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Plant Physiol. (1999) 120: 237-244
Purification and Partial Characterization of a Dehydrin Involved
in Chilling Tolerance during Seedling
Emergence of
Cowpea1
Abdelbagi M. Ismail,
Anthony E. Hall, and
Timothy J. Close*
Department of Botany and Plant Sciences, University of California,
Riverside, California 92521-0124
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ABSTRACT |
Dehydrins are a family of proteins
(LEA [late-embryogenesis
abundant] D11) commonly induced by environmental stresses
associated with low temperature or dehydration and during seed
maturation drying. Our previous genetic studies suggested an
association of an approximately 35-kD protein (by immunological
evidence a dehydrin) with chilling tolerance during emergence of
seedlings of cowpea (Vigna unguiculata) line 1393-2-11. In the present study we found that the accumulation of this protein in
developing cowpea seeds is coordinated with the start of the
dehydration phase of embryo development. We purified this protein from
dry seeds of cowpea line 1393-2-11 by using the characteristic
high-temperature solubility of dehydrins as an initial enrichment step,
which was followed by three chromatography steps involving cation
exchange, hydrophobic interaction, and anion exchange. Various
characteristics of this protein confirmed that indeed it is a dehydrin,
including total amino acid composition, partial amino acid sequencing,
and the adoption of  -helical structure in the presence of sodium dodecyl sulfate. The propensity of dehydrins to adopt -helical structure in the presence of sodium dodecyl sulfate, together with the
apparent polypeptide adhesion property of this cowpea dehydrin,
suggests a role in stabilizing other proteins or membranes. Taken
together, the genetic, physiological, and physicochemical data are at
this stage consistent with a cause-and-effect relationship between the
presence in mature seeds of the approximately 35-kD dehydrin, which is
the product of a single member of a multigene family, and an increment
of chilling tolerance during emergence of cowpea seedlings.
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INTRODUCTION |
A range of molecules have been found to accumulate during seed
development and are thought to play a role in preventing damage to
embryos during desiccation. These include soluble sugars (Koster and
Leopold, 1988 ; Chen and Burris, 1990) and proteins, among which the LEA
(late-embryogenesis abundant)
proteins are typical (Blackman et al., 1991, 1995; Dure, 1993; Close,
1996; Ingram and Bartels, 1996). For example, studies with
soybean indicated that accumulation of LEA proteins during
embryogenesis might reduce the extent of desiccation-induced
electrolyte leakage in immature seeds (Blackman et al., 1995).
The LEA D11 family (Dure, 1993), also known as dehydrins, includes some
of the most commonly observed proteins induced by environmental
stresses associated with dehydration or low temperature and that
comprise an immunologically distinct family (Close, 1997). These
proteins accumulate in dehydrating plant tissue, such as in seeds that
are becoming mature or in leaves due to drought, salinity, or extracellular freeze-thaw cycles. Also, some specific genes in the dehydrin multigene family typically are induced by cold
temperatures (Close, 1997). Distinct subclasses of dehydrins have been
noted (Houde et al., 1995), and a "YSK" nomenclature scheme
within the dehydrin family has been developed (Close, 1997). Several
lines of evidence are consistent with a role of dehydrins in
membrane interactions, including immunolocalization data that imply an
endomembrane association of a basic YSK2 maize
dehydrin in the cytoplasm (Egerton-Warburton et al., 1997), a plasma
membrane association of an acidic SK3 wheat
dehydrin (Danyluk et al., 1998), and adoption of an -helical
structure by several cereal dehydrins in the presence of SDS (T.J.
Close, unpublished data). Dehydrins can also be present in nuclei
(Asghar et al., 1994), which may require phosphorylation (Jensen et
al., 1998). A role in protein stabilization has been proposed. It has
been hypothesized that dehydrins function as surfactant molecules,
acting synergistically with compatible solutes to prevent coagulation
of colloids and a range of macromolecules (Close, 1997).
