).
Affinity Purification of the INH Antiserum
The recombinant Nt-inh1 protein was used for affinity purification of the previously prepared antiserum directed against purified plant INH protein (Krausgrill et al., 1996). Five-hundred micrograms of purified recombinant Nt-inh1 protein was diluted in 10 mL
of transfer buffer (48 mm Gly, 40 mm Tris base,
0.038% [v/v] SDS, and 20% [v/v] methanol) and bound to a
60-cm2 Immobilon P membrane (Millipore). After a
15-min incubation the membrane was washed in 50 mL of TBST
(Tris-buffered saline plus Tween-20) for 5 min, blocked with 5%
defatted milk powder in TBST, and washed again three times in TBST.
Thereafter, INH antiserum, diluted 1:100 in 1% defatted milk
powder/TBST, was bound to the Nt-inh1 protein-coated
membrane for 1 h at room temperature. The membrane was then washed
three times with TBST, and bound antibodies were eluted with 2 mL of
Gly buffer (0.1 m Gly, 0.5 m NaCl, and 0.05%
Tween 20, adjusted to pH 2.8 with 1 N HCl) during a 3-min incubation.
The eluate was immediately mixed with 300 µL of 1 m
Tris-HCl, pH 8.1, and the elution procedure was repeated. To the
combined eluted fractions BSA and sodium azide were added at 1.5% and
1 mm final concentrations, respectively.
Chromatography of Total Cell Wall Protein on Ni-NTA-Bound Nt-inh1 Protein
Expression of Nt-inh1 in E. coli, binding of the solubilized recombinant Nt-inh1 protein to the Ni-NTA column, and on-column renaturation were performed as described above. To the Ni-NTA column with bound renatured inhibitor protein we applied 100 µg of total cell wall protein, diluted in 4 volumes of TBS and adjusted to pH 6.5, and the flow-through was passed through the column two additional times. After this binding step, the column was first washed with at least 25 column volumes of TBS and then eluted with 2 volumes of TBS including 50 mm EDTA. The different fractions were analyzed by SDS-PAGE and Coomassie staining for their polypeptide patterns. For immunoblot analysis all fractions were adjusted to 100 µg protein/mL by adding BSA to ensure complete protein precipitation by TCA prior to SDS-PAGE.Assay for Inhibitor Function of Recombinant Nt-inh1
Tobacco CWI and tomato fruit VI were prepared as previously described (Weil and Rausch, 1994; Sander et al., 1996). Inhibition assays followed the protocol of Weil et al. (1994). Invertase preparations and Nt-inh1 protein preparations were mixed and
incubated for 60 min at 37°C in the absence or presence of 20 mm Suc. After this preincubation 20 mm Suc was
also added to the minus-Suc sample, and the Glc released during a
subsequent 60-min incubation at 37°C was determined enzymatically.
The concentration of the recombinant Nt-inh1 protein was
determined according to the method of Peterson (1977).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cloning of a Tobacco INH-Encoding cDNA
The INH protein expressed in transformed tobacco cells (Weil et al., 1994) was purified from a cell wall protein fraction obtained by
nondisruptive salt elution as previously described (Weil and Rausch,
1994). After fractionated ammonium sulfate precipitation and
ion-exchange chromatography on sulfopropyl Sephadex using sequentially
pH-gradient and NaCl-gradient elution, the purified INH protein was
electroeluted from the gel (Fig. 1). The
homogeneous INH protein was then subjected to a tryptic digest.
Four peptides could be separated by HPLC and their sequences were
determined via Edman degradation. The locations of all peptide
sequences in the Nt-inh1 full-length clone are
indicated in Figure 2 (see below).
|
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). With cDNA from transformed tobacco cells as the template,
the longest specific amplification product obtained had a size of 300 bp. The sequence of this cDNA fragment contained a continuous open
reading frame comprising all five peptide sequences obtained directly
from the INH protein.
