Plant Physiol. (1998) 116: 1539-1549
Genes Involved in Osmoregulation during Turgor-Driven Cell
Expansion of Developing Cotton Fibers Are Differentially
Regulated1
Lawrence B. Smart2, 3,
Fakrieh Vojdani3,
Masayoshi Maeshima, and
Thea A. Wilkins*
Department of Vegetable Crops (L.B.S.), and Department of Agronomy
and Range Science (F.V., T.A.W.), University of California, Davis,
California 95616; and University of California, Davis,
California 95616Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, Japan (M.M.)
 |
ABSTRACT |
Cotton
(Gossypium hirsutum L.) fibers are single-celled
trichomes that synchronously undergo a phase of rapid cell
expansion, then a phase including secondary cell wall deposition, and
finally maturation. To determine if there is coordinated regulation of gene expression during fiber expansion, we analyzed the expression of
components involved in turgor regulation and a cytoskeletal protein by
measuring levels of mRNA and protein accumulation and enzyme activity.
Fragments of the genes for the plasma membrane proton-translocating
ATPase, vacuole-ATPase, proton-translocating pyrophosphatase (PPase),
phosphoenolpyruvate carboxylase, major intrinsic
protein, and
-tubulin were amplified by polymerase chain reaction
and used as probes in ribonuclease protection assays of RNA from a
fiber developmental series, revealing two discrete patterns of mRNA
accumulation. Transcripts of all but the PPase accumulated to highest
levels during the period of peak expansion (+12-15 d postanthesis
[dpa]), then declined with the onset of secondary cell wall
synthesis. The PPase was constitutively expressed through fiber
development. Activity of the two proton-translocating-ATPases peaked at
+15 dpa, whereas PPase activity peaked at +20 dpa, suggesting that all
are involved in the process of cell expansion but with varying roles.
Patterns of protein accumulation and enzyme activity for some of the
proteins examined suggest posttranslational regulation through
fiber development.
 |
INTRODUCTION |
Plant cell expansion occurs through the interaction of multiple
influences, including cell wall-yield properties; the opposing force of
turgor pressure; the biosynthesis of new membrane lipids, cell wall
components, and proteins; and proper trafficking of these newly
synthesized materials to their final cellular destination. During
cell expansion the force of turgor pressure is related to the osmotic
potential and to the transport coefficient for water uptake (Cosgrove,
1986
). A vital component of rapid and sustained cell expansion is the
maintenance of sufficient osmoticum to compensate for dilution effects
resulting from the influx of water. In plant cells, osmoticum and water
accumulate primarily in the vacuoles, although osmoticum in the
cytoplasm also contributes to cellular osmotic potential. The driving
force for the transport and accumulation of ions in the vacuole is
provided by two types of electrogenic, proton-translocating
pumps, one that hydrolyzes ATP (V-ATPase) and another that
hydrolyzes PPi (PPase). Membrane potential across the PM can be
generated by a H+-ATPase, which is
structurally quite different from the V-ATPase. Movement of anions,
which serve as osmoticum, across the tonoplast and the PM occurs
through various carriers and channels (Barkla and Pantoja, 1996
).
Experimental and theoretical approaches to gain a better
understanding of the factors regulating cell expansion and tissue
growth are often confounded by the varying properties of different cell
types in a tissue (Silk, 1984
; Cosgrove, 1986
). Thus, dissection of the
mechanisms controlling cell expansion in a single cell type may provide
a foundation for understanding the regulation of tissue
growth.
The fiber cells of cotton (Gossypium hirsutum L.) represent
a single cell type that undergoes a period of very rapid elongation. Cotton fibers also represent an agricultural commodity of major economic importance in the United States. Fiber cells are single-celled trichomes, which arise in near synchrony from the epidermis of the
ovule and may elongate at peak rates in excess of 2 mm
d
1 during the rapid polar-expansion phase of
development (Schubert et al., 1973
; Basra and Malik, 1984
).
Ultrastructural evidence indicates that expansion occurs through a
diffuse growing mechanism, albeit with some bias for deposition of
newly synthesized cell wall materials at the tip (Tiwari and Wilkins,
1995
). To gain insight into the processes responsible for rapid fiber
growth, we initially focused on characterizing the V-ATPase in cotton fiber cells at the molecular level (Wilkins, 1993
; Wan and Wilkins, 1994a
; Hasenfratz et al., 1995
).
In this work we have assayed the expression of several components,
outlined below, involved in cell expansion at the levels of RNA
accumulation, protein accumulation, and enzymatic activity throughout
fiber development. The V-ATPase and PPase, as mentioned above, are
responsible for driving solute movement into the vacuole, which is
important for maintaining the osmotic potential necessary to generate
turgor pressure. The PM H+-ATPase translocates
protons out of the cytosol, acidifying the apoplast, which is theorized
to effect a change in cell wall extensibility (Rayle and Cleland,
1992
). Proteins in the MIP superfamily have been shown to act as
aquaporins (Maurel et al., 1993
), which may reduce the resistance to
water transport across the tonoplast and/or the PM. Malate, synthesized
in the cytoplasm through the activity of the highly regulated enzyme
PEPCase, accumulates as osmoticum in the vacuole (Barkla and Pantoja,
1996
). Finally,
-tubulin is a protein subunit of microtubules, which
appear to function by coordinating the organellar organization in the
cytoplasm (Goddard et al., 1994
) and are related to the orientation of
cellulose microfibrils in the cell wall (Cyr and Palevitz, 1995
). By
comparing the patterns and peak levels of expression of these various
components, we hope to gain insight into the role of each through fiber
expansion. Our results, especially when considered together with data
describing expression of other genes in fibers, suggest that there are
at least three discrete patterns of gene expression that can be related to the period of organellar biogenesis, a period of rapid expansion, and primary and secondary cell wall deposition. In addition to coordination of transcript accumulation for vacuolar, PM, and cytoplasmic components during fiber expansion, we present evidence supporting the posttranslational regulation of enzyme activity throughout fiber development.