Cowpea (Vigna unguiculata) is a warm-season annual crop that
is sensitive to chilling temperature during seedling emergence. Typically, soil temperature below 20°C can cause substantial
reduction in seedling emergence under field conditions. Two closely
related cowpea lines were found to vary in maximal emergence under
chilling field conditions (Ismail et al., 1997). Seeds of the
chilling-tolerant line 1393-2-11 were shown to contain a substantial
quantity of an approximately 35-kD protein using immunoblot analysis
(Ismail et al., 1997) with antibodies specific for the consensus
K-segment of dehydrin proteins (Close et al., 1993), whereas this
protein was not detected in seeds of the chilling-sensitive line. Based on studies with F1 hybrids and their parents,
Ismail et al. (1997) hypothesized that this protein confers an
increment of chilling tolerance during emergence of cowpea that is not
related to line differences in electrolyte leakage. Dehydrin
purification methods have been described previously (Plana et al.,
1991; Ceccardi et al., 1994; Kazuoka and Odeda, 1994; Houde et
al., 1995; Jepson and Close, 1995; Lisse et al., 1996). In the present
study we examined the developmental expression of the approximately
35-kD protein in developing cowpea seeds. We also purified it from dry seeds of cowpea line 1393-2-11 using the method of Ceccardi et al.
(1994) with some modifications. We examined various characteristics of
this protein, including total amino acid composition, partial amino
acid sequence, and the effect of SDS on secondary structure. The
results established that this protein is a dehydrin with properties that may be relevant to its physiological function.
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MATERIALS AND METHODS |
Developmental Expression of the Approximately 35-kD Dehydrin
Cowpea (Vigna unguiculata L. Walp.) line 1393-2-11 was
sown in a greenhouse with day/night temperatures of 35°C/20°C.
After anthesis pods were tagged daily until the oldest pod was fully mature and dry. Tagged pods were harvested and quickly moved to the
laboratory for further analysis. From each pod, one to two seeds were
removed and used for total protein extraction and dehydrin assay using
SDS-PAGE and western blotting, as described by Ismail et al. (1997).
The remaining seeds in each pod were weighed, dried at 105°C, and
reweighed to determine their seed moisture content on a fresh weight
basis.
Protein Purification
Dehydrin purification was carried out following the procedure of
Ceccardi et al. (1994) with some modifications. Protein concentration throughout the purification was determined by a dye-binding assay (Harlow and Lane, 1988) using bovine  -globulin (Bio-Rad) as a standard. Seeds of cowpea line 1393-2-11 were obtained from plants grown in field conditions during the summer of 1996 at Riverside, California.
About 250 g of dry seeds (1050 seeds) was ground to the
consistency of flour using a coffee grinder (model IDS-50, Mr. Coffee, Bedford Heights, OH). The ground material was then mixed into 1.5 L of
prechilled 25 mM Mes (2-[morpholino]-ethane sulfonic acid) buffer, pH 6.0, 20 mM NaCl, and 1 mM PMSF
and stirred for 3 h at 4°C. The mixture was then blended for 1 min using a blender (model 31BL92, Waring) and stirred overnight at
4°C. The mixture was then centrifuged at 6000g for 20 min
at 4°C and the supernatant was decanted and filtered through four
layers of cheesecloth.
The supernatant was heated to 70°C in a boiling water bath with
stirring, held for 10 min at 68°C to 72°C, cooled on ice, and
filtered through a Whatman filter paper no. 1. The filtrate was
concentrated to a final volume of about 200 mL using a Centriprep 10 concentrator with a 10,000 Mr cutoff
(Amicon, Beverly, MA), and a clarified supernatant was produced by
centrifugation at 30,000g for 1 h at 4°C.