Characteristics of the INH Protein Predicted by the
Nt-inh1 Sequence
) yielded five independent positive clones hybridizing with
the 300-bp partial cDNA obtained by reverse-transcriptase PCR (see
above). The cDNA sequence of one of the clones, Nt-inh1, has
a total length of 631 bases, excluding the
poly(A+) tail, and predicts an open reading frame
of 182 amino acids (Fig. 2). The cDNA fragment used as a probe extends
from position 134 to 433 of the full-length clone. All other cDNA
clones showed the identical sequence except for the length of the 5
untranslated region, which was 49 bases long in four of the clones and
13 bases shorter in one of the clones. Southern-blot analysis performed with the same 300-bp coding region probe indicated that INH is encoded
by a single- or low-copy gene (Fig. 3),
making the existence of closely related isoforms unlikely.
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Figure 3.
Southern-blot analysis of INH sequences in the
tobacco genome. Ten micrograms of genomic DNA was digested with
BamHI (lane 1), DraI (lane 2),
EcoRI (lane 3), EcoRV (lane 4), and
HindIII (lane 5). The blot was hybridized with a 300-bp
Nt-inh1-coding region probe.
). Furthermore, when E. coli cells harboring the
pBK-CMV-Nt-inh1 plasmid were induced with IPTG, a protein of
approximately 18 kD was induced that strongly reacted with the
antiserum directed against the purified INH protein (data not shown).
Heterologous Expression in E. coli and Proof of
Nt-inh1 Function
Specificity of Nt-inh1 Protein Binding to CWI
Expression of CWI and Nt-inh1 during Plant Development
Cloning of the First Plant INH
Regulation of CWI-INH Complex Formation and Physiological Role(s)
of INH during Plant Development
), the
C. unshiu EST clone produced a potential signal peptide with
only a weak score. The most likely cleavage sites, as deduced from the
-1/-3-rule (Von Heijne, 1986), are indicated in Figure 4; however, only
for the Nt-inh1 clone was the cleavage site confirmed by
N-terminal sequencing of the mature protein.
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Figure 4.
Protein sequence alignment of
Nt-inh1 with putative homologs from A. thaliana (A.t.) and C. unshiu (C.u.). The
A. thaliana cDNA clone At-inhh (A.t.;
EMBL nucleotide sequence database, accession no. Y12807) is the
full-length clone corresponding to the A. thaliana EST
clone with the accession no. T88450. The partial C. unshiu cDNA sequence (C.u.) represents an EST clone with the accession no. C22245. The putative signal sequences are underlined, and
the most likely predicted cleavage sites are marked by upward arrows.
Note that the putative signal peptide of the C. unshiu EST clone shows a very low score (Von Heijne, 1986 ). Conserved Cys
residues are indicated by downward arrows. Amino acid residues identical between at least two sequences are in bold type. Except for
the N-terminal sequences (upstream from Asn-20 in
Nt-inh1), the predicted mature protein sequences were
aligned using the CLUSTAL program from PC Gene (Intelligenetics,
Mountain View, CA).
) would yield an N
terminus of the mature protein identical to the sequence obtained from
INH protein isolated from tobacco suspension-cultured cells (Weil et
al., 1994). The protein contains four Cys residues, the locations of
which are conserved when compared with the At-inhh clone.
Recently, a tobacco cDNA encoding a putative cytosolic homolog of
Nt-inh1 was isolated, which also had four Cys residues at
the same positions (S. Greiner and T. Rausch, unpublished data). Thus,
it appears that these Cys residues are essential for Nt-inh1 function, an assumption supported by the observation that treatment with DTT alleviates the inhibitory action of the INH protein (R. Vogel
and T. Rausch, unpublished results). It is noteworthy that the equally
conserved Thr-42, neighboring the first Cys residue, is predicted to be
a phosphorylation site for protein kinase C.
