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Plant Physiol. (1998) 118: 907-916
Acid-Growth Response and
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured
cells possess an expansin-mediated acid-growth mechanism was examined
by multiple approaches. BY2 cells grew three times faster upon
treatment with fusicoccin, which induces an acidification of the cell
wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold.
Protein extracted from BY2 cell walls possessed the expansin-like
ability to induce extension of isolated walls. In western-blot analysis
of BY2 wall protein, one band of 29 kD was recognized by anti-expansin
antibody. Six different classes of 
-expansin mRNA were identified in
a BY2 cDNA library. Northern-blot analysis indicated moderate to low
abundance of multiple -expansin mRNAs in BY2 cells. From these
results we conclude that BY2 suspension-cultured cells have the
necessary components for expansin-mediated cell wall enlargement.
Plant cells are surrounded by a cell wall composed of cellulose
microfibrils embedded in a complex polysaccharide matrix. The plant
cell wall has substantial mechanical strength, which must either be
overcome or reduced to allow wall extension and cell growth (Cosgrove,
1987 Plant cell suspensions have been favorite subjects for studies of cell
growth and wall biochemistry, in part because their walls are more
homogeneous than those of complex tissues (McNeil et al., 1984 Although plant cell cultures are sensitive to pH, an acid-growth
response has not been demonstrated, and we do not know if expansins
function in this cell type. Published evidence suggests that
cell-suspension cultures do not show acid-growth responses (Nesius and
Fletcher, 1973 Cell Culturing
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Carpita and Gibeaut, 1993). Our laboratory has identified a group
of proteins, named expansins, that enable the growing cell wall to
extend, apparently by weakening noncovalent bonding between the matrix
and cellulose microfibrils (McQueen-Mason et al., 1992; Cosgrove,
1996). Expansins have an acidic pH optimum and are prime candidates for
the agents mediating the "acid-growth" response of plant cell walls
(Hager et al., 1971; Rayle and Cleland, 1992). Recently, a second group
of proteins, previously know as group-1 allergens from grass pollen,
was identified as a second family of expansins (Cosgrove et al., 1997).
These proteins are called 
-expansins to distinguish them from the
original class of expansins, referred to as -expansins. Both -
and -expansins comprise large multigene families (Shcherban et al.,
1995; Cosgrove et al., 1997). Judging from the number of expressed
genes identified to date in the Arabidopsis and rice expressed sequence
tag databases, it appears that -expansins may have assumed
specialized roles for cell wall loosening during the evolutionary
divergence of the grasses (Cosgrove et al., 1997). Despite the growing
multitude of expansin genes being discovered, questions remain about
the generality of expansin-mediated growth. To address this question, we have studied the growth of Bright Yellow 2 (BY2) tobacco
(Nicotiana tabacum L) suspension-cultured cells.
; Carpita
and Gibeaut, 1993). Because cell suspensions are in constant contact
with the medium, exogenous proteins have ready access to the cell
walls. This is an important advantage for testing the ability of
exogenous wall enzymes to modulate plant cell expansion.
; Smith and Krikorian, 1992; Roberts and Haigler, 1994).
In this investigation we examined the possibility that BY2 tobacco cell
cultures possess an expansin-mediated growth mechanism. If this were
the case, we would predict that: (a) BY2 cell growth would be
responsive to fusicoccin-induced wall acidification and to application
of expansins; (b) BY2 cell walls would contain active expansins, and
(c) BY2 cells would express one or more expansin genes. The results of
these tests were found to support the involvement of expansins in BY2
cell enlargement.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
).