 |
MATERIALS AND METHODS |
Growth Conditions
Cotton (Gossypium hirsutum L. cv Acala SJ-2) was grown
in greenhouse conditions as previously described (Hasenfratz et al., 1995
). Staged ovules were collected based on the phyllotactic arrangement of flowering nodes and the proximity to open flowers on the
day of anthesis. Developing ovules were excised from bolls collected
3 d before anthesis (
3 dpa),
1 dpa, on the day of anthesis (0 dpa), and at +1, +3, +5, +10, +15, +20, +25, +30, and +35 dpa for
the isolation of protein or RNA.
Isolation of Cotton DNA and RNA
Genomic DNA was isolated from young, expanding leaves of cotton as
described (Wilkins et al., 1994
). For RNA, developing fibers were
collected from bolls by freezing the excised ovules in liquid N2, and then brushing off the protruding fiber cells. The
isolated fiber cells from +5, +10, +15, +20, +25, and +30 dpa were used for RNA extraction (Wan and Wilkins, 1994b
; Wilkins and Smart, 1996
).
Purified total RNA was divided into aliquots, precipitated with
ethanol, and stored at
80°C under 70% (v/v) ethanol. For RPAs, the
RNA pellets were collected by centrifugation and the pellet was dried
under vacuum, and then resuspended to a final concentration of 0.2 mg
mL
1 in diethyl pyrocarbonate-treated water.
PCR Amplification and Cloning
PCR was used to amplify partial cDNAs encoding the PM
H+-ATPase, PEPCase, MIP, and
-tubulin from
approximately 1 × 109 plaque-forming units
of a cotton +10-dpa fiber cDNA library constructed in the phagemid
vector
ZAPII (Stratagene). First-strand cDNA from
reverse-transcribed RNA isolated from +10-dpa cotton ovule and fiber
tissue was used as the template for PCR amplification of the PPase.
Oligonucleotide PCR primer sequences, expected product size, and source
targets are listed in Table I. PCR
mixture was 1× PCR buffer (10 mm Tris-HCl [pH 8.3] and
50 mm KCl), 1.5 mm MgCl2,
0.8 mm dNTPs, 1.0 µm of each oligonucleotide
primer, and 2.5 units of Taq DNA polymerase. Reactions (50 µL) were performed in either a Perkin-Elmer model 480 or an Ericomp
thermal cycler (San Diego, CA) using the annealing temperatures listed
in Table I. A portion of the cDNA CVA69.24 (Wilkins, 1993
), which
encodes the 69-kD subunit of the V-ATPase from cotton, was PCR
amplified from plasmid DNA containing the cloned cDNA using the above
conditions. A portion of the 18S rRNA gene was amplified from cotton
genomic DNA using the above conditions. Amplification products from all of the reactions described above were subcloned into the plasmid vector
pCRII (Invitrogen, San Diego, CA). Cloned products encoding PEPCase and
18S rRNA were further subcloned into the plasmid vector pBluescript II
(Stratagene) to facilitate synthesis of in vitro-transcribed probes for
RPAs. The DNA sequences of the cloned fragments to be used as probes
were determined from both strands using the Sequenase version II
dideoxy sequencing kit according to the manufacturer (Amersham) or
using an automated sequencing system (Applied Biosystems). Sequence
analysis was performed using either MacDNASIS (Hitachi Software, Palo
Alto, CA) or PCGene (IntelliGenetics, Campbell, CA).
RPAs
RPAs were performed using the HybSpeed RPA kit essentially as
described by the manufacturer (Ambion, Austin, TX). RNA probes were
synthesized by in vitro transcription from linearized plasmid DNA in
the presence of 50 µCi of
-[32P]UTP (3000 Ci mmol
1) (Dupont/NEN) using either
T7 or SP6 RNA polymerase and were labeled to a
specific activity of approximately 1 × 109
cpm µg
1. The 18S rRNA probe was synthesized
using 1 µCi of
-[32P]UTP together with the
T7 Megashortscript kit as described by the
manufacturer (Ambion) and was labeled to a specific activity of
approximately 5 × 104 cpm
µg
1. Full-length transcripts for probes were
purified after electrophoresis in a 5% (w/v) denaturing polyacrylamide
vertical slab gel. Trial reactions using 0.2, 1.0, and 2.5 µg of RNA
were performed, and a linear increase was observed when comparing the
signal from the 1.0-µg sample to that of the 2.5-µg sample,
indicating that the reactions were not saturated, even for the 18S rRNA
probe (data not shown). One microgram of total cotton fiber RNA was hybridized with 2 × 104 cpm of radiolabeled
probe for each reaction, except for the 18S rRNA probe, in which 3 × 103 cpm were added. RPA reaction products were
resolved by electrophoresis in a 5% denaturing polyacrylamide gel
poured using an analytical comb and electrophoresed using an S2 gel
electrophoresis apparatus for optimum resolution (Gibco-BRL). These
gels were treated with a solution of 10% (v/v) ethanol, 10% (v/v)
acetic acid, transferred to chromatography paper, and then vacuum
dried. Protected products were detected by autoradiography at
80°C
using reflection film and intensifying screens (DuPont/ NEN), and a
radioactive signal was quantified by phosphor imagery (Fujix BAS1000,
Fuji Film Co., Tokyo, Japan). If the RPA yielded a doublet of bands,
then the combined intensity of both bands was quantified. The sizes of protected products were compared with the migration of radiolabeled products generated from the Century RNA Size Marker kit (Ambion). Assays were repeated at least three times.