), one of the putative
N-glycosylation sites of At-inhh (Asn-73) is
located in a sequence motif of 20 amino acids (Asp-58 to Thr-77), which
is highly conserved (80% identities) between At-inhh and the C. unshiu EST clone.
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Figure 5.
Expression of the recombinant
His-tagged Nt-inh1-encoded protein in E. coli and purification by Ni-affinity chromatography. The open
reading frame of the Nt-inh1 cDNA was amplified by PCR and cloned into the pQE-30 vector (Qiagen). Expression of the fusion
protein was induced with IPTG. A, Coomassie-stained SDS-PAGE gel. Lane
M, Marker proteins; lane 1, protein from uninduced bacteria; lane 2, protein from IPTG-induced bacteria; lane 3, purified recombinant protein after Ni-affinity chromatography. B, Immunoblot of fractions 1 to 3 from A developed with the polyclonal antiserum directed against
the INH protein (Krausgrill et al., 1996 ).
; Sander et al., 1996). To further confirm the
specificity of invertase inhibition, we analyzed the effect of the
recombinant inhibitor on other plant and fungal invertase preparations.
CWI from C. rubrum cells and VI from tomato fruits were also
strongly inhibited at similar INH concentrations, but no substrate
protection was observed for these invertase preparations (Table
I). In marked contrast to plant CWI and
VI preparations, fungal invertases from S. cerevisiae and
C. utilis were not affected by INH (Table I).
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Figure 6.
In vitro inhibition of tobacco CWI by the
recombinant Nt-inh1 protein. Partially purified CWI (Weil and Rausch,
1994 ) from transformed tobacco cells was preincubated with the
recombinant Nt-inh1 protein in the absence (
) or presence (
) of
20 mm Suc for 1 h in a volume of 200 µL, and its
activity was determined in a subsequent 1-h incubation.
View this table:
Table I.
Effect of the recombinant Nt-inh1 protein on plant
and fungal invertases
Invertase preparations were preincubated with the recombinant Nt-inh1
protein in the absence (+INH-Suc) or presence (+INH+Suc) of 20 mm Suc for 1 h in 200 µL, and their activities were
determined in a subsequent 1-h incubation. Enzyme amounts were adjusted
to give 140 pKat per assay, and 133 pmol of recombinant Nt-inh1 protein was added. Fungal invertases (Inv) were obtained from Sigma.
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Figure 7.
Specific binding of CWI to recombinant Nt-inh1
protein immobilized on a Ni-affinity column. A total cell wall protein
fraction from tobacco cells was passed through a column packed with
recombinant Nt-inh1 protein bound to the Ni-NTA matrix (Qiagen). After
extensive washing the recombinant Nt-inh1 protein with bound CWI was
eluted with EDTA buffer. A, Analysis by SDS-PAGE and Coomassie
staining. Total cell wall protein (lane 1) was passed through Ni-NTA
columns with bound Nt-inh1 protein (lanes 2-5) or without Nt-inh1
protein (lanes 6-9); lanes 2 and 6, flow-through; lanes 3 and 7, EDTA eluate; lanes 4 and 8, washing fractions before EDTA elution; lane M,
marker proteins. B, Immunoblot analysis of the same fractions as in A
developed with an antiserum directed against CWI. The 28-kD splitting
product results from intrinsic CWI lability (Weil and Rausch, 1994 ).
). Using
cDNA probes for CWI (Greiner et al., 1995) and Nt-inh1 we have now compared the expression of CWI and INH at the transcript level in different organs of adult tobacco plants (Fig.