Cell-Growth Assays
Cell enlargement was monitored with a video camera attached to an inverted microscope. Cells were attached to coverslips by two methods. For the first method, 15 mL of a 6-d-old culture was centrifuged for 5 min at 800g and 1 mL of liquid was drawn from the cell-liquid interface. For the agarose-embedding technique, the cells were mixed with 1 mL of BY2 medium containing 1.2% agarose (premelted and cooled to 40°C; Sigma no. A-9539) and spread thinly over a sterile coverslip (48 × 65 mm) on a 40°C heat block. The coverslip was placed in a Petri dish and allowed to harden for 30 min, and 1 mL of "conditioned" medium from the centrifuged culture was spread over the top of the agarose, creating a liquid layer 0.32 mm deep. The remaining conditioned medium was transferred to a new tube and stored at 4°C for use later in the assay. Five cell groups, each containing 5 to 15 cells lying in the focal plane, were chosen for microscopic analysis at 64× magnification. After a 24-h recovery period we recorded images of the cells on videotape. Twelve hours later we recorded their images once each hour for 8 h to determine their basal growth rate. The liquid over the agarose was then removed and replaced with 1 mL of conditioned medium plus the appropriate treatment: 1 µL of 1 mM fusicoccin in 50% ethanol or 20 µL of C3 purified cucumber expansins (approximately 1.0 µg/µL in 50 mM sodium acetate, pH 4.5; see below for C3 protein preparation). Controls were given either 1 µL of 50% ethanol or 20 µL of 50 mM sodium acetate, pH 4.5. We recorded images once an hour for an additional 8 h and recorded a final image 20 h after the treatment.Weight Assay
Three 25-mL aliquots from a culture were placed in separate 125-mL Erlenmeyer flasks. Two of the aliquots (the first and the last) were used as controls. To the second aliquot was added 12.5 µL of 1 mM fusicoccin in 50% ethanol (giving a final concentration of 500 nM). An equal amount of ethanol was added to the controls. The three aliquots were grown for 5 h. The contents were transferred to 50-mL tubes, weighed, and centrifuged for 10 min at 800g. After removal of the free culture medium, the packed cells were weighed. Cell density was calculated as a simple weight percentage of the culture.Protein Extraction and Expansin Activity Assay
Wall proteins were extracted as described previously (McQueen-Mason et al., 1992), except that 1 mM DTT was
added to the grind and extraction buffers to maintain expansin
activity. Six-day-old BY2 cells were collected by centrifugation in
50-mL tubes for 10 min at 800g, suspended in grind buffer
(25 mM Hepes, pH 7.0, 2 mM sodium
metabisulfite) to make 100 mL, and homogenized for 30 s in a
chilled Waring blender. The mixture was allowed to settle for 1 min on
ice, homogenized again for 30 s, and centrifuged at
800g for 10 min. The pellet was washed by resuspension in
grinding buffer and centrifugation, then resuspended in twice its
volume of extraction buffer (20 mM Hepes, 2 mM
EDTA, 3 mM sodium meta-bisulfite, and 1 M
NaCl, pH 7.0), and extracted for 30 min with agitation at 4°C. The
mixture was centrifuged to pellet the walls and additional debris,
which were discarded. Some proteins were removed from the supernatant
by centrifugation after addition of 113 g/L ammonium sulfate.
Subsequent centrifugation after addition of another 277 g/L ammonium
sulfate yielded a crude wall protein fraction containing expansin. This
was resuspended in 1 mL of 50 mM sodium acetate, pH 4.5, plus 1 mM DTT and immediately tested for expansin activity by reconstitution assays with heat-inactivated walls from cucumber hypocotyls, as described previously (Wu et al., 1996).
; Keller and Cosgrove, 1995). The
ammonium sulfate precipitate of wall protein was desalted on a
Centricon 30-kD spin column and the proteins were separated by HPLC on
a C3 hydrophobic interactions column (ISCO Synchropak C-3/65 µm,
Lincoln, NE). The relevant fractions were collected and
electrophoresed into a 15% SDS polyacrylamide gel. Prestained markers
(6-60 kD, GIBCO-BRL) were used for molecular mass standards. The gels
were electroblotted onto nitrocellulose membranes, which were probed
with a rabbit polyclonal antibody made to cucumber S1 expansin (Li et
al., 1993).