Preparation of Microsomal Membranes and Tonoplast
Microsomal membranes were prepared essentially as described
(Bennett et al., 1984
) with some modification to enhance the microsomal preparation, particularly at +10 and +15 dpa. Cotton ovules were isolated and blended in homogenization medium (0.35 mm Suc,
70 mm Tris-HCl [pH 8.0], 10% [v/v] glycerol, 3 mm Na2EDTA, 0.15% [w/v] BSA, 1.5%
[w/v] PVP-40, 4 mm DTT, and 1.5 mm PMSF) with a chilled Waring blender for a few seconds. Ovules of +15 dpa and older
stages were cut with scissors into small pieces before homogenization.
The tissue homogenate was set aside at 0°C for 10 to 15 min to allow
foam to settle and was then filtered through three layers of Miracloth
(Calbiochem). The filtrate was centrifuged for 15 min at
15,000g at 4°C, and the resulting pellet was discarded. A
2-mL portion of the supernatant was saved as the enzyme extract for
PEPCase activity determination. The remainder of the filtrate was
centrifuged at 100,000g at 4°C for 40 min, and the
microsomal pellet was resuspended in RM (0.35 m Suc, 10 mm Tris-Mes [pH 7.0], 2 mm DTT, and 1.5 mm PMSF). The microsomal suspensions were frozen in liquid
nitrogen and saved at
80°C until the next day for enzyme assays or
for further membrane purification. Resuspended microsomal membranes
were centrifuged in an Eppendorf centrifuge at 16,000g at
4°C for 5 min, and the resulting supernatant was used as the soluble
protein fraction for enzyme assays.
To purify vacuolar membrane vesicles (tonoplast), the microsomal
suspension was layered onto a 16/27% (w/w) discontinuous Suc gradient
made in 10 mm Tris-Mes (pH 7.0), 2 mm DTT, and
1.5 mm PMSF; and centrifuged at 100,000g at
4°C for 35 min in an SW28 rotor (Beckman). The tonoplast fraction,
which is at the 16/27% Suc interface (Bennett and Spanswick, 1984
),
was collected with a Pasteur pipet and diluted in an equal volume of 10 mm Tris-Mes (pH 7.0), 2 mm DTT, and 1.5 mm PMSF. The tonoplast was subsequently centrifuged again
at 100,000g at 4°C for 30 min; and the pellet was
resuspended in RM, the protein content was assayed, and then the
solution was diluted to a concentration of 1 to 5 mg protein mL
1. The tonoplast suspension was further
diluted to approximately 200 µg protein mL
1
for enzyme assays.
Purification of Cotton Ovule PM
The PM of cotton ovules were purified by two-phase partitioning
(Kjellbom and Larsson, 1984
; Larsson, 1985
). The microsomal pellet, as
prepared above, was resuspended in 0.33 m Suc, 5 mm KCl, and 5 mm potassium phosphate (pH 7.8).
One gram of the suspension containing about 6 mg of protein was added
to the 7-g phase mixture to give an 8-g phase system with a final
composition of 6.5% (w/w) PEG (Mr = 3350),
6.5% (w/w) dextran T-500, 0.33 m Suc, 5 mm
KCl, and 5 mm potassium phosphate (pH 7.8). The phase
system was thoroughly mixed by 20 to 30 inversions of the tube, then
phase separation was facilitated by centrifugation in a swinging bucket
rotor at 2000g, at 4°C for 5 min. The PM partitioned into
the PEG-rich upper phase, whereas the intracellular membranes
partitioned at the interface and in the dextran-rich lower phase
(Larsson and Anderson, 1979
). The upper phase was removed and
repartitioned two additional times with fresh lower phase. The final
upper phases were diluted at least 2-fold with RM, and the PM was
collected by ultracentrifugation at 100,000g at 4°C for 20 min. The pellet was washed twice with RM, and the pure PM pellet was
resuspended in RM for use in enzyme assays.
Protein Assays
Protein concentration was measured by the dye-binding method
(Bradford, 1976
) using BSA as a protein standard. Membrane protein in
RM was first solubilized in 0.05% (v/v) Triton X-100 before the
addition of the dye reagent (Bio-Rad). The final concentration of
Triton X-100 in the protein assay mixture was 0.005% (v/v).
Enzyme Assays
ATPase activity was assayed as the liberation of Pi from either
ATP or PPi and was detected colorimetrically (Ames, 1966
; Bennett et
al., 1988
). ATP and PPi were purchased as disodium salts (Sigma). To
deplete the ATP and PPi of sodium ions, they were first treated with
HCl, and then desalted with Dowex 50W resin. Finally, the pH was
adjusted to 6.5 with BTP (bis-Tris propane
[1,3-bis(Tris-[hydroxymethyl]methylamino) propane]). The reaction
was initiated by the addition of 8 to 12 µg of membrane protein to a
0.4-mL reaction volume, and the reaction was incubated at 30°C for 30 min. The reactions were terminated and measured spectrophotometrically
at 820 nm, as described (Ames, 1966
).
