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Plant Physiol. (1998) 118: 1307-1316
Expression Analysis of a Ripening-Specific, Auxin-Repressed
Endo-1,4-
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A cDNA (Cel1) encoding
an endo-1,4-
-glucanase (EGase) was isolated from ripe fruit of
strawberry (Fragaria × ananassa).
The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one
potential N-glycosylation site. Southern- blot analysis
of genomic DNA from F. × ananassa, an
octoploid species, and that from the diploid species Fragaria
vesca indicated that the Cel1 gene is a member
of a divergent multigene family. In fruit, Cel1 mRNA was
first detected at the white stage of development, and at the onset of
ripening, coincident with anthocyanin accumulation, Cel1
mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent.
Antibodies raised to Cel1 protein detected a protein of 62 kD only in
ripening fruit. Upon deachenation of young white fruit to remove the
source of endogenous auxins, ripening, as visualized by anthocyanin
accumulation, and Cel1 mRNA accumulation were both
accelerated. Conversely, auxin treatment of white fruit repressed
accumulation of both Cel1 mRNA and ripening. These
results indicate that strawberry Cel1 is a
ripening-specific and auxin-repressed EGase, which is regulated during
ripening by a decline in auxin levels originating from the achenes.
Fruit ripening is a complex developmental program in which
senescing tissues undergo programmed changes in firmness, texture, coloration, flavor, and susceptibility to microbial infection. Changes
in firmness and texture are largely attributed to alterations in the
composition and structure of cell wall polysaccharides. Because these
modifications influence the postharvest properties (i.e. storage time
and expense, handling damage, and desirability to the consumer) of
important food crops and, consequently, are of great commercial
importance, research in recent years has focused on identifying enzyme
activities that are rate limiting in the promotion of fruit
deterioration. In the climacteric species, which are characterized by
the autocatalytic production of the ripening hormone ethylene and a
ripening-related transient burst in CO2
evolution, the antisense suppression of ACC synthase (Oeller et al.,
1991 In nonclimacteric species such as strawberry (Fragaria × ananassa), much less is known about the ripening process.
Because these plants lack a respiratory climacteric and because
ethylene appears to play a minimal, if any, role in fruit ripening,
there is growing interest in identifying the factor(s) that mediates ripening. Strawberry fruit (actually an enlarged receptacle rather than
a true fruit) exhibit a low level of ethylene production, which is
rather constant during ripening (Knee et al., 1977 Efforts to reveal the molecular basis of changes in firmness, which are
a major contributing factor to fruit quality, have focused on cell
wall-associated enzymes, which are believed to mediate and/or
contribute to cell wall breakdown. The most studied of these
activities, endopolygalacturonase, is absent or below the limit of
detection in ripening strawberry fruit (Neal, 1965 Although largely correlative, there is considerable evidence for the
importance of EGases in a wide variety of physiological processes
involving changes in cell wall architecture, which range from cell wall
expansion to disassembly (for review, see Brummell et al., 1994 Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
) and ACC oxidase (Picton et al., 1993) in tomato has provided
fruit in which ripening and softening are retarded and can be
controlled by the application of ethylene. Similar approaches have been
taken in efforts to diminish the activities of cell wall-associated
hydrolases (Sheehy et al., 1988; Smith et al., 1988), which may play a
central role in fruit cell wall breakdown during ripening (Brady,
1987).
), and there is no
observable stimulation of ripening upon application of exogenous
ethylene (Iwata et al., 1969). Although there is no evidence for a
ripening-related role for ethylene, strawberry fruit ripening has been
shown to be negatively regulated by auxins that originate in the
receptacle-borne achenes (Given et al., 1988b; Manning, 1994). As auxin
levels decline, fruit exhibit a characteristic ripening profile, one of
the major hallmarks of which is rapid deterioration once fruit achieve
the red-ripe stage. In general, strawberry fruit ripening is typified
by the induction of enzyme markers for anthocyanin pigment biosynthesis (e.g. Phe ammonia-lyase), a concomitant decrease in chlorophyll and
increase in anthocyanin pigments, and a progressive decrease in tissue
firmness (Woodward, 1972; Given et al., 1988a).