To prepare for cation-exchange chromatography, the sample was dialyzed
in a 6000 to 8000 Mr cutoff dialysis
membrane (Spectra/Por, Spectrum, Laguna Hills, CA) against prechilled
25 mM Mes, pH 6.0, and 20 mM NaCl at 4°C. Two buffer changes were made
with a minimum of 6 h for each dialysis. The sample was then
filtered through a 0.2-µm filter (Nalge, Rochester, NY) and passed
over a source 15S fast protein liquid chromatography column (Pharmacia
LKB Biotechnology, Uppsala, Sweden) that had been equilibrated with 25 mM Mes, pH 6.0, and 20 mM
NaCl. Under these conditions the approximately 35-kD protein became
bound to the column. An NaCl concentration gradient was applied in the
same buffer, and the approximately 35-kD protein eluted between 20 and
320 mM NaCl. Fractions of 10 mL were collected
and stored at  20°C until completion of immunoblot analysis of the
fractions. Fractions containing the approximately 35-kD protein were
pooled.
For hydrophobic-interaction chromatography, pooled fractions were
dialyzed against 50 mM
KH2PO4/K2HPO4,
pH 7.0, and 0.8 M (NH4)2SO4,
with two changes of buffer. Samples were filtered using a 0.2-µm
filter (Nalge) and passed over a Phenyl Superose HR 10/10 fast protein
liquid chromatography column (Pharmacia LKB Biotechnology) that had
been equilibrated with 50 mM potassium phosphate, pH 7.0, and 0.8 M
(NH4)2SO4.
The approximately 35-kD protein was retained on the column under these
conditions and was eluted by a gradient of decreasing ammonium sulfate
concentration from 0.8 to 0.0 M over 222 mL. Fractions of
1.5 mL each were collected and analyzed by immunoblotting, as before.
Fractions containing the approximately 35-kD protein were pooled and
stored at 80°C until the subsequent purification step.
For anion-exchange chromatography, pooled samples from
hydrophobic-interaction chromatography were dialyzed in 6000 to 8000 Mr cutoff dialysis tubing against 20 mM Tris, pH 8.8, with two changes of the dialysis
buffer for a minimum of 6 h each. Following dialysis, the samples
were filtered using a 0.2-µm filter (Nalge) and then passed over a
Source 15Q fast protein liquid chromatography column (Pharmacia LKB
Biotechnology) previously equilibrated with 20 mM
Tris, pH 8.8. Under these conditions the approximately 35-kD protein
was retained on the column. The protein was then eluted by applying a
gradient of NaCl from 0 to 300 mM. Fractions of 1.5 mL were collected and analyzed by immunoblotting with anti-dehydrin antibody as before and by 13% SDS-PAGE with colloidal Coomassie Brilliant Blue G250 staining (Harlow and Lane, 1988). Fractions containing the immunopositive approximately 35-kD protein were stored
at 80°C.
Amino Acid Composition Analysis
To prepare for amino acid composition analysis, the protein in a
sample of about 500 µL was concentrated three times using a
Centricon-3 concentrator (Amicon) with a 3000 Mr cutoff to about 250 µL and then
rediluted to 500 µL. Deionized water was used for dilution in the
first cycle, and 10 mM Tris, pH 8.0, was used in
the second and last cycles. These washes decreased the concentration of
NaCl in the sample from 200 to about 25 mM. The
sample was then packed on dry ice and submitted to Beckman Research
Institute of the City of Hope, Division of Immunology (City of Hope,
CA) for total amino acid composition analysis.