V-ATPase activity (Cl
stimulated, vanadate
insensitive, and NO3
inhibited) was measured in the presence of 25 mm BTP-Mes
(pH 8.0), 3 mm magnesium sulfate, 50 mm
ammonium molybdate, 50 mm sodium vanadate, 50 mm sodium azide, 0.1% (w/v) LPC, 3 mm ATP-BTP, and 50 mm KCl or 50 mm
KNO3. V-ATPase activity, expressed as micromoles per minute per milligram of protein, was calculated as the difference in Pi released assayed in the presence of Cl
or
NO3
ions.
PM H+-ATPase activity (vanadate sensitive) was
measured in the presence of 25 mm BTP-Mes (pH 6.5), 3 mm magnesium sulfate, 50 mm ammonium molybdate,
50 µm sodium azide, 0.1% LPC (w/v), 3 mm
ATP-BTP, and 50 mm KCl, with or without 50 µm
sodium vanadate. PM H+-ATPase activity was
calculated as the difference in Pi released assayed in the presence or
absence of vanadate (Bennett et al., 1984
; Gibrat et al., 1989
). Azide
and molybdate were added to the reaction mixture to inhibit the
mitochondrial and phosphatase activities, respectively (Gallagher and
Leonard, 1982
).
PPase activity was measured in the presence of 25 mm
BTP-Mes (pH 8.0), 3 mm magnesium sulfate, 50 mm
ammonium molybdate, 0.1% (w/v) LPC, 3 mm PPi-BTP, and 50 mm KCl. The enzyme activity was expressed as
K+-stimulated (difference in the activity assayed
in the presence and absence of KCl) and total PPase activity (Rea and
Poole, 1985
). PPase activity was calculated as one-half of the rate of
Pi liberation because the hydrolysis of 1 mol of PPi yields 2 mol of
Pi.
PEPCase (EC 4.1.1.31) activity was assayed spectrophotometrically at
340 nm at 24°C. The reaction was enzymatically coupled to malate
dehydrogenase (EC 1.1.1.37), and the rate of NADH oxidation was
monitored in the presence of 25 mm BTP-Mes (pH 8.0), 10 mm magnesium chloride, 10 mm
NaHCO3, 0.2 mm NADH, 5 mm
DTT, 3 mm PEP, and 10 units of malate dehydrogenase (Meyer
et al., 1988
; Marczewski, 1989
; Schuller et al., 1990
). The reaction
was started by the addition of the enzyme extract, which was prepared
as described above.
Denaturing Gel Electrophoresis and Immunoblotting
SDS-PAGE was performed using 10% (w/v) polyacrylamide gels with
Laemmli buffers (Laemmli, 1970
) and the Mini Protean II electrophoresis system (Bio-Rad). Antibodies raised against V-ATPase, PM
H+-ATPase, PPase, PEPCase, MIP, or
-tubulin
(Table II) were used for estimating the
relative abundance of each protein during development of the cotton
ovules. Protein resolved by SDS-PAGE was immediately transferred to
PVDF membrane (Millipore) in a Trans-Blot Cell (Bio-Rad) (Towbin et
al., 1979
). The blots were blocked with 1% (w/v) nonfat dry milk and
0.4% (v/v) Tween 20 in PBS for 2 h at 23°C, and probed with the
appropriate antisera (Table II) for 16 h at 23°C. Nonbinding
primary antibody was removed by rinsing with PBS. Positive antibody
reaction was detected with protein A-alkaline phosphatase conjugate
reacted with 5-bromo-4-chloro-3 indolyl phosphate and nitroblue
tetrazolium. Relative intensities of the colorimetric reaction products
on the immunoblots blots were determined by densitometric scanning
using an imaging densitometer (model GS-670, Bio-Rad).
 |
RESULTS |
PCR Amplification and Cloning
To obtain probes to assay transcript accumulation throughout
development of fiber cells, PCR was used to amplify portions of genes
encoding the PM H+-ATPase, the 69-kD subunit of
the V-ATPase, PPase, PEPCase,
-tubulin, and MIP from developing
cotton fibers. All of the amplified products were ligated into the
plasmid vector pCRII (Invitrogen) and transformants were screened for
the presence of the expected size insert (Table I). A portion of those
plasmids with the expected size insert were used to obtain partial
nucleotide sequence to verify the identity of those clones. Individual
clones chosen to be used as probes were sequenced completely, and the
sequences not previously reported, excluding the primer sequences, were
deposited in GenBank and given the following accession numbers:
PEPCase, AF008940;
-tubulin, AF009565; PM
H+-ATPase, AF009566; MIP, AF009567; and PPase,
AF009568. Determination that a clone encodes a particular protein was
based on sequence comparison with published gene sequences.
Nucleotide sequences of the five previously uncharacterized clones,
encoding the PM H+-ATPase, PPase, PEPCase, MIP,
and
-tubulin, were aligned and compared over the corresponding
regions, excluding the primer sequences, with individual nucleotide
sequences downloaded from the GenBank database. The fragment of the PM
H+-ATPase is 85 and 81% identical in nucleotide
sequence to the corresponding regions of the genes AHA10 from
Arabidopsis thaliana (accession no. S74033) and PHA2 from
potato (Solanum tuberosum L., accession no. X76535),
respectively. The PPase fragment cloned from cotton ovule RNA is 84%
identical over the same region to the tobacco (Nicotiana
tabacum) TVP9 PPase clone and 82% identical to the red beet
(Beta vulgaris) PPase clone P1 (accession nos. X83730 and
L32792, respectively). The cotton
-tubulin clone is highly identical
(87% over the same region) to the TUA6 gene from A. thaliana (accession no. M84699) and to the TubA1 gene (85% over
the same region) from pea (Pisum sativum, accession no.