; Barnes and
Patchett, 1976; Huber, 1984). Although strawberry
fruit is a rich source of pectin, this observation is consistent with cell wall studies that have shown that total extractable polyuronides remain constant as a proportion of cell wall material during ripening and do not show detectable depolymerization (Huber, 1984). In contrast
to these findings, however, the hemicellulosic fraction of cell walls
prepared from ripening fruit demonstrates a progressive shift from
high- to low-Mr polymers (Huber, 1984). Whereas
there is no discernible change in the neutral sugar composition of
hemicelluloses isolated from the small-sized green to red-ripe stages,
the average net Mr change is quite dramatic,
suggestive of an active, developmentally regulated endohydrolyase. It
is interesting that this observed hemicellulose depolymerization
correlates well with a soluble CMCase activity measured in extracts
prepared from ripening strawberry fruit (Barnes and Patchett, 1976). In
ripening fruit of avocado (Hatfield and Nevins, 1986) and pepper
(Harpster et al., 1997), CMCase activity is attributed to an EGase
(EC 3.2.1.4).
). For
example, in abscission-zone formation the infusion of antiserum raised
against an abscission-zone-related EGase into explants, which had been
induced to abscise by ethylene, was observed to inhibit cell separation
(Sexton et al., 1980). Furthermore, the induction of EGase gene
expression in fruit of tomato (Lashbrook et al., 1994), avocado
(Christoffersen et al., 1984), and pepper (Ferrarese et al., 1995;
Harpster et al., 1997) correlates well with the onset and development
of ripening. Recently, a partial cDNA showing homology to EGases was
isolated from strawberry fruit and shown to be expressed in a
ripening-related manner (Manning, 1998). As a first step toward
determining whether the in vivo suppression of EGase gene expression is
a viable strategy for enhancing firmness in harvested strawberry fruit,
we describe the cloning of a full-length strawberry EGase cDNA
(Cel1), an analysis of the hormonal and developmental
regulation of Cel1 gene expression, and the identification
and quantitation of Cel1 protein.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
2°C.
80°C. Fruit were staged by size and color. Color readings were
conducted with a colorimeter (model CR-300, Minolta, Ramsey, NJ) and
are described by the a* value on the Commission Internationale de
l'Eclairnge L*a*b* scale, which is a measure of green
(negative) to red (positive) reflectance of the visible spectrum.
Although there was some variability between fruit, the average times
taken for fruit to attain a specific stage of development in the
ripening process and their color readings (means of at least four
readings ± SD) were as follows: small green (14 dpa,
a* = 14.4 ± 0.4), large green (20 dpa, a* = 12.8 ± 0.5), small white (28 dpa, a* = 11.3 ± 1.5), large white (35 dpa, a* = 9.4 ± 1.1), turning (40 dpa, a* = 1.7 ± 8.1),
red ripe (45 dpa, a* = 24.8 ± 1.2), and overripe (>55 dpa, a* = 21.5 ± 2.9).
cDNA Cloning
The isolation of EGase cDNAs was conducted by screening a Uni-Zap XR cDNA library (Stratagene) constructed from red fruit poly(A+) mRNA. Hybridization conditions were empirically determined by probing replicate northern blots of red fruit total RNA over a range of temperatures with end-labeled ([
-32P]ATP, >5000 Ci
mmol
1) degenerate oligonucleotides
corresponding to the conserved amino acid domain CWERPEDM (see Fig.
1B; for sequences, see Harpster et al.,
1997). Hybridization at 55°C in 7% (w/v) SDS, 0.25 M
sodium phosphate (pH 7.4), 1 mM EDTA, and 1% (w/v) BSA
(type V) provided the recognition of a single mRNA species of the
appropriate size for EGase (approximately 1.8 kb). Using these same
conditions library lifts were then hybridized and washed in 2× SSC
(1× SSC is 0.15 M NaCl and 15 mM sodium
citrate, pH 7.0) and 0.1% (w/v) SDS at 55°C. After the purification
of phage from hybridizing plaques and the in vivo excision of cloned
inserts into phagemids, double-stranded DNA minipreps were partially
characterized by dideoxy sequencing (Sanger et al., 1977) and
restriction endonuclease mapping. The two longest cDNA clones were
completely sequenced on both strands.