RNA Extraction, PCR Amplification, and cDNA Library Screening
BY2 cells were collected by centrifugation at 800g for 5 min and placed on ice. Packets of 1.2 g of packed cells were weighed out and frozen in liquid nitrogen. RNA was extracted with TRIzol reagent (GIBCO-BRL) using the manufacturer's protocol. Poly(A+) RNA was isolated using the PolyAtract system (Promega) and used for either northern-blot analysis or reverse-transcription PCR. We used Superscript II (GIBCO-BRL) for reverse transcription and degenerate primers (sense primer, GGHGGNTGGTGYAAYCC; antisense primer, ACCDBNAARCCDGTYTG) to amplify expansin fragments from the tobacco first-strand cDNA. Two fragments were isolated, cloned using TA cloning (Invitrogen, San Diego, CA), and sequenced using standard methods (Sambrook et al., 1989). The larger
fragment was used to screen a BY2 cDNA library obtained from Lee
McIntosh (Michigan State University, East Lansing) (Vanlerberghe and
McIntosh, 1994). Once sequences for the tobacco expansins were
obtained, they were used to predict protein sequences, including signal peptides. The cleavage sites of the signal peptides were predicted using the SignalP program available on the Internet at The Center for
Biological Sequence Analysis, the Technical University of Denmark
(www.cbs.dtu.dk/services/signalp/) (Henrik et al., 1997). Tobacco
sequences were assigned accession nos. AF049350 to AF049355.
-expansin sequences by first using the PileUp program (Genetics Computer Group, Madison, WI) (gap penalty = 3.00, extension
penalty 0.10). The ESEE (Eyeball Sequence Editor, version 2.0) program was then used to create a corresponding alignment of the nucleotide sequences (Cabot and Beckenbach, 1989). We generated a phylogenetic tree from this alignment using the MEGA program with the
neighbor-joining method and Kimura two-parameter distances with the
complete deletion option (Kumar et al., 1993).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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BY2 Cells Grow Faster in Response to Fusicoccin
Figure 1 shows that BY2 cells selected for analysis grew as cell files under our culture conditions. To determine if BY2 cells have an acid-growth response, we measured their growth response to fusicoccin. This fungal toxin activates the H+-ATPase in the plasma membrane, causing strong acidification of the cell walls and thereby inducing rapid cell elongation (Marré, 1979; Kutschera and
Schopfer, 1985). The size of BY2 cell clusters was measured at 1-h
intervals before and after treatment with fusicoccin. Figure
2A shows that BY2 cells enlarged faster,
by 2- to 3-fold, after treatment with fusicoccin.
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), which is mediated via expansins (Cosgrove, 1996),
we infer from these results that BY2 cells possess an endogenous,
expansin-mediated acid-growth mechanism.
BY2 Cells Grow Faster after Application of Expansins
Cell-growth assays were also used to determine the effect of exogenous expansins on cell expansion. Figure 2B shows that BY2 cells increased their growth rate by 2- to 3-fold after treatment with exogenous cucumber expansins. To quantify and compare this effect, we calculated average growth rates for the 8 h before and the 8 h after treatment. The ratios of these two rates are shown in Figure 2C. Expansin increased the growth rate by 337% (n = 34, SE = 86.2%), an effect nearly the same as the growth stimulation by fusicoccin, 339% (n = 14, SE = 28.2%). Whereas the average growth stimulation was the same for fusicoccin and expansin, the SE was much larger for expansins.
BY2 Cell Walls Contain Expansin Proteins
BY2 Cells Express Multiple
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Figure 3.
Fusicoccin- and expansin-growth responses as a
function of initial growth rate. A, Growth response as a function of
pretreatment growth rate. The increase in growth rates for cell groups
(growth rate after treatment divided by growth rate before treatment)
was plotted against the initial (pretreatment) growth rate. The solid
and dotted lines represent least-squares fits for the expansin and
fusicoccin data, respectively. The line for the expansin data is an
exponential decay fit. The dashed line for the fusicoccin data is a
first-order fit. B, The growth rate after treatment with expansins is
plotted against the pretreatment growth rate. The solid diagonal line
(slope = 1) represents where the data would fall if expansin
treatment had no effect. The dashed line is a least-squares fit of the
data and has a slope of 0.145 ± 0.252, and a y
intercept of 2.71%/h. All growth rates were based on change in the
optical cross-sectional area versus time.
-expansin protein (Fig. 5). The anti-expansin antibody
recognized a band with similar molecular mass as cucumber -expansin.
The relatively weak signal found with the BY2 proteins could be caused
by low relative abundance of extractable expansins in the BY2 cell
walls, weak recognition of BY2 expansins by the antibody, or a
combination of these two effects. The western-blot data, together with
the results of the fusicoccin and wall-extension assays, indicate that
BY2 cultures produce their own active -expansins.