U12589). The clone of a MIP family gene from cotton is most similar to
a root-specific aquaporin from tobacco (78% identity, accession no.
X54855) and to
-tonoplast intrinsic protein from A. thaliana (75% identity, accession no. U39485), which has been
demonstrated to be localized to the tonoplast (Daniels et al., 1996
).
Finally, the fragment of PEPCase amplified from cotton fibers is 78%
identical to genes from both soybean (Glycine max, accession
no. D10717) and alfalfa (Medicago sativa, accession no.
M83086).
The PCR strategy utilized to amplify portions of genes encoding the PM
H+-ATPase, the PPase,
-tubulin, MIP, and
PEPCase included the potential for the amplification of members of
multigene families. Partial sequence data from multiple clones of the
PM H+-ATPase, PEPCase, and MIP indicated that the
clones analyzed apparently represent the same genes in all three cases
(data not shown). However, sequences from different
-tubulin clones
indicated that they encoded four unique members of a multigene family
(data not shown).
Assays of Transcript Accumulation
RPAs were utilized to measure the relative transcript abundance of
the aforementioned genes during the expansion of developing cotton
fibers (Fig. 1). Accumulation of
transcripts encoding the PM H+-ATPase was highest
during the phase of rapid elongation, from +5 dpa through +15 dpa. In
fibers +20 dpa and older, PM H+-ATPase gene
transcript abundance dropped to one-fourth of the level of that in
fibers earlier in development. A similar pattern was observed for
transcripts encoding the 69-kD subunit of the V-ATPase, confirming
results of another study (T.A. Wilkins, C.-Y. Wan, W. Kim, F. Vojdani,
and M.-P. Hasenfratz, unpublished data). Message levels for
-tubulin
followed a similar pattern as for the ATPases, with peak accumulation
at +10 and +15 dpa, although the rise and decline of message levels
appeared to occur at a slower rate. This peak period of mRNA
accumulation corresponds to the period of most rapid cell expansion
(Basra and Malik, 1984
; T.A. Wilkins, C.-Y. Wan, W. Kim, F. Vojdani,
and M.-P. Hasenfratz, unpublished data). The accumulation of mRNA
encoding PEPCase and MIP was highest in the earlier stages (+5 and +10
dpa), but then declined after +15 dpa. The detection of MIP mRNA
accumulation required much longer exposures, even though all of the
mRNA probes were labeled to approximately the same specific activity.
Exposure in the +30 dpa lane in Figure 1A was not due to hybridization of the MIP probe, but rather was a signal from a radiolabeled size
ladder in the adjacent lane (not shown). In contrast to the two
ATPases, the accumulation of transcript encoding the PPase was
constitutive through all stages of fiber development.

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| Figure 1.
A, Autoradiographs of representative results of
RPAs of cotton fiber RNA isolated throughout development. Developmental
stage in dpa is noted above each lane (DPA). Probes used for RPAs are described in the text. Exposure times varied for each probe. Each assay
contained 1 µg of cotton fiber total RNA. B, Quantification of RPAs
by phosphor imagery. Bars represent the averages of at least three
assays quantified by a Fuji phosphor imager and expressed relative to
the highest value. Lines extending above the bars represent the
sd. Developmental stage in dpa is noted at the bottom of
each lane (DPA). Intensities of the 18S rRNA RPA products are included
for comparison in A, but were not used to normalize the values for the
other probes.
|
|
Use of the 18S rRNA gene probe for RPAs gave consistently equal levels
of hybridization signal between the samples in the developmental
series, except for a slightly lower signal in samples from +20-dpa
fibers. The consistency of signal from this probe indicates that the
differences in accumulation observed using the other probes is not due
simply to experimental error or to improper RNA quantification. RPAs
using probes encoding the PM H+-ATPase, MIP, and
18S rRNA resulted in doublet bands, one of which migrates at
the predicted size of the correct protected fragment. These doublets,
which are not unusual for this procedure, may result from secondary
structure in the mRNA-probe duplex or from base-pairing mismatches that
are digested in a fraction of duplexes, either due to PCR errors in the
probes used or by cross-hybridization to transcripts from very similar
gene family members.
Immunoblot Analysis of Protein Accumulation
To assay protein accumulation throughout development,
immunoblotting was performed using protein isolated from developing ovules and fibers harvested starting at
3 dpa through +35 dpa (Figs.
2 and 3).
Initially, protein was also extracted from only fibers at the later
developmental stages and used for comparison with protein from ovules
and fibers. Immunoblots probed for V-ATPase protein indicated
comparable patterns of accumulation throughout development, except for
a slightly higher level of V-ATPase protein in the ovule plus fiber
extract compared with fiber protein between +15 and +20 dpa (data not
shown), which is the period when the embryo enlarges (Wilkins and
Jernstedt, 1997
).

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| Figure 2.
Immunoblots of protein purified from cotton
locules throughout development. Developmental stage in dpa is noted
above each lane (DPA). The V-ATPase blot was probed with a mixture of
antibodies raised to the 69-kD subunit and antibodies to the 57-kD
subunit. Protein for the PM H+-ATPase blot was from
microsomes (14 µg of protein per lane). Protein for the V-ATPase,
PPase, and MIP blots was from tonoplast-enriched membranes (8 µg of
protein per lane). Total soluble protein was used for blots probed with
PEPCase and -tubulin antibodies (50 µg of protein per lane).
|
|

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| Figure 3.
