Plant Physiol. (1999) 120: 93-104
Pvlea-18, a Member of a New
Late-Embryogenesis-Abundant Protein Family That Accumulates during
Water
Stress and in the Growing Regions of
Well-Irrigated Bean
Seedlings1
José M. Colmenero-Flores,
Liz P. Moreno,
Claudia E. Smith2, and
Alejandra A. Covarrubias*
Departamento de Biología Molecular de Plantas, Instituto de
Biotecnología, Universidad Nacional Autónoma de
México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, México
 |
ABSTRACT |
Pvlea-18
is a novel stress gene whose transcript is present in the dry embryo
and the endosperm from bean (Phaseolus vulgaris) seeds.
It accumulates in vegetative tissues in response to water deficit and
abscisic acid application (J.M. Colmenero-Flores, F. Campos, A. Garciarrubio, A.A. Covarrubias [1997] Plant Mol Biol 35: 393-405).
We show that the Pvlea-18 gene encodes a 14-kD protein
that accumulates during late embryogenesis. Related proteins have been
detected in both monocots and dicots, indicating that PvLEA-18 is a
member of a new family of LEA (Late
Embryogenesis Abundant) proteins. We also show
that the PvLEA-18 transcript and protein accumulate not only in
different organs of the bean seedlings during water stress but also in
well-irrigated seedlings. This accumulation occurs in seedling regions
with more negative values of water and osmotic potentials, such as the
growing region of the hypocotyl. This phenomenon has not previously
been described for LEA proteins. Immunohistochemical localization
showed that the PvLEA-18 protein is present in the nucleus and
cytoplasm of all cell types, with a higher accumulation in the
epidermis and vascular cylinder tissues, particularly in protoxylem
cells and root meristematic tissues. We found a similar localization
but a higher abundance in water-stressed seedlings.
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INTRODUCTION |
LEA (Late Embryogenesis
Abundant) proteins are a broad family of plant proteins
that are stored in the dry seed. Their presence in vegetative tissues
is restricted to osmotic stress situations. Characteristically, they
are very hydrophilic and seem to be structured in an extended form
instead of being folded in a stable globular manner, as can be deduced
from the randomly coiled moieties of some LEA proteins and from their
boiling resistance (heat stability). These properties (which are
consistent with a role in binding water), together with their high
intracellular concentration and their expression patterns, have led to
the suggestion that LEA proteins can protect specific cellular
structures or ameliorate the effects of drought stress by maintaining a
minimum cellular water requirement (for review, see Dure, 1993a
; Ingram
and Bartles, 1996). Other hypothetical roles assigned to the LEA
proteins are sequestration of ions (Dure, 1993b), molecular chaperone
activity (Close, 1996), and transport of nuclear-targeted
proteins during stress (Goday et al., 1994).
Based on regions of significant similarity between
LEA proteins of different species (Dure, 1993a), five classes of
LEA proteins have been established (Ingram and Bartels, 1996). Two of
these have a functional role in stress tolerance: HVA1, a group 3 LEA protein from barley, and LE25, a group 4 LEA protein from tomato. Overexpression of HVA1 improves drought and salinity resistance in
transgenic rice plants (Xu et al., 1996). The LE25 protein was
expressed in yeast and found to confer improved resistance to high
salinity and freezing (Imai et al., 1996). During normal seed
development, most of the characterized LEA proteins have been located
in all embryo cell types (Close et al., 1993; Roberts et al., 1993;
Goday et al., 1994). However, tissue-specific and temporal-dependent
expression has also been observed in Rab28 (which is restricted to the
meristem and vascular elements of the plumule) and the root and
scutellum of the developing maize embryo (Niogret et al., 1996). Little
information is available concerning the localization of LEA proteins in
the vegetative tissues of plants subjected to water deficit. When
localization was investigated, LEA proteins were found predominantly in
the cytosol and nucleus of meristematic, vascular, and provascular cells (Mundy and Chua, 1988; Close et al., 1993; Godoy et al., 1994;
Houde et al., 1995; Niogret et al., 1996).
We previously described the water-stress and ABA inducibility of
Pvlea-18, a novel lea-like gene whose transcript
is stored in dry seeds (Colmenero-Flores et al., 1997). Here we report
evidence that the Pvlea-18 gene encodes a 14-kD protein that
establishes a new group of LEA proteins in plants. Additionally, we
show that the PvLEA-18 transcript and protein accumulate not
only in response to stress conditions but also during normal
development, particularly in the growing regions of bean seedlings.
Immunolocalization experiments indicate that this protein is present in
the cytosols and nuclei of different