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Figure 4.
Results of extension assays using wall protein
extracted from BY2 cells. Heat-inactivated wall specimens from cucumber
hypocotyls were placed in the extensometer under 20 g of tension,
and bathed in 200 µL of 50 mM sodium acetate at pH 4.5. Their initial creep rates were recorded for 45 min, after which time
the buffer for two of the cuvettes was replaced with 200 µL of the
BY2 wall protein extract, which was resuspended in 50 mM
sodium acetate at pH 4.5. Total wall protein concentration was
approximately 170 µg/mL. Initial length of the wall sample was 5 mm.
Extension assays were done on four separate occasions with similar
results.
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Figure 5.
Western blot of partially purified cucumber and
tobacco cell wall proteins probed with antibodies to cucumber
-expansin. Wall proteins from the expansin-containing fractions of
an HPLC C3 hydrophobic interactions column were electrophoresed into a
15% SDS-PAGE gel, blotted, and cross-reacted with a polyclonal
antibody raised against cucumber S1 -expansin (Li et al., 1993).
Lane C, One microgram of cucumber hypocotyl wall protein; lane T, 10 µg of tobacco proteins. Western-blot analysis was done on five
separate occasions with similar results.
-Expansin Genes
-expansins expressed by BY2 cells, we used a
degenerate oligonucleotide primer pair to amplify a fragment of a
tobacco expansin cDNA by reverse-transcription PCR. This produced two
unique fragments that were cloned, sequenced, and confirmed to code for
-expansins. One fragment was 331 bp and the other was 377 bp. The
377-bp fragment was used to probe a cDNA library made from 3-d-old BY2
cells. An initial screening of 60,000 plaques produced 24 positives.
Restriction mapping and partial sequencing demonstrated that these
corresponded to six unique classes of -expansins. Class I (NTEXP1)
contained 11 clones; class II (NTEXP2), 2 clones; class III (NTEXP3), 6 clones; class IV (NTEXP4), 2 clones; class V (NTEXP5), 2 clones; and
class VI (NTEXP6), 1 clone. A full-length or nearly full-length
sequence was obtained by sequencing the largest member of each class.
The cDNAs ranged in size from 1.17 to 1.45 kb. We isolated only partial clones for NTEXP5 and NTEXP6. The largest clone for NTEXP5 appeared to
be missing the starting Met, whereas the largest clone for NTEXP6 was
missing about 60 amino acids at the N terminus, as well as the signal
peptide. The 377-bp PCR fragment is from NTEXP1, whereas the 331-bp PCR
fragment is identical to a portion of NTEXP6.
To our knowledge, acid growth has not been previously reported in
cell cultures. Other investigators have looked at the long-term effect
of pH on cultures and reported results apparently at odds with the
acid-growth hypothesis. For zinnia mesophyll cultures (Roberts and
Haigler, 1994 Received April 9, 1998;
accepted August 17, 1998.
We acknowledge Dr. Simon Gilroy for help with cell imaging, Dr.
Richard Cyr for the BY2 cell lines, Dr. Lee McIntosh (Michigan State
University) for the gift of the BY2 cDNA library, Dr. Mark Guiltinan
for advice on screening the cDNA library, Daniel M. Durachko and Melva
Perich for technical assistance, Jennie Hay for help with
phylogenetics, and Mark Shieh and Tatyana Shcherban for many helpful
suggestions.
Akehurst BC (1970) The botany, genetics and the development of
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An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls.
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[Abstract]
McQueen-Mason S,
Durachko DM,
Cosgrove DJ
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Two endogenous proteins that induce cell wall expansion in plants.
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Michael AJ
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A cDNA from pea petals with sequence similarity to pollen allergen, cytokinin-induced and genetic tumour-specific genes: identification of a new family of related sequences.
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[Abstract]
-expansin cDNAs encode
proteins containing a signal sequence of approximately 23 amino acids
and a 233-amino acid conserved region ending with a stop codon. All of
the cloned sequences contain the conserved domains described previously
for -expansins (Shcherban et al., 1995; Cosgrove, 1996).