Peptide Sequencing
CNBr digestion and peptide separation were performed following the
procedures of Promega (Promega Technical Manual, 1993). A Centricon-3
concentrator (Amicon) was used to concentrate a sample of about 100 µg of protein in a volume of about 500 µL and to lower the
concentration of Tris and NaCl by serial washes with 10 mM
Tris (pH 8.0). The sample was then divided into five microcentrifuge
tubes, each containing 100 µL, and lyophilized in a SpeedVac
concentrator (model SVC 200, Savant, Farmingdate, NY). Two hundred
microliters of CNBr solution (10 mg CNBr/mL in 70% formic acid) was
added to each tube and incubated overnight at room temperature. Samples
were then dried in a SpeedVac, dissolved in 45 µL of water, and
redried. Thirty microliters of sample buffer was added to each tube,
and the samples were combined and electrophoresed in a Tricine SDS-PAGE
system (Schägger and von Jagow, 1987). Transfer of the fragmented
peptides to a ProBlot PVDF sequencing membrane and staining were
carried out following the protocol of Promega (Promega
Technical Manual, 1993). One of the fragments was excised from the
membrane and used for N-terminal amino acid- sequencing using a protein
sequencer (Procise-492, Perkin-Elmer/Applied Biosystems, Foster City,
CA) at the University of California, Riverside.
CD Analysis
A spectropolarimeter (model J715, Jasco, Easton, MD; laboratory of
Carl Frieden, Washington University School of Medicine, St. Louis, MO)
was utilized. Fraction 34, which was one of the three major
chromatogram peaks following the final (anion-exchange) step of the
purification, was analyzed. The sample was dialyzed against 20 mM
KH2PO4/K2HPO4,
pH 7.0, using Slide-A-Lyzer dialysis cassettes with a 3500 Mr cutoff membrane (Pierce). The buffer was
changed four times with a minimum of 8 h during each
dialysis. The sample was then concentrated using a Centricon
concentrator to about 0.2 mg/mL and was subsequently dialyzed against
50 mM NaCl in preparation for CD analysis in the
presence of SDS.
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RESULTS |
Accumulation of Approximately 35-kD Protein during Seed Development
The temporal accumulation of the approximately 35-kD protein was
determined by western analysis of proteins extracted from developing
seeds. The moisture content of developing cowpea seeds decreased
progressively with time as shown in Figure
1. A sharp reduction in seed moisture
content was observed 24 d after anthesis, when the seed moisture
decreased to about 15% (on a fresh weight basis), approaching the
typical moisture content of mature seed at harvest (Fig. 1). Just prior
to this (21-22 d after anthesis) when the seed moisture content had
decreased to about 60%, the dehydrin protein level rose considerably
from a low initial level attained about 5 d earlier and continued
to rise steadily as the seeds became mature (Fig. 1). The commencement
of the later phase of accumulation coincided with the start of the
color-break stage of pod development. Initially pods were green, but
they began to develop a yellow color 21 d after anthesis, and by d
22 about 30% of the pod surface area was yellow. Coordination of
maximal dehydrin accumulation with the dehydration phase of seed
development is a unifying property of LEA proteins (Hughes
and Galau, 1989; Dure, 1993). All cowpea genotypes that have been
examined produce several dehydrin proteins during seed development
(A.S. El-Kholy, A.E. Hall, and T.J. Close, unpublished data), but the
approximately 35-kD protein is not detectable in many genotypes, and it
is the major species detected by anti-dehydrin antibodies in genotype 1393-2-11 (Ismail et al., 1997).
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| Figure 1.
Changes in seed moisture content and dehydrin
expression during seed development of cowpea line 1393-2-11.
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Purification of the Approximately 35-kD Protein
Mature seeds were used as a source of the approximately 35-kD
protein. The characteristic retention of solubility of dehydrins at
high temperature was used in an initial purification step to obtain a
dehydrin-enriched sample. Approximately 80% of the total soluble
protein was precipitated by heating the sample to 70°C (Table
I), whereas the approximately 35-kD
protein remained in solution.
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Table I.
Total protein and yield during purification of an
approximately 35-kD cowpea dehydrin from dry seeds of line 1393-2-11
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Three column chromatography steps were used to further purify the
approximately 35-kD protein. The first step involved the use of a
Source 15S cation-exchange chromatography column, where the
approximately 35-kD protein eluted from the column over a wide range
between 20 and 320 mM NaCl (Figs.
2A and 3).