8). In both 7-week-old nonflowering and
15-week-old flowering plants, transcript amounts for CWI and INH
appeared to be independently regulated. Whereas in plants of both ages
CWI transcripts were abundant in roots, this was not the case for INH
transcripts. In internodes, petals, ovaries, and stamens of 15-week-old
plants INH transcript amounts were high, whereas CWI transcripts were abundant only in the stamen. It is noteworthy that in total RNA from
stamen the Nt-inh1 probe apparently hybridized with two
transcripts, one of which had the expected size and the other appeared
to be approximately 200 bases larger. Only in leaves did the transcript amounts for both proteins show some correlation, with the highest amounts in senescent leaves and the lowest amounts in young sink leaves. Immunoblot analysis of leaf protein samples indicated that both
proteins are expressed in mature and senescent leaves (Fig.
9).
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Figure 8.
Transcript amounts for CWI and
Nt-inh1 in different tissues of tobacco plants as
determined by RNA-blot analysis. Total RNA was isolated from different
tissues of 7- and 15-week-old tobacco (cv SNN) plants. For 15-week-old
plants leaves were numbered starting from the oldest (senescent) leaf.
For each tissue 10 µg of total RNA was loaded per lane. Blots were
hybridized with coding region probes for CWI (Krausgrill et al., 1996 )
and Nt-inh1.
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Figure 9.
Expression of CWI and INH protein in tobacco leaf
tissue as determined by immunoblot analysis. Cell wall protein
fractions from source (lane 1) and senescent (lane 2) leaves were
separated by SDS-PAGE and subsequently analyzed by immunoblot. The
polyclonal antisera were directed against CWI and the recombinant
Nt-inh1 protein (affinity-purified antiserum).
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Krausgrill et al.,
1996; this paper). The perfect identity of five different peptide
sequences independently obtained from the plant INH protein leaves no
doubt that Nt-inh1 encodes the previously characterized tobacco INH expressed in A. tumefaciens-transformed cells
(Fig. 2; Weil et al., 1994; Krausgrill et al., 1996). In particular, the predicted N terminus of the mature Nt-inh1 protein is identical to
the N terminus determined for a homogeneous INH preparation of tobacco
cells (Weil et al., 1994). Further confirmatory features are the
absence of glycosylation sites (Weil et al., 1994) and the
cross-reaction of the Nt-inh1 protein with an antiserum directed against INH isolated from tobacco cells (Krausgrill et al., 1996, 1997). The high stability of the tobacco INH protein with respect to
high temperature and low pH may result from intramolecular disulfide
bridges, since the Nt-inh1 sequence predicts four Cys residues, the positions of which are conserved between
Nt-inh1 and At-inhh (Fig. 4). The presence of
structure-stabilizing disulfide bridges is a common feature of other
plant apoplasmic proteins, such as peroxidases and the chitin-binding
domain of several defense-related proteins (Raikhel et al., 1993).
Whether the conserved Cys residues are also involved in the regulation
of INH activity is not known; however, preliminary evidence suggests
that the activity of at least some INHs may be reduced by treatment
with DTT (R. Vogel and T. Rausch, unpublished results). The presence of
a putative phosphorylation site, a conserved Thr residue (Thr-42 in
Nt-inh1), is intriguing, but to our knowledge, regulation of
apoplasmic proteins by phosphorylation has not yet been demonstrated.
), (c) an EST clone from C. unshiu, and (d) a putative cytosolic tobacco homolog of
Nt-inh1 (not shown) reveals a new protein family with
limited sequence conservation except for specific sequence motifs,
including the four Cys residues at almost identical positions (Fig. 4).
However, at present only the Nt-inh1-encoded protein and the
tomato protein have been characterized as INHs, whereas the function(s)
of the other members of this new protein family remain as yet unknown.
),
it is not yet known whether apoplasmic and/or vacuolar isoforms exist
for Nt-inh1. Although the tobacco INH has been shown to
inhibit both VI and CWI isoforms in vitro (Sander et al., 1996), the
existence of VI inhibitors cannot be excluded. Whereas Southern-blot
analysis (Fig. 3) suggested that Nt-inh1 may not have
closely related isoforms, the RNA blot of tobacco stamen (Fig. 8)
revealed transcripts of different sizes. Whether the different
transcripts detected in stamen result from differential splicing,
different transcription initiation, or polyadenylation sites, or,
alternatively, represent bona fide isoforms, is not yet known.