-Expansins contain eight Cys residues, which are conserved in
NTEXP1-NTEXP5; we do not have a sequence for this portion of NTEXP6.
Expansins have four conserved Trp residues near the C terminus. These
are shown at positions 190, 197, 201, and 234 in Figure 6. They are
similarly spaced to Trp residues in the cellulose-binding domain of
cellulases (Gilkes et al., 1991; Poole et al., 1993; Shcherban et al.,
1995). These Trp residues are conserved in all of the tobacco expansins
except for NTEXP3, in which the third Trp is substituted with Phe.
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Figure 6.
Amino acid alignment of previously published expansin sequences
with predicted sequences from the tobacco cDNAs. Areas with dots are
identical to the consensus sequence, and dashes are used to indicate
gaps in the alignment. The order of sequences matches the order on the
phylogenetic tree in Figure 7. The large letters along the right margin
indicate the -expansin subfamily shown in Figure 7. Changes in amino
acids that appear to be conserved among subfamilies are boxed. The
sequence for NTEXP6 is missing at least the first 60 amino acids.
Dashes were inserted in this area to align it with the other
sequences.
-expansins. This degree of
identity is similar to that found in -expansin sequences of
Arabidopsis and cucumber. The exceptions to this rule are NTEXP1 and
NTEXP2, which differ from each other at only 5 amino acids within this
same region and share 95% identity at the nucleotide level. Overall,
the sequence divergence of tobacco -expansins is as large as that of
-expansins among all species.
-expansins distinguishable by
their sequence. Some of the subfamilies have identifying domains in their 5
nonconserved regions. In addition to the 5 identifiers, some
changes within the expansins appear to be conserved within a subfamily.
These changes are boxed in Figure 6 and may indicate significant
subdomains. NTEXP6 is not shown on the tree because it is a partial
clone, and when it was included, it biased the estimation of branch
lengths. When it was included in the analysis, it branched with the
group A -expansins.
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Figure 7.
Phylogenetic tree based on the nucleotide
sequences and the alignment of expansins presented in Figure 6. The
third position of each codon was excluded, because it is likely to be
saturated with mutations. The tree was produced by the MEGA program
using the neighbor-joining method, with Kimura two-parameter distances
with the complete deletion option. Bootstrap values are given above the
branches and confidence probability values are indicated below. The
tree was rooted using Phleum pollen allergen (PHL P1).
Accession numbers are given after the gene names. The names are given
according to our current naming convention, in which the letters EXP
designate expansin and the first two letters indicate the genus and
species: AT for Arabidopsis, CS for cucumber (Cucumis
sativus), LE for tomato (Lycopersicon
esculentum), NT for tobacco (Nicotiana tabacum),
OS for rice (Oryza sativa), and PS for pea (Pisum
sativum). Lengths of the branches represent nucleotide
distances.
-expansins are expressed in the BY2 cell culture, a
northern blot of 2 µg of poly(A+) RNA was probed with
NTEXP1 and washed under stringent conditions (Fig.
8). A diffuse band or an overlapping
series of bands was observed between 1.1 and 1.5 kb, corresponding to
the range in size of the cloned cDNAs. The high homology of
-expansins would allow the probe to cross-hybridize with all of the
known members of the -expansin family. The results of the
northern-blot analysis confirm that BY2 cells transcribe -expansins.
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Figure 8.
Northern-blot analysis of BY2 mRNA. Two micrograms
of BY2 poly(A+) RNA was loaded for each lane. The blot was
probed with a probe made to the full-length NTEXP1 cDNA. The numbers
above the lanes indicate the ages of the cells in days. The numbers on
the right indicate the sizes of the RNAs in kilobases. Northern-blot
analysis was repeated on at least five separate occasions with similar
results.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
), carrot embryo cultures (Smith and Krikorian, 1992), and
rose cell-suspension cultures (Nesius and Fletcher, 1973), low-pH
values promoted cell division, whereas cell expansion and elongation
seemed to require a slightly higher pH. In zinnia mesophyll cultures
the optimal pH for cell elongation was 5.5 to 6.0, whereas cell
differentiation occurred when the pH of the medium decreased to 4.8. Smith and Krikorian (1992) showed that embryonic carrot cells elongate
only when the pH of the medium is greater than 4.5, and that elongated
cells appear in the culture even if the pH is buffered to 5.8. Based on
these results they concluded that some mode of growth other than acid growth may be responsible for cell elongation in cell cultures.