In the fractions containing the highest concentration of approximately 35-kD protein, the approximately 35-kD protein seemed to adhere to
other proteins in a manner that was not fully disrupted by the Laemmli
sample buffer and SDS-PAGE, as shown for fractions 21 to 35 in Figure
3. Fractions containing the approximately 35-kD protein were pooled and
further separated on a Phenyl Superose HR 10/10 hydrophobic-interaction
column. After this purification step, there was no further evidence of
apparent adhesion to other proteins, and the approximately 35-kD
protein eluted at approximately 270 to 540 mM ammonium
sulfate (Figs. 2B and 4). Fractions 27 to
59 were then pooled.
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| Figure 2.
Chromatograms showing the different steps of
dehydrin purification from cowpea seeds. Arrows indicate the fractions
containing the dehydrin. A, Cation exchange; B, hydrophobic
interaction; and C, anion exchange. Dashed lines indicate the salt
gradient.
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| Figure 3.
Immunobot of the fractions following
cation-exchange chromatography, with 5 µL of each fraction loaded per
lane. Fractions 22 to 35 were pooled and used for the next step. The
arrow indicates the position of the approximately 35-kD dehydrin. Lane
MWT, Low-range Mr markers (Bio-Rad).
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| Figure 4.
Immunobot of the fractions following
hydrophobic-interaction chromatography, with 5 µL of each fraction
loaded per lane. Fractions 27 to 59 were pooled and used for the next
step. Lane MWT, Low-range Mr markers
(Bio-Rad).
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The final purification step involved the use of a Source 15Q
anion-exchange column. The approximately 35-kD protein eluted at
approximately 150 to 280 mM NaCl (Figs. 2C and
5). Fractions 34 to 54, which covered
this range, were stored at 80°C. Fractions collected were subjected
to both Coomassie Blue staining and immunoblot analysis (Fig. 5), which
showed that the approximately 35-kD protein was free of detectable
contaminants. Figure 2C shows the anion-exchange chromatogram, in which
four peaks were observed. These peaks may reflect different levels of
phosphorylation of the same polypeptide. An illustration of the
relative purity at each major step is given in Figure
6. Total protein content and protein
yield following each purification step are summarized in Table I.
Approximately 0.04% of the extracted soluble protein was recovered as
pure approximately 35-kD protein.
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| Figure 5.
Fractions after anion-exchange chromatography with
5 µL of each fraction loaded per lane. A, 13% SDS-PAGE stained for
total protein using colloidal Coomassie Brilliant Blue G250. B,
Imunoblot analysis using anti-dehydrin antibodies as a probe. Lane MWT,
Low-range Mr markers (Bio-Rad).
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| Figure 6.
Steps in dehydrin purification. Lanes correspond
to: 1, crude extract; 2, heat-treated; 3, cation-exchange; 4, hydrophobic-interaction; and 5, anion-exchange peaks. Lanes MWT,
Low-range Mr markers (Bio-Rad). A,
Colloidal Coomassie Brilliant Blue staining. Protein loaded was: lanes
1 and 2, 10 µg each; lane 3, 4.0 µg; lane 4, 2.8 µg; and lane 5, 1.9 µg. B, Immunoblot analysis using anti-dehydrin antibodies as a
probe. Protein loaded was: lanes 1 and 2, 10 µg each; lane 3, 2.0 µg; lane 4, 1.0 µg; and lane 5, 0.6 µg.
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Peptide Sequencing and Amino Acid Composition
Efforts to perform N-terminal amino acid sequence analysis on the
purified protein were not successful, presumably because of N-terminal
blocking. Purified protein was then partially fragmented using CNBr,
which cleaves the protein at the peptide bond involving the carboxyl
group of Met (Gross, 1967). Cleavage products were separated using
Tricine SDS-PAGE, as described by Schägger and von Jagow (1987),
and transferred to a ProPlot PVDF sequencing membrane. After staining,
three bands were observed as shown in Figure
7. The fragment with the lowest
Mr was excised and used for N-terminal
amino acid sequencing, and a sequence of 22 amino acids was obtained.