Quantification of signal from immunoblots by
scanning densitometry. The intensity of bands of the correct size on
immunoblots was quantified using a scanning densitometer (Bio-Rad) and
is expressed as a percentage of the highest value, which was set at
100% for each repetition. Values are the averages of quantifications of at least three blots for each antibody, and bars above and below
each point represent one sd. Values in the V-ATPase panel represent quantification of the 69-kD subunit ( ) and the 57-kD subunit ( ). In the PEPCase panel, quantifications of the 100-kD band
( ) and the 95-kD band ( ) are shown. Developmental stage in dpa is
noted on the x axis (DPA).
|
|
From these results we concluded that the major contribution of protein
in the fiber and ovule extract was from the fiber cells. Polyclonal
antibodies raised to the A. thaliana PM
H+-ATPase (Parets-Soler et al., 1990
) detected a
band of the expected size of approximately 100 kD at greatest levels in
microsomes from tissues harvested closest to the peak period of
expansion, at around +15 dpa. The PM H+-ATPase
protein level declined in samples taken at later points in development
(+20 dpa and beyond). The 57-kD and 69-kD subunits of the V-ATPase were
detected using a mixture of antibodies raised to the comparable
proteins purified from mung bean (Matsuura-Endo et al., 1992
) and
displayed changes in accumulation in the tonoplast that paralleled each
other throughout development. The levels of V-ATPase subunit
accumulation were highest in early stages (
3 through +1 dpa), and
then dropped to less than one-half of those levels through the period
of expansion, except for a small peak at +25 dpa. Antibodies raised to
the PPase from mung bean (Maeshima and Yoshida, 1989
) detected many
proteins, but the most prominent bands were of the expected size
(approximately 60-65 kD). As with the V-ATPase, the PPase protein
accumulated to highest levels in samples from
1 through +1 dpa, after
which the levels declined except for a minor peak at +25 dpa. An
additional band of approximately 24 kD was also detected, which
comigrates with the band detected by the TIP antibody and probably
represents reaction of the PPase polyclonal antibodies to cotton TIP
protein. This reaction is probably the result of TIP contamination of
the PPase protein preparation used to raise the antibodies, since antibodies raised to a PPase peptide did not cross-react to a 24-kD
band on comparable immunoblots (data not shown).
Antibodies raised to maize PEPCase gave complex banding patterns at
different stages of development. In all samples a doublet of bands was
detected migrating at the expected sizes of approximately 100 and 110 kD. The relative intensities of cross-reaction to these bands varies
throughout development, with peak accumulation of the lower band in
samples from +3 dpa on, whereas the upper band showed greatest
accumulation at +20 dpa, decreasing progressively in the samples from
the last three time points. Bands of unexpected sizes detected by the
PEPCase antibodies varied in accumulation throughout development and
may represent nondenatured multimers or breakdown products. Antibodies
raised to the VM23 MIP protein of radish (Maeshima and Yoshida, 1989
)
cross-reacted with a protein of approximately 24 kD in
tonoplast-enriched membranes at nearly equal levels throughout
development, with a small peak at +15 dpa. Antibodies recognizing
-tubulin (Sigma) detected a protein of approximately 53 kD in total
protein from +10 dpa and later, with peak abundance at +25 dpa, then
decreasing in samples from +30 and +35 dpa. One should note that the
changes in specific protein accumulation detected in purified membrane
fractions reflect differences relative to the total amount of membrane
in the cell and to the amount of that membrane purified at each
developmental stage. The samples were not standardized to allow
determination of the absolute amount of any specific protein in a cell
throughout development.
Enzyme Activity Assays
The ATPase activity of the PM H+-ATPase and
the V-ATPase were assayed in microsomes (data not shown) and in PM- or
tonoplast-enriched fractions (Fig. 4)
using a published procedure that relies on the differential sensitivity
of the V- and P-type ATPases to inhibitors (Gallagher and Leonard,
1982
; Bennett et al., 1984
). The pyrophosphatase activity of the PPase
was also measured in microsomes (data not shown) and tonoplast-enriched
fractions (Fig. 4) as described (Rea and Poole, 1985
). Care was taken
to measure enzyme activity as soon as possible after protein
purification and to use samples that had experienced minimal
freeze/thaw cycles, since activity does decrease slightly over time and
after a freeze/thaw event (data not shown). The activity of the PM
H+-ATPase displays the lowest levels of activity
early and late in fiber development (<+10 dpa and >+25 dpa), whereas
the highest activity was measured in samples from +15 dpa. The activity
of the PM H+-ATPase was highly enriched in PM
purified by two-phase partitioning (Fig. 4) as compared with activity
in total microsomes (data not shown). The activity of the V-ATPase
displayed a very similar pattern through development, with peak
activity at +15 dpa. As with both H+-ATPases, the
PPase displayed rising levels of activity in samples from +3 through
+15 dpa but, in contrast, the peak of PPase activity was at +20 dpa
rather than +15 dpa. Likewise, the activities of the vacuolar enzymes
were greater in tonoplast-enriched fractions isolated by Suc
density-gradient fractionation (Fig. 4) than in total microsomes (data
not shown). The patterns of enzyme activity in total microsomes were
essentially equivalent to the patterns observed in enriched membrane
preparations, indicating that enrichment for the membrane fractions was
performed with equivalent efficiency throughout the developmental
series. The activity of PEPCase was measured in total protein (Fig. 4).