;
Kutschera, 1994).
; McQueen-Mason et al., 1992), the pH of
6-d-old medium for our BY2 cultures is typically around 5.5, a value
more than 1 pH unit above the optimum for expansin activity. Nevertheless, exogenous expansins increased cell growth rates in these
cultures. The concentration of expansins added to the BY2 cultures in
these experiments was not likely to be excessive (unphysiological)
because (a) it was equivalent to the concentration required to give
moderate extension responses in isolated walls, and (b) at this
concentration the amount of exogenous expansin that binds to cell walls
is approximately equivalent to the amount of native expansin
extractable from growing cucumber hypocotyl walls (i.e. about 1 part
expansin to 5000 parts wall on a dry-weight basis [McQueen-Mason and
Cosgrove, 1995]). The fact that expansin can stimulate cell
enlargement in cultures with a bulk pH of 5.5 suggests that it can
induce wall extension in vivo at a pH above the optimum observed in
vitro. The likeliest explanation for this difference is that proton
pumping, acting at the plasma membrane, reduces the local pH in the
inner part of the cell wall. The high concentration of fixed carboxyl
groups in the wall would give the wall a strong buffering capacity,
thus allowing steep pH gradients within the wall. It is also possible
that the BY2 cells are able to modify the optimal pH at which expansin
functions in muro.
). Inclusion of the tobacco sequences in the
phylogenetic tree suggests that there are at least four subgroups of
-expansins. When NTEXP6 is included on the tree there is a tobacco
expansin in each of the subgroups. Each subgroup has conserved changes
away from the consensus at particular sites within the protein and has
members from two or more species. This suggests that each group may be
tailored for a specialized function, e.g. to act on walls of differing
compositions or under different wall conditions. These groupings may
help us to identify orthologous expansins from different species, as
well as aid us in identifying key domains or residues within the
protein.
), whereas the pea expansin (shown as PSEXP1)
is most highly expressed in flower petals (Michael, 1996).
). It is possible that these two clones
represent orthologous genes from each of the parent species.
Alternatively, they may be the products of an evolutionarily recent
duplication and divergence of an expansin gene.
), and ripening tomato fruit (Rose et al., 1997). BY2 cells
typically grow at rates of 2%/h, in comparison with 10%/h for the
fastest-growing cells of the cucumber hypocotyl. The results of our
northern- and western-blot analyses indicate that expansin gene
expression is relatively low in BY2 cells, consistent with the
relatively low growth rates in these cells.
-expansins
were highly represented in a BY2 cDNA library. Very few (60,000)
plaques were screened, but six different cDNAs were identified. The
number of expansins expressed in the suspension culture is remarkable.
We suggest that the BY2 culture cells are in a developmentally confused
state, and that individual -expansins are probably expressed in very
specific patterns in planta. When more than 500,000 plaques were
screened from a cDNA library made from cucumber hypocotyls, only two
expansins were identified (Shcherban et al., 1995).
-expansin cDNAs from a BY2 library. Tobacco
-expansins have high sequence similarity to Arabidopsis and cucumber
-expansins. Addition of the tobacco sequences to the phylogenetic
tree indicates that there are at least four groups of -expansins.
Previous work from our laboratory has shown that -expansins are part
of an ancient gene family, hinting that they are central to plant cell
growth. Having established that expansins do play a role in the growth of suspension cultures, we are now in a position to begin to test directly how essential a mechanism they provide.
1
This work was supported by grants from the
National Aeronautics and Space Administration and the National Science
Foundation.
FOOTNOTES
*
Corresponding author; e-mail dcosgrove{at}psu.edu; fax
1-814-865-9131.
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-1,4-glucanases: sequence conservation, function, and enzyme families.
Microbiol Rev
55:
303-315
Copyright Clearance Center: 0032-0889/98/118//10
© 1998 American Society of Plant Physiologists
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-Expansins in Suspension Cultures of
Bright Yellow 2 Tobacco