Comparison of this sequence with other protein sequences in the
National Center for Biotechnology Information database is shown in
Figure 8. The greatest similarity was
observed between this amino acid sequence and the deduced sequences of four previously identified LEA D11 proteins. One of these proteins is
the CPRD22 dehydrin identified in leaves of cowpea plants subjected to
drought (Iuchi et al., 1996). Two others, MAT1 (accession no. L00921;
Y.J. Chyan, R.W. Rinne, L.O. Vodkin, and A.L. Kriz, unpublished
data) and MAT9 (accession no. M94012; Chyan and Kriz, 1992), are
maturation-associated proteins from soybean seeds and the fourth is a
protein (accession no. U10111; N. Maitra and J.C. Cushman, unpublished
results) from soybean leaves. The amino acid sequence of the latter
protein is identical to that of MAT9, suggesting that these represent
very similar or identical alleles of the same gene.
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| Figure 7.
PVDF membrane stained with colloidal Coomasie
Brilliant Blue G250 after the transfer of the protein from a Tricine
gel using the western-blot technique. Lane MWt, left, Bio-Rad low-range
Mr marker; lane CNBr digest, the fragments
of the dehydrin after CNBr digestion; and lane MWt, right, the Promega
low-range Mr markers. The large arrow shows
the peptide used for the N-terminal amino acid sequence
determination.
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| Figure 8.
Comparison of the amino acid sequence of the
cowpea dehydrin fragment with other dehydrin sequences in the National
Center for Biotechnology Information database. CPRD22 is a
drought-inducible protein produced in cowpea leaves. MAT1 and MAT9 are
maturation-associated proteins from soybean seeds. U10111 is a
drought-induced protein from soybean leaves. Boxes indicate conserved
regions.
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Total amino acid composition of the purified dehydrin showed that this
protein is rich in Gly and polar and charged amino acids such as Thr,
Asn, Gln, Ser, Asp, and Glu, suggesting that it is highly hydrophilic.
It is also devoid of both Cys and Trp (Table
II). This composition is in agreement
with the common properties of dehydrin proteins (Close, 1997). A
comparison was made between the amino acid composition of the purified
cowpea approximately 35-kD protein and the deduced amino acid sequence
of cowpea CPRD22 cDNA (Iuchi et al., 1996). A high level of similarity,
but apparently not identity, was observed between these two dehydrins
(Table II). It is not possible from this information to decipher
whether these two cowpea dehydrins represent alleles of a single gene or of two different cowpea dehydrin genes.
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Table II.
Comparison of the total amino acid composition of
the purified approximately 35-kD cowpea dehydrin with the deduced amino
acid sequence of cowpea CPRD22 cDNA from Iuchi et al. (1996)
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CD Analysis
The induction of -helical structure by SDS, which has been
observed previously for several cereal dehydrins (T.J. Close, unpublished data), was examined for the approximately 35-kD cowpea dehydrin. Far-UV CD was performed to estimate the structural
conformation of the purified dehydrin in the presence and absence of 10 mM SDS. In the absence of SDS, the spectrum for fraction 34 contained a strong negative band near 197 nm and a weak band near 220 nm, which are characteristic of peptides lacking a well-defined
secondary structure (Woody, 1992). The CD spectrum of fraction 34 was
altered in the presence of SDS, such that the difference spectrum is
typical of -helical structure: positive bands in the range of 195 to 198 nm and a broad negative band in the range of 205 to 235 nm (Fig.
9).
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| Figure 9.
Effect of 10 mM SDS on secondary
structure of the cowpea approximately 35-kD dehydrin. Path length, 0.1 cm; step, 0.2 nm; scan speed, 20 nm/min, six accumulations
(noise-reduced and smoothed); bandwidth, 2 nm; sensitivity, 10 millidegrees; response, 2 s; and concentration = 0.19 mg/mL in 50 mM NaCl, pH 7.0, at 23°C. Solid line, 0 mM SDS; dashed line, 10 mM SDS; and stippled
line, CD value in 0 mM subtracted from CD value in 10 mM SDS ("difference spectrum").