; Sander et al., 1996)
could be faithfully reproduced by the recombinant Nt-inh1 protein
expressed in E. coli (Figs. 5 and 6; Table I). In
particular, the specific effect of strong substrate protection, which
was confined to tobacco CWI and absent for tomato VI (Sander et al., 1996), was also observed for the recombinant Nt-inh1 protein. Tobacco
CWI and tomato fruit VI preparations of identical enzyme activity were
completely inactivated with similar amounts of Nt-inh1 protein. Thus,
if we assume similar specific activities for both invertases, the molar
ratio of inhibitor protein to invertase would be identical. It is
interesting that a CWI preparation from C. rubrum cells was
completely inhibited in the presence of 20 mm Suc, showing
no substrate protection (Table I). For several plant species, different
CWI isoforms have been cloned with sometimes considerable structural
differences (Weber et al., 1995). This discrepancy with tobacco CWI may
indicate that different CWI isoforms are differentially affected.
Alternatively, the substrate protection phenomenon may depend on
specific structural features of the protein-protein interaction in the
homologous system. The observation that two fungal invertases were not
affected by INH indicates that an inhibitory effect on fungal
pathogen invertases is at least unlikely.
),
co-chromatography of CWI and INH on concanavalin A-Sepharose, and
native cathodic PAGE (Krausgrill et al., 1998), appears to be specific
as demonstrated by the results of affinity chromatography of a complex
cell wall protein fraction on a recombinant Nt-inh1 protein-bearing
Ni-NTA resin (Fig. 7). Although it is not yet clear why CWI shows some weak binding to the control Ni-NTA resin, resulting in its delayed elution, the EDTA-induced co-release of Nt-inh1 protein and CWI leaves
little doubt about the specificity of CWI binding to INH. Since the
percentage of the on-column renaturation of the Nt-inh1 protein is not
known, the binding ratio of both proteins is yet to be determined.
) had shown that the concentrations of Suc and
divalent cations, as well as the pH in the apoplasmic space, were all
likely to affect CWI inhibition in vivo. When partially purified INH
protein was added to intact suspension-cultured tobacco cells, an in
vivo inhibition of CWI was observed (Sander et al., 1996), suggesting
that the INH protein could freely move in the cell wall to reach CWI,
which is thought to be ionically bound to the cell wall matrix. Both
the charge of Nt-inh1 protein (pI 8.2) and its size (17 kD) support the
assumption that the inhibitor is mobile in the apoplasmic space
(Gogarten, 1988).
), transcript amounts are very low. However, the high transcript levels of Nt-inh1 in internodes and in different
parts of the flower with low or not detectable CWI transcript levels have been confirmed by immunoblot analysis (Krausgrill et al., 1998),
suggesting that expression of the Nt-inh1-encoded protein may indeed be up-regulated independently from CWI. This phenomenon could be explained in different ways: (a) INH may inhibit other CWI
isoforms going undetected with the probe/antiserum used in this study;
(b) because the Nt-inh1-encoded protein may interact with
CWI and VI, INH may be required in these tissues for regulation of VI;
or (c) INH may have additional, as yet unknown functions.
). Transgenic tobacco plants with
constitutive Nt-inh1 overexpression have been raised to test
this hypothesis.
). These data indicate that the nutritional
status (feast versus famine [Koch, 1996]) of the cells affects CWI
inhibition by INH.
| |
FOOTNOTES |
|---|
Received October 6, 1997;
accepted November 7, 1997.
| |
ABBREVIATIONS |
|---|
Abbreviations:
CWI, cell wall invertase(s).
EST, expressed
sequence tag.
INH, invertase inhibitor.
IPTG, isopropylthio-
-galactoside.