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Phylogenetic Analysis
Mature EGase protein sequences (i.e. after removal of signal sequences) were aligned using Clustal V (Higgins et al., 1992), followed by analysis using PAUP (Swofford, 1993) with a bacterial cellulase (accession no. X60545) as the outgroup. A heuristic search
was employed using the random stepwise addition of taxa with 100 replicates and global (tree bisection and reconnection) branch
swapping.
Southern-Blot Analysis
Strawberry genomic DNA was isolated using a small-scale extraction procedure that uses urea as a denaturant (Greene et al., 1994).
Contaminating pectins were removed by selective precipitation with
2-butoxyethanol (Sigma). DNA digestion, gel and electrophoresis conditions, and probe hybridization conditions have been described elsewhere (Harpster et al., 1997). High-stringency blot washes were
done at 65°C in 0.1× SSC and 0.1% (w/v) SDS.
RNA Isolation and Expression Analysis
Total RNA was isolated from different tissues of strawberry plants using a hot borate/phenol method (Wan and Wilkins, 1994) with several
modifications. Strawberry tissue (1-2 g) was first ground to a fine
powder in liquid nitrogen and then transferred to a 15-mL polypropylene
tube containing 5 mL of borate extraction buffer (0.2 M
borax, 30 mM EGTA, 1% [w/v] sodium deoxycholate, 1%
[w/v] SDS, and 10 mM DTT) adjusted to 85°C. The sample
was vortexed for 10 s, after which an equal volume of
phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) was added and the
sample vortexed for an additional 30 s. The sample was then
transferred to a 15-mL Corex tube and centrifuged at 10,000g
for 10 min at 4°C. After removal of the aqueous layer and
reextraction with an equal volume of chloroform:isoamyl alcohol (24:1,
v/v), the sample was centrifuged at 1,500g for 5 min and the
aqueous layer transferred to a Corex tube containing an equal volume of
4 M lithium acetate. The sample was incubated at 4°C
overnight, centrifuged at 10,000g for 20 min, and the
supernatant discarded. To remove pigments, the pellet was resuspended
in 0.5 mL of cold 2 M lithium acetate, transferred to a
microcentrifuge tube, and then repeatedly pelleted (16,000g for 5 min at 4°C) and resuspended in 2 M lithium acetate
until the supernatant was clear of pigmentation (twice for fruit RNA and three to four times for leaf RNA). After the last precipitation, the pellet was resuspended in 0.3 mL of water with vigorous vortexing, adjusted to 0.2 M potassium acetate (pH 5.5), and vortexed
for 20 to 30 s with an equal volume of phenol:chloroform:isoamyl
alcohol (25:24:1, v/v/v). Finally, the sample was centrifuged (5 min at 4°C), the aqueous layer was transferred to a microcentrifuge
tube containing 2.5 volumes of ethanol, and the RNA was precipitated and resuspended in RNase-free water. Based on
A260 readings, yields of total RNA were 600 to 1200 µg g1 fresh weight from leaf tissue
and 40 to 100 µg g1 fresh weight from fruit
tissue. Northern-blot analysis was conducted as described elsewhere
(Harpster et al., 1997), and high-stringency washes of blots were
performed as described for Southern blots. To ensure that equal amounts
of RNA per lane were loaded for each northern blot, parallel blots were
stained with ethidium bromide and individual lanes evaluated for
comparable levels of fluorescence upon exposure to a short-wave UV
light source (data not shown).
Hormone Treatment
Deachenation was conducted by removing the achenes from longitudinal halves of green/small-white fruit (on the vine) with needle-nose forceps. For hormone treatment, NAA was applied to longitudinal halves of similarly staged but otherwise intact fruit (possessing achenes) at a concentration of 0.2 mM in a lanolin paste containing 1% (v/v) DMSO. Seven days after deachenation or auxin treatment, tissue encompassing the zone separating treated from untreated halves (i.e. deachenated/achenated and +NAA/NAA) was
discarded (slice of approximately 5 mm in thickness) and the remaining
halves were wiped clean of the lanolin, frozen separately, and stored
at 80°C. Color readings of the individual fruit halves on the
L*a*b* scale were recorded as described above.