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DISCUSSION |
Our previous studies with closely related cowpea lines and their
F1 hybrids (Ismail et al., 1997) indicated that
an approximately 35-kD protein detected by anti-dehydrin antibodies
confers an increment of chilling tolerance during seedling emergence.
In this work we have established by partial amino acid sequencing that
this protein is indeed a dehydrin. A high level of similarity between
this protein and the deduced amino acid sequence of the cowpea dehydrin
CPRD22 cDNA (Iuchi et al., 1996) was observed (Table II). Comparison of
the 22-amino acid sequence of the fragment obtained after digestion
with CNBr to other protein sequences in the National Center for
Biotechnology Information database showed substantial similarity with
four Fabaceae LEA D11 proteins (Fig. 8) and a lack of similarity to any
other enzymes or proteins. The amino acid composition is also typical
of dehydrins. We demonstrated that accumulation of this protein in seed
of cowpea line 1393-2-11 is coordinated with the dehydration phase of
embryo development. Detectable amounts were observed beginning when the
moisture content of the developing embryo had decreased to about 65%
on a fresh weight basis, about 5 d prior to the pod color-break
stage (Fig. 1). At the pod color-break stage, when the moisture content
had declined to about 60%, a further increase in abundance began, continuing to a maximal level of the approximately 35-kD protein at
full-seed maturity. This coordinated expression between embryo maturation and desiccation is a typical property of LEA proteins (Hughes and Galau, 1989; Dure, 1993) and may be related to a function in protecting the embryo and cotyledons from damage during desiccation at maturity or during rehydration at germination. In this case, the
consequences are apparent under chilling conditions during seedling
emergence, particularly in seeds of low moisture content (Ismail et
al., 1997).
Cowpea seeds contained several proteins detectable by anti-dehydrin
antibodies, in addition to the approximately 35-kD dehydrin, which is
typical of the multigene nature of this family of proteins. The
approximately 35-kD protein appears to be the most abundant of these in
genotype 1393-2-11 by our western-blot assay. Some of the other
dehydrin cross-reactive proteins were collected from the chromatography
steps and may be studied in the future. However, the approximately
35-kD dehydrin is of particular interest because of the genetic
association that we demonstrated with seedling emergence under chilling
conditions. We have previously mapped the genetic determinant, which
controls the presence of the approximately 35-kD dehydrin in mature
cowpea seeds (data included in Menéndez et al., 1997). However,
it is not yet known whether this mapped locus is a structural gene for
the approximately 35-kD protein or a regulatory locus that controls
expression of the approximately 35-kD dehydrin. To differentiate
between these two possibilities, it is first necessary to obtain the
cDNA that matches the approximately 35-kD protein and then determine
its map position.
In the current study the approximately 35-kD protein was successfully
purified using the characteristic high-temperature solubility of
dehydrins as an initial enrichment step, followed by three sequential
chromatography steps involving cation-exchange,
hydrophobic-interaction, and anion-exchange chromatography. Compared
with the G50 maize dehydrin, which has been purified by Ceccardi et al.
(1994), the cowpea approximately 35-kD protein seems to be more
hydrophobic. Its elution from the Phenyl Superose column required a
gradient concentration of 0.54 to 0.27 M ammonium sulfate,
which is much less than the concentration for elution of the G50 maize
dehydrin, which eluted at about 1.0 M ammonium sulfate
(Ceccardi et al., 1994). Hydrophobic interactions were also considered
to be a possible explanation of the apparent adhesion of the
approximately 35-kD dehydrin to other proteins in fractions from
cation-exchange chromatography (Fig. 3). One possible explanation of
this apparent protein-protein adhesion is that, at high dehydrin
concentration and specific salt conditions, protein-protein complexes
may form that are not disrupted by SDS,  -mercaptoethanol, and
elevated temperature. Once the approximately 35-kD dehydrin has been
bound to and eluted from a hydrophobic-interaction chromatography
column, these putative protein-protein complexes are no longer evident.