Ni-NTA, Ni-nitrilotriacetic acid.
VI, vacuolar invertase(s).
| |
ACKNOWLEDGMENT |
|---|
The antiserum against CWI was a gift from Arnd Sturm.
| |
LITERATURE CITED |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Bracho GE,
Whitaker JR
(1990)
Purification and partial characterization of potato (Solanum tuberosum) invertase and its endogenous proteinaceous inhibitor.
Plant Physiol
92:
386-394
Cheng WH, Taliercio EW, Chourey PS (1996) The miniature1 seed locus of maize encodes a cell wall invertase required for normal development of endosperm and maternal cells in pedicel. Plant Cell 8: 971-983 [Abstract]
Delmer DP, Amor Y (1995) Cellulose biosynthesis. Plant Cell 7: 987-1000 [CrossRef][Web of Science][Medline]
Ehness R, Roitsch T (1997) Co-ordinated induction of mRNAs for extracellular invertase and a glucose transporter in Chenopodium rubrum by cytokinins. Plant J 11: 539-548 [CrossRef][Web of Science][Medline]
Eschrich W (1989) Phloem unloading of photoassimilates. In DA Baker, JA Milburn, eds, Transport of Photoassimilates. Longman Scientific & Technical, New York, pp 206-263
Frommer WB,
Sonnewald U
(1995)
Molecular analysis of carbon partitioning in solanaceous species.
J Exp Bot
46:
587-607
Gogarten JP (1988) Physical properties of the cell wall of photoautotrophic suspension cells from Chenopodium rubrum L. Planta 174: 333-339
Greiner S, Weil M, Krausgrill S, Rausch T (1995) A tobacco cDNA coding for cell-wall invertase. Plant Physiol 108: 825-826 [CrossRef][Web of Science][Medline]
Herbers K, Meuwly P, Frommer WB, Metraux JP, Sonnewald U (1996) Systemic acquired resistance mediated by the ectopic expression of invertase: possible hexose sensing in the secretory pathway. Plant Cell 8: 793-803 [Abstract]
Jang JC, Leon P, Zhou L, Sheen J (1997) Hexokinase as a sugar sensor in higher plants. Plant Cell 9: 5-19 [Abstract]
Jaynes TA,
Nelson OE
(1971)
An invertase inactivator in maize endosperm and factors affecting inactivation.
Plant Physiol
47:
629-634
Koch KE (1996) Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 509-540 [CrossRef][Web of Science]
Krausgrill S, Greiner S, Köster U, Vogel R, Rausch T (1998) In transformed tobacco cells the apoplasmic invertase inhibitor operates as a regulatory switch of cell wall invertase. Plant J (in press)
Krausgrill S, Sander A, Greiner S, Weil M, Rausch T (1996) Regulation of cell wall invertase by a proteinaceous inhibitor. J Exp Bot 47: 1193-1198
Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16-20 [CrossRef][Web of Science][Medline]
Löw R, Rausch T (1996) Detection of nucleic acids with biotinylated PCR-amplified probes. In T Meier, F Fahrenholz, eds, A Laboratory Guide to Biotin-Labelling in Biomolecule Analysis. Birkhäuser Verlag, Basel, Switzerland, pp 201-213
Miller ME,
Chourey PS
(1992)
The maize invertase-deficient miniature-1 seed mutation is associated with aberrant pedicel and endosperm development.
Plant Cell
4:
297-305
Murray MG,
Thompson WF
(1980)
Rapid isolation of high molecular weight plant DNA.
Nucleic Acids Res
8:
4321-4325
Ovalle R, Keyes AC, Ewing EE, Quimby FW (1995) Purification and characterization of the acid-stable proteinaceous inhibitor of potato tuber invertase by nonideal size exclusion chromatography. J Plant Physiol 147: 334-340
Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 346-356 [CrossRef][Web of Science][Medline]
Pressey R
(1968)
Invertase inhibitors from red beet, sugar beet, and sweet potato roots.