Protein Extraction and CMCase Activity Measurements
Soluble-protein extracts used for both CMCase activity measurements and analysis by SDS-PAGE were prepared from strawberry tissues by first grinding frozen samples to a fine powder in liquid nitrogen. The powders were then ground with a mortar and pestle to a thick slurry by the addition of cold 100% acetone, which was gravity filtered using Whatman no. 1 filter paper, and extensively washed with acetone (50 mL g1 fresh weight of tissue) until
the filtrates were clear of pigment. After air drying the retentates to
a powder, samples were frozen in liquid nitrogen and stored at 80°C
until further use, or processed immediately. To extract soluble
proteins, acetone powders were first ground in liquid nitrogen and then
ground further in extraction buffer (0.1 M sodium phosphate
[pH 6.0], 9.2 mM borax, 13 mM boric acid, 5 mM EDTA, 5 mM DTT, 0.15 M NaCl, and
0.5% Triton-X) containing a cocktail of proteinase inhibitors (see
Harpster et al., 1997). The resultant slurry was centrifuged at
10,000g (4°C) for 15 min to remove insoluble material and
the supernatant assayed directly for relative CMCase activity by
viscometry (Harpster et al., 1997). For preparation of samples for
electrophoresis, the protein concentration of supernatants was
determined using the dye-binding method of Bradford (1976), after which
the desired amount of protein was adjusted to 80% (v/v) acetone and
incubated on ice for a minimum of 30 min. Samples were then centrifuged
(16,000g for 10 min at 4°C) and the protein pellets dried
and resuspended in a sample loading buffer (20 mM Tris-Cl
[pH 6.8], 3% [w/v] SDS, 10% [v/v] glycerol, and 0.01% [w/v]
bromophenol blue) by boiling for 10 min. To prevent band smearing
during electrophoresis, residual insoluble material was removed from
samples by centrifugation at 16,000g for 10 min before
loading. Molecular-mass markers were purchased from BRL.
Antibody Preparation and Western-Blot Analysis
Polyclonal antiserum was raised against strawberry EGase by popliteal lymph node injections of New Zealand White rabbits with a protein A/Cel1 fusion protein. The fusion consisted of a 1.53-kb NarI-HincII fragment of the Cel1 cDNA containing the entire ORF (except for a 189-bp deletion at the 5
end
deleting 63 amino acids at the amino terminus of the Cel1 protein) and
224 bp of the 3 untranslated sequence, which was translationally fused to a protein A fusion-protein vector (Harpster et al., 1997). The
resultant construct produces a fusion protein that is localized in
inclusion bodies and inefficiently binds IgG-Sepharose (Pharmacia). Therefore, partial purification of the fusion protein for antibody production was performed by preparative SDS-PAGE, as described previously (Harpster et al., 1997).
and provided a complex of several
cross-reacting polypeptide species, despite the observation that
parallel blots treated with preimmune serum demonstrated an absence of
background hybridization (data not shown). Previous work using
antiserum raised against a ripening-related pepper EGase suggests that
the majority of these cross-reacting polypeptides are cell
wall-associated proteins with shared antigenic epitopes (Harpster et
al., 1997). To enrich for antibodies specific to strawberry Cel1,
contaminating antibodies were selectively removed by passing antiserum
over an affinity column containing covalently coupled protein isolated
from small, green fruit tissue, which shows an absence of
Cel1 expression. This was accomplished by first desalting a
soluble-protein extract from small, green fruit on a column (PD-10,
Pharmacia) and then covalently coupling the protein to a HiTrap column
according to the manufacturer's instructions (Pharmacia). Finally,
antiserum was loaded onto the column, which was then sealed and
incubated overnight at 10°C. Unbound antiserum was collected from the
column according to the manufacturer's instructions. When used in
subsequent western-blot analysis of fruit protein extracts, the
antiserum showed the selective enrichment of antibodies recognizing a
single 62-kD polypeptide species.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Isolation of EGase cDNAs from Strawberry and Characterization of Encoded Protein
Using degenerate oligonucleotides corresponding to a conserved amino acid domain shared by plant EGases (see ``Materials and Methods''), 55 putative EGase cDNAs were identified in the screening of a phage cDNA library constructed from red fruit poly(A+) mRNA. Because this library was not amplified and 80,000 plaques were screened, we estimate that strawberry EGase expression accounts for 0.07% of poly(A+) mRNA isolated from red fruit. Plasmid cross-hybridization experiments using all cDNA clones and dideoxy sequencing analysis at the 5 and 3
ends of a subset of these clones demonstrated that they were all
derived from the expression of a single gene, designated strawberry
Cel1. Analysis at the 5 end of the longest cDNA (see Fig.