Highest PEPCase activity levels were measured in samples from
1 dpa,
which represents a sharp increase from the activity at
3 dpa.
Activity declined through +10 dpa, with another peak at +15 dpa, then
declined again through fiber development.

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| Figure 4.
Enzyme activity in protein extracts from
developing cotton locules. Each point represents the average of at
least three independent assays, with bars above and below representing
one sd. Purified PM was used for the PM
H+-ATPase assays, whereas tonoplast-enriched membranes were
used for V-ATPase and PPase assays. Total soluble protein was assayed for PEPCase activity. The average specific activity of PM
H+-ATPase at 15 dpa was 152.4 ± 26.6 nmol Pi
mg 1 min 1, of V-ATPase at 15 dpa was
103.7 ± 23.4 nmol Pi mg 1 min 1, of
PPase at 20 dpa was 142.3 ± 27.4 nmol PPi mg 1
min 1, and of PEPCase at 1 dpa was 0.1 ± 0.01 unit
mg 1, where 1 unit equals the oxidation of 1 µmol NADH
min 1 using a molar extinction coefficient of 6.22 × 103.
|
|
 |
DISCUSSION |
The regulation of cell expansion is a vital process of plant
growth and development and is subject to control by environmental and
hormonal factors. Cotton fibers afford us the opportunity to study the
development of a single cell type that undergoes very rapid, virtually
synchronized cell elongation (Basra and Malik, 1984
; Tiwari and
Wilkins, 1995
) and that can be harvested in pure preparations for RNA
and protein purification. Here we have characterized the expression of
the proton pumps, a putative water channel, an enzyme that contributes
to the production of osmoticum, and a cytoskeletal protein by assaying
mRNA accumulation, protein accumulation, and enzyme activity. To
measure RNA accumulation, we chose to use the RPA because it is very
sensitive, quantitative, and requires only a small amount of RNA. These
experiments were performed using tissue isolated from carefully staged
greenhouse-grown plants, which, in our hands, showed very predictable
developmental timing, even from season to season (Hasenfratz et al.,
1995
). Accumulation of 18S rRNA transcript was assayed throughout
development for comparison with accumulation of the mRNAs tested.
Protein samples for immunoblots were standardized to the amount of
total protein in each sample. To make comparisons between levels of mRNA and protein accumulation, one must assume equal variation in the
accumulation of 18S rRNA and cellular protein throughout development.
PCR-based strategies were used to isolate novel clones to serve as
probes for PM H+-ATPase, PPase, PEPCase, MIP, and
-tubulin. Cotton is an allotetraploid, making complete
characterization of large gene families, such as the PM
H+-ATPase (Ewing and Bennett, 1994
; Harper et
al., 1994
) and MIP (Weig et al., 1997
) gene families, intractable. In
other plants, the PPase and PEPCase have been found to be encoded by
low-copy-number genes or small gene families (Kim et al., 1994
; Chollet
et al., 1996
), whereas
-tubulin is encoded by a large gene family in Arabidopsis (Kopczak et al., 1992
). For all but PEPCase, degenerate oligonucleotide primers were used, not in a strategy designed to
isolate all of the potential gene family members, but rather with the
expectation of isolating perhaps the most predominantly expressed gene
family member. Of the PCR clones isolated and characterized by sequence
analysis, only clones of
-tubulin appeared to originate from
multiple genes (data not shown), suggesting that the recovered PM
H+-ATPase and PPase clones may represent the
predominantly expressed isoform. The cotton MIP clone isolated is most
similar to the
-TIP gene from A. thaliana (Daniels et
al., 1996
). Notably, it is 99% identical over the region cloned to
another MIP isolated from cotton fibers (Ferguson et al., 1997
)
(GenBank accession no. U62778), and thus may represent an allele of
that gene. MIP proteins are encoded by large multigene families in
plants (Chrispeels and Agre, 1994
; Weig et al., 1997
), so it is
difficult to say whether this gene represents the predominant isoform
expressed in fibers. The very low accumulation of transcript, compared
with relatively abundant protein detected by immunoblotting, suggests that this gene may not be the predominant MIP expressed during cotton
fiber development.
This and previous work suggest that there are multiple developmental
programs controlling gene expression through cotton fiber development
(Wilkins and Jernstedt, 1997
). One pattern is represented by those
genes expressed at their highest levels slightly before and during the
period of peak fiber expansion, followed by a sharp decline in message
accumulation by +20 dpa, when the rate of expansion slows sharply
(Basra and Malik, 1984
). Transcripts arising from the PM
H+-ATPase, V-ATPase, PEPCase,
-tubulin, and
MIP genes accumulate to their highest levels in the days just prior to
the peak period of expansion (+12-15 dpa; Basra and Malik, 1984
).
Other genes display this same pattern of expression throughout cotton
fiber development. A gene encoding a fiber-specific lipid-transfer
protein is most highly expressed at about +15 dpa and is probably
involved in cutin deposition on the surface of the fiber cell (Ma et
al., 1995
). Likewise, genes encoding expansin and
endo-1,4-
-glucanase are expressed at highest levels between +9 and
+15 dpa (Shimizu et al., 1997
). These proteins act by loosening or
cleaving bonds in the cell wall to allow cell expansion (Fry, 1995
;
Cosgrove, 1997
). In addition, the E6 gene displays this same
pattern of gene expression, verified to be regulated at the level of
transcription (John and Crow, 1992
; Rinehart et al., 1996
). As with the
genes we have analyzed, the accumulation of transcripts from these
three genes declines by +20 dpa. This program of gene expression is
consistent with proteins that are involved in the process of rapid
cell expansion and primary cell wall deposition, which
occurs during this period of cotton fiber development.