Plant Physiol
43:
1430-1434
Pressey R (1994) Invertase inhibitor in tomato fruit. Phytochemistry 36: 543-546 [CrossRef]
Raikhel NV, Lee HI, Broekaert WF (1993) Structure and functions of chitin-binding proteins. Annu Rev Plant Physiol Plant Mol Biol 44: 591-615 [CrossRef][Web of Science]
Roitsch T, Bittner M, Godt DE (1995) Induction of apoplastic invertase of Chenopodium rubrum by d-glucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation. Plant Physiol 108: 285-294 [Abstract]
RuanYL, Patrick JW (1995) The celllular pathway of postphloem sugar transport in developing tomato fruit. Planta 196: 434-444 [Web of Science]
Sander A, Krausgrill S, Greiner S, Weil M, Rausch T (1996) Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors. FEBS Lett 385: 171-175 [CrossRef][Web of Science][Medline]
Sauer N, Stadler R (1993) A sink-specific H+/monosaccharide cotransporter from Nicotiana tabacum: cloning and heterologous expression in baker's yeast. Plant J 4: 601-610 [CrossRef][Web of Science][Medline]
Schwimmer S,
Makower RU,
Romem ES
(1961)
Invertase and invertase inhibitor in potato.
Plant Physiol
36:
313-316
Stitt M, Sonnewald U (1995) Regulation of metabolism in transgenic plants. Annu Rev Plant Physiol Plant Mol Biol 46: 341-368 [CrossRef][Web of Science]
Sturm A,
Chrispeels MJ
(1990)
cDNA cloning of carrot extracellular -fructosidase and its expression in response to wounding and bacterial infection.
Plant Cell
2:
1107-1119
Sturm A,
Sebkova V,
Lorenz K,
Hardegger M,
Lienhard S,
Unger C
(1995)
Development- and organ-specific expression of the genes for sucrose synthase and three isoenzymes of acid -fructofuranosidase in carrot.
Planta
195:
601-610
Towbin H,
Staehelin T,
Gordon J
(1979)
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354
Truernit E, Schmid J, Epple P, Illig J, Sauer N (1996) The sink-specific and stress-regulated Arabidopsis STP4 gene: enhanced expression of a gene encoding a monosaccharide transporter by wounding, elicitors, and pathogen challenge. Plant Cell 8: 2169-2182 [Abstract]
Von Heijne G
(1986)
A new method for predicting signal sequence cleavage sites.
Nucleic Acids Res
14:
4683-4690
Weber H, Borisjuk L, Heim U, Buchner P, Wobus U (1995) Seed coat-associated invertases of fava bean control both unloading and storage functions: Cloning of cDNAs and cell type-specific expression. Plant Cell 7: 1835-1846 [Abstract]
Weil M, Krausgrill S, Schuster A, Rausch T (1994) A 17 kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase. Planta 193: 438-445 [Web of Science][Medline]
Weil M,
Rausch T
(1990)
Cell wall invertase in tobacco crown gall cells: enzyme properties and regulation by auxin.
Plant Physiol
94:
1575-1578
Weil M, Rausch T (1994) Acid invertase in Nicotiana tabacum crown-gall cells: molecular properties of the cell-wall isoform. Planta 193: 430-437
Zhang L, Cohn NS, Mitchell JP (1996) Induction of a pea cell-wall invertase gene by wounding and its localized expression in the phloem. Plant Physiol 112: 1111-1117 [Abstract]
Zrenner R (1993) Klonierung und funktionelle Analyse von Genen kodierend für am Saccharosestoffwechsel der Kartoffel beteiligte Proteine. PhD thesis. Freie Universität Berlin, Berlin, Germany
Zrenner R, Salanoubat M, Willmitzer L, Sonnnewald U (1995) Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant J 7: 97-107 [CrossRef][Web of Science][Medline]
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