1A for a schematic map) showed that it contained a single ORF encoding
496 amino acids and 14 nucleotides of sequence upstream of the
presumptive ATG initiation codon. Analysis of the 3 ends of all of the
cDNAs identified an untranslated region of approximately 260 nucleotides and a minimum of three alternative, closely spaced polyadenylation sites.
, and the subsequent mature protein
sequences were used to construct a phylogenetic tree (Fig. 1C). Based
on their phylogenetic relatedness, there are three major branches of
plant EGases, with one branch containing EGases possessing a
membrane-spanning domain such as Arabidopsis Cel3 and Cel4, and tomato
Cel3 (Brummell et al., 1997b), another branch containing Arabidopsis
Cel2 alone, and the third branch containing all of the remaining
sequences. Strawberry Cel1 is within this last major branch and, with
pepper Cel3, tomato Cel2, and Arabidopsis Cel1 (all show 80%-82%
amino acid identity with strawberry Cel1), forms a subgroup.
Genomic Southern-Blot Analysis of Cel1
F. × ananassa Duch. cv Chandler is a highly cultivated octoploid variety, which is an interspecific hybrid of two diploid species, Fragaria chiloensis and Fragaria virginiana (Hancock et al., 1996). Consequently, because of the
polyploid nature of the F. × ananassa genome,
genomic Southern-blot analysis of Cel1 yields a complex
banding pattern that does not provide an accurate assessment of the
gene copy number (Fig. 2). To estimate
the number of genes closely related to Cel1, genomic DNA was
analyzed from Fragaria vesca, a diploid line. Based on the
hybridization profile seen after washing the blot at high stringency,
and the observation that all of the bands revealed in the digestion of
F. vesca appeared to be a subset of the bands provided by
the digestion of F. × ananassa, we estimate that
strawberry Cel1 is probably encoded by a single gene per
diploid genome. Although the hybridization patterns provided by digests
with BclI, EcoRI, HindIII, and
NcoI showed two strongly hybridizing bands, their sizes are
more consistent with a single gene and can be accounted for by invoking
the presence of these sites within introns. The presence of faint bands
in both blots after washing at high stringency indicates the presence of related sequences that collectively constitute a multigene family
for EGase in strawberry.
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Expression of Cel1 in Strawberry Tissues
Northern-blot analysis of total RNA isolated from developing fruit revealed Cel1 expression as a 1.8-kb transcript, which was first detectable in small-sized white fruit (Fig. 3A). Thereafter, Cel1 transcript levels increased as fruit enlarged, and attained maximum size at the red-ripe stage, with a gradual increase in Cel1 mRNA in white fruit being followed by a larger increase coincident with the development of red color. In red-ripe fruit steady-state Cel1 mRNA levels were at their highest, and we observed no significant change in expression levels during ripening, even in fruit that had evident signs of deterioration (i.e. tissue rotting and liquefaction; see Fig. 3A, lane over ripe 3). Although the pattern of Cel1 mRNA accumulation generally showed an abrupt increase in steady-state levels between large-sized white and red-ripe fruit, we occasionally observed higher levels in large-sized white fruit and a more gradual increase in transcript accumulation (data not shown). This suggests that the increase in Cel1 mRNA abundance and the development of red coloration are not completely coupled. Autoradiograph exposure of the blot shown here in excess of 2 weeks revealed an apparent absence of Cel1 mRNA expression in developing green fruit (data not shown).