In general, the patterns of transcript and protein accumulation
correlate with the patterns of activity for the PM
H+-ATPase and PEPCase, although the peaks of mRNA
accumulation precede the peaks of protein accumulation and enzyme
activity by a few days. In contrast, the V-ATPase protein level is
highest early in fiber development, when enzyme activity is relatively
low. The peak of V-ATPase activity at +15 dpa, with no corresponding change in protein accumulation, suggests posttranslational activation of this enzyme, perhaps at the level of assembly. Accumulation of
-tubulin message peaks sharply at +15 dpa, which is 10 d prior to the peak of protein accumulation and could reflect a long half-life for
-tubulin turnover. The constant level of MIP protein
accumulation throughout development may indicate that the protein is
quite stable or that the VM23 antibodies detected protein(s) other than the one assayed by RPAs, the mRNA of which declined dramatically beyond
+10 dpa.
Another pattern of gene expression is displayed by the PPase gene, for
which message accumulation was constitutive through fiber development.
Likewise, the 18S rRNA gene was constitutively expressed. Genes
encoding actin, endoxyloglucan transferase, and Suc synthase also
display constitutive mRNA accumulation through fiber development
(Shimizu et al., 1997
).
It is interesting that the PPase enzyme activity changes
dramatically during expansion, with a peak at +20 dpa, a few days after
the peak rate of fiber expansion, whereas the greatest protein accumulation occurs at +1 dpa, when the levels are severalfold higher
than those at +15 or +20 dpa. As with the V-ATPase, the highest levels
of PPase activity simultaneous with relatively low levels of protein
accumulation are indicative of posttranslational activation of the
PPase enzyme. The peak of PPase activity occurred at about +20 dpa, a
few days later than the peaks of activity of the two
H+-ATPases, which may suggest that the PPase
plays more of a role in the process of secondary wall deposition.
An additional pattern of gene expression through fiber development is
apparent from analysis of other cotton genes. This pattern is
characterized by highest levels of mRNA accumulation later in fiber
development, during the phase of secondary cell wall formation,
approximately +17 dpa through +35 dpa. The genes encoding cellulase
synthase, celA1 and celA2, display greatest
accumulation beyond +17 dpa (Pear et al., 1996
). Likewise, the
H6 gene and FbL2A genes are expressed primarily
during the later stages of fiber development (John and Keller, 1995
;
Rinehart et al., 1996
). None of the genes characterized in our study
displayed low levels of expression early in development, which is
indicative of this class of genes.
The defined patterns of mRNA accumulation presented here suggest that
gene expression through fiber development may be controlled by common
regulatory elements, and that the patterns are consistent with the
presumed functions of these enzymes in the major cellular processes of
turgor regulation, cell wall deposition, and cytoskeleton formation.
Preliminary research suggests that genes encoding turgor control
enzymes and cell wall-related proteins respond differently to hormonal
signals, perhaps indicating coordinated responses to multiple signaling
cascades (W. Kim and T.A. Wilkins, unpublished data). Discrepancies
between protein accumulation and enzyme activity, as in the cases of
the V-ATPase and PPase, suggest posttranslational mechanisms of
regulation, which are the topics of current research. When considering
molecular genetic approaches to manipulation of fiber properties, it
will be important to know the contributions of posttranscriptional and
posttranslational regulation of enzyme activity, in addition to the
tissue-specific and developmental patterns of transcription. It is
clear that only after gaining a thorough understanding of the biology
of fiber development will predictable manipulation of fiber properties
be accomplished.
 |
FOOTNOTES |
1
This work was supported by funding from Cotton
Incorporated and the U.S. Department of Energy. L.B.S. was supported by
a National Science Foundation postdoctoral fellowship in plant biology
(no. BIR-9203665).
2
Present address: State University of New York
(SUNY) College of Environmental Science and Forestry, Faculty of
Environmental and Forest Biology, One Forestry Drive, 302 Illick Hall,
Syracuse, NY 13210.
3
These authors contributed equally to the paper.
*
Corresponding author; e-mail tawilkins{at}ucdavis.edu; fax
1-916-752-4361.
Received November 14, 1997;
accepted December 8, 1997.
 |
ABBREVIATIONS |
Abbreviations:
dpa, days postanthesis.
H+-ATPase, proton-translocating ATPase.
LPC, l-
-lysophosphatidylcholine.
MIP, major intrinsic protein.
PEPCase, phophoenolpyruvate
carboxylase.
PM, plasma membrane.
PPase, proton-translocating
pyrophosphatase.
RM, resuspension medium.
RPA, RNase protection assay.
V-ATPase, vacuolar H+-ATPase.
 |
ACKNOWLEDGMENTS |
The authors are grateful to Dr. Alan B. Bennett for allowing
some of this work to be performed in his laboratory and to Dr. Ching-Yi
Wan for isolating the
-tubulin clone. We acknowledge the generous
gifts of antibodies from Dr. Ramon Serrano, Valencia, Spain, and Dr.
Carole Bassett, U.S. Department of Agriculture-Agricultural Research
Service, Kearneysville, WV.
 |
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