The hydrophobic amino acid residues of the approximately 35-kD protein
constitute only approximately 10% of the total amino acid composition
(Table II). The capability of this protein for in vitro hydrophobic
interactions may involve the formation of an amphipathic -helix by
the K-segment, analogous to the lipid-binding domain of exchangeable
apolipoproteins, as suggested previously (Close, 1996). Evidence in
favor of the formation of lipid-bound amphipathic -helices was
obtained by measuring the CD spectrum of the approximately 35-kD
dehydrin in the presence of 10 mM SDS. As has been observed
in other studies where it was shown that several cereal dehydrins form
amphipathic -helices in association with SDS (T.J. Close,
unpublished data), the approximately 35-kD cowpea dehydrin also seems
to share this propensity. In the absence of SDS, the CD spectrum of the
approximately 35-kD cowpea dehydrin shows a strong negative band near
197 nm and a weak band at approximately 222 nm, which are
characteristic of polypeptides that lack a well-defined secondary
structure. These SDS-free CD data are equivalent to those obtained for
a recombinant Craterostigma plantagineum dehydrin purified
from an Escherichia coli expression strain studied in an
SDS-free aqueous solution, from which the authors concluded that the
native protein is generally unstructured (Lisse et al., 1996). However,
the apparent structure-promoting effect of 10 mM
SDS on the approximately 35-kD cowpea dehydrin (and others) suggests
that dehydrins in vivo may contain -helical structure(s) in a
lipid-bound state.
Several proteins contain lipid-binding class A amphipathic -helices
(Segrest et al., 1990) resembling the dehydrin K-segment. In addition
to exchangeable apolipoproteins, a more recently discovered analogy is
-synuclein. This protein has a role in both Alzheimer's and
Parkinson's diseases, in the former case as the nonamyloid component
of amyloid plaques and in the latter as a component of Lewy bodies. The
-synuclein protein binds to acidic phospholipids and vesicles with
small diameters, which is accompanied by pronounced -helicity
(Davidson et al., 1998). There are numerous additional examples of
proteins that appear to be "natively unfolded" in pure form but are
structured in association with ligands of various types, including
lipids, tubulin, and other proteins (for example, see table I of
Weinreb et al., 1996). Perhaps by exploring hydrophobic interactions between dehydrins and their ligands, the physiological roles of what have often been referred to as "extremely
hydrophilic" LEA and COR proteins (Thomashow, 1998) can also become
better understood.
Further genetic and biochemical studies are currently underway to
continue to test the apparent cause-and-effect relationship between the
approximately 35-kD dehydrin and seedling emergence under chilling
conditions and to define the interactions of the approximately 35-kD
protein with other molecules, whether they be free fatty acids,
membrane surfaces, proteins, or some combination.
| |
FOOTNOTES |
1
This research was partially supported by the
U.S. Department of Agricuture-National Research Initiative Competitive
Grants Program (award no. 94-37100-0688 to A.E.H.) and by the National Science Foundation (IBN 92-05269) to T.J.C.
*
Corresponding author; e-mail timclose{at}citrus.ucr.edu; fax
1-909-787-4437.
Received September 14, 1998;
accepted January 15, 1999.
| |
ABBREVIATIONS |
Abbreviations:
CD, circular dichroism.
CNBr, cyanogen
bromide.
| |
ACKNOWLEDGMENTS |
We thank Raymond D. Fenton for his excellent technical
assistance, Dr. Carl Frieden for advice on CD and for use of the
spectropolarimeter (Jasco) by T.J. Close while on sabbatical leave, and
Dr. A. Clay Clark for first pointing out the parallels between LEA
proteins and -synuclein.
| |
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Top
Abstract
Introduction