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Auxin Effects on Cel1 Expression
As with other nonclimacteric fruit, ethylene production levels in ripening strawberry were exceptionally low and have been reported to decrease on a per unit fresh weight basis when harvested fruit turn from white to red (Knee et al., 1977). To investigate the role of auxin
in Cel1 expression, three auxins were tested for their
capacity to inhibit ripening: IAA and the two auxin analogs 2,4-D and
NAA. Based on a demonstrable reduction in visible anthocyanin
accumulation relative to control fruit, NAA was almost completely
effective in retarding ripening when applied to small-sized white fruit
on the vine, 2,4-D was moderately effective, and IAA performed poorly
(data not shown). Presumably, the higher percentage of fruit showing a
substantial decrease in anthocyanin accumulation after NAA treatment is
a consequence of enhanced hormone uptake and/or stability relative to
2,4-D and IAA.
Cel1 Protein and EGase Activity in Mature Strawberry Plants
We have isolated and characterized full-length cDNAs encoding a
ripening-related EGase (Cel1) from strawberry, which correspond to a
partial-length EGase cDNA recently reported by Manning (1998) Received July 2, 1998;
accepted August 25, 1998.
Abbreviations:
CMCase, carboxymethylcellulase.
dpa, days
postanthesis.
EGase, endo-1,4- We thank Dr. Rita Teutonico for her efforts in the construction
of the cDNA library, Malini Nag for assistance with the isolation and
characterization of cDNA clones, Dr. Paul Oeller for careful review of
the manuscript, and Jay Maddox and Vincenzo Pirozzi of the greenhouse
staff for maintenance of the plants.
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Reduction of polygalacturonase activity in tomato fruit by antisense RNA.
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PAUP: Phylogenetic Analysis Using Parsimony, Version 3.1.1.
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The expression of cellulase gene family members during avocado fruit abscission and ripening.
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View larger version (51K):
[in a new window]
Figure 4.
Flesh (receptacle) color readings and
northern-blot analysis of Cel1 expression in
longitudinal halves of whole fruit after deachenation (A) or treatment
with auxin (B). Whole fruit on the vine were either deachenated on one
half and the other half left untreated, or one half of otherwise intact
fruit was left untreated and the other half coated with lanolin alone
or lanolin containing 0.2 mM NAA. After 7 d, two fruit
per treatment were harvested, dissected longitudinally, and untreated
and treated halves analyzed separately. Color readings (a* on the
Commission Internationale de l'Eclairnge L*a*b* scale) are
given on a green (negative) to red (positive) scale, and each value is
the mean of two readings ± SD. For northern-blot
analysis, each lane contained 10 µg of total RNA, and the probe used
was the same as described in the legend of Figure 3. Autoradiograph
exposure time was 22 h.
; Given et al., 1988b), these results
provide corollary evidence that Cel1 expression is inhibited
by auxins. Clearly, the removal of endogenous auxin through
deachenation facilitates the premature initiation of the ripening
program, which is accompanied by the accumulation of Cel1
mRNA. More work is required, however, to determine whether auxin
control of Cel1 expression is at the transcriptional or the
posttranscriptional level.
; Kanellis and Kalaitzis, 1992). In an extreme
case, tomato Cel3 encodes a predicted 68.5-kD polypeptide, which is detected as 88- and 93-kD polypeptide species upon SDS-PAGE (Brummell et al., 1997b).
View larger version (86K):
[in a new window]
Figure 5.
Western blot of soluble-protein extracts prepared
from different tissues of flowering strawberry plants probed with a
1:5000 dilution of antiserum raised to a Cel1 fusion protein.
Each lane contained 100 µg of acetone-precipitated protein
electrophoresed on a 10% SDS-PAGE gel.
View larger version (25K):
[in a new window]
Figure 6.
Histogram of relative CMCase activities measured
in soluble-protein extracts prepared from various strawberry tissues.
Relative specific activities are an average of three independent
measurements (for a definition of activity units as provided here, see
Harpster et al., 1997 ). SD values were less than 10% of
the values shown.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
. As
observed for cell wall-localized EGases from a wide variety of plant
species, the amino-terminal sequence of the predicted protein (32 amino
acids) is predominantly hydrophobic and shows homology with eukaryotic
signal sequences (von Heijne, 1986). Although the sequence alignment of
all EGases to date reveals several conserved amino acid domains, it is
becoming clear that amino acid sequence homology and phylogenetic
similarity do not necessarily predict mRNA expression pattern. This is
illustrated by the observation that the closest related EGases to
strawberry Cel1 are tomato Cel2, Arabidopsis
Cel1, and pepper Cel3, three genes that exhibit
quite different patterns of expression. Whereas tomato Cel2
mRNA is most similar to strawberry Cel1 in that it accumulates predominantly during fruit ripening (Lashbrook et al.,
1994), Arabidopsis Cel1 mRNA is expressed mainly in
expanding vegetative tissues (Shani et al., 1997) and pepper
Cel3 mRNA is expressed in abscissing leaf-abscission zones
(Ferrarese et al., 1995). Two other EGases with mRNA-accumulation
patterns that are predominantly ripening related, avocado
Cel1 (Christoffersen et al., 1984) and pepper
PCEL1 (Harpster et al., 1997), encode proteins that are less
related and that are found in other branches or subbranches of the
phylogenetic tree (Fig. 1C).
. As shown in previous studies (Given et al.,
1988a; Abeles and Takeda, 1990), the onset of fruit softening in
strawberry begins during the white stage and slightly precedes the
appearance of anthocyanin pigments. Thereafter, fruit progressively
exhibit a loss of firmness, which nears a maximum at the red stage.
Although correlative, these findings raise the possibility that Cel1
activity may contribute to fruit softening. This association between
strawberry Cel1 expression and fruit softening is further
supported by the absence of Cel1 mRNA (Fig. 3B) and Cel1
protein (Fig. 5) in other tissues. In other species in which a
ripening-related EGase has been described, either significant mRNA
levels were also found in abscission zones (e.g. tomato Cel2 [Lashbrook et al., 1994] and avocado Cel1 [Tonutti et
al., 1995]) or low levels were also detected in stems and petioles
(e.g. pepper Cel1 [Harpster et al., 1997]). Because levels
of strawberry Cel1 mRNA are either absent or below the limit
of detection in tissues other than fruit, strawberry Cel1
appears to show the most ripening-specific pattern of mRNA accumulation
for an EGase described to date.
; Milligan and Gasser,
1995; del Campillo and Bennett, 1996; Brummell et al., 1997a, 1997b;
Catala et al., 1997).
), the pea EGL1 gene in epicotyls (Wu et al.,
1996), and the tomato Cel7 gene in hypocotyls (Catala et
al., 1997), and decreases in the expression of the ethylene-regulated, abscission-related EGase genes of bean (BAC1; Tucker et al.,
1988) and tomato (Cel1 and Cel5; del Campillo and
Bennett, 1996). In ripening avocado fruit discs, suppression of
ethylene-induced EGase gene expression by auxin is observed at the
posttranscriptional level (Buse and Laties, 1993). Whereas the
antagonistic control of EGase expression by auxin and ethylene is
observed in many plant species, this relationship may not necessarily
hold true in all cases. In the nonclimacteric plant species, for
instance, attempts to demonstrate ethylene-mediated effects on gene
expression and physiological processes have been largely inconclusive.
; Abeles and Takeda, 1990), and
treatment with ethylene antagonists such as 2,5-norbornadiene,
aminoethoxyvinylglycine, or silver did not affect ripening, as measured
by anthocyanin accumulation (Given et al., 1988a). Furthermore,
exposing strawberry fruit to high concentrations of exogenous ethylene
had no obvious effect on ripening (Iwata et al., 1969). Whereas these
findings question the importance of ethylene to nonclimacteric fruit
ripening, the well-documented yet minor promotion by ethylene of EGase
expression and fruit reddening in nonclimacteric pepper (Ferrarese et
al., 1995; Harpster et al., 1997) suggests that ethylene may play some role in the ripening of nonclimacteric fruit. In most nonclimacteric ripening fruit, however, the potential role of ethylene is generally obscured by technical difficulties in monitoring the physiological consequences of exceptionally low-level endogenous ethylene production, and by the possibility that the low levels of endogenous ethylene production are saturating, in which case the application of exogenous ethylene would be without observable effect. Whatever the precise role
ethylene may have, the main regulation of the ripening process in
strawberry and grape appears to occur through declining levels of auxin
in the fruit (Given et al., 1988b; Manning, 1994; Davies et al., 1997).
).
Achenes produce large amounts of free and conjugated IAA, with peak
levels achieved at 10 to 14 dpa, depending on the cultivar (Dreher and
Pooviah, 1982; Archbold and Dennis, 1984). Removal of achenes from
white fruit mimics the in vivo decline of endogenous auxin levels,
albeit at an accelerated rate, in that there is a rapid accumulation of
anthocyanins and a concomitant loss in chlorophyll content and tissue
firmness, all of which are typical of ripening in strawberry fruit
(Given et al., 1988a, 1988b; Manning, 1994). Because the application of
the synthetic auxin NAA prevents these changes, it is concluded that
strawberry ripening is regulated in part by a decline in the amount of
auxin produced by the achenes (Given et al., 1988b; Manning, 1994).
, the removal of achenes from immature-green strawberry
fruit and the subsequent analysis of in vitro translation products by
two-dimensional PAGE showed the induction of several mRNAs that
increase in abundance in normally ripening fruit. Additional studies
with deachenated fruit have demonstrated the auxin-mediated repression
of Phe ammonia-lyase and total anthocyanin accumulation in ripening
fruit (Given et al., 1988b) and the isolation of an auxin-repressed
cDNA of unknown function (Reddy and Pooviah, 1990). Medina-Escobar et
al. (1997) have characterized a strawberry pectate-lyase gene that
shares identical spatial, temporal, and hormonal patterns of regulation
with those shown here for Cel1. In future experiments transgenic Cel1-suppressed plants will be monitored for
phenotypic effects, and a reduction in the activity of a single enzyme
in a complex developmental program such as ripening will have to be
evaluated with the understanding that the activity of several genes
may contribute to overall fruit firmness.
*
Corresponding author; e-mail harpster{at}dnap.com; fax
1-510-547-2817.
FOOTNOTES
The accession number for the sequence reported in this article is
AF074923.
ABBREVIATIONS
-glucanase.
NAA, naphthalene acetic
acid.
ORF, open reading frame.
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-glucanase expressed at high levels in rapidly expanding tissues.
Plant Mol Biol
33:
87-95
[CrossRef][Web of Science][Medline]-glucanase is localized on Golgi and plasma membranes of higher plants.
Proc Natl Acad Sci USA
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4794-4799
-glucanases.
In
ME Himmel,
JO Baker,
RP Overend,
eds, Enzymatic Conversion of Biomass for Fuels Production. ACS Symposium Series 566.
American Chemical Society, Washington, DC, pp 100-129-D-glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls.
Plant J
12:
417-426
[CrossRef][Web of Science][Medline]-glucanase from pepper (Capsicum annuum L.).
Plant Mol Biol
33:
47-59
[CrossRef][Web of Science][Medline]-glucanase genes exhibit overlapping expression in ripening fruit and abscissing flowers.
Plant Cell
6:
1485-1493
[Abstract]-glucanase.
Plant Cell Physiol
36:
1229-1235
-glucanase (cel1) from Arabidopsis thaliana.
Plant Mol Biol
34:
837-842
[CrossRef][Web of Science][Medline]-glucanase gene induced by auxin in elongating pea epicotyls.
Plant Physiol
110:
163-170
[Abstract]
Copyright Clearance Center: 0032-0889/98/118//10
© 1998 American Society of Plant Physiologists
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-Glucanase Gene in Strawberry
-32P]dCTP, 800 Ci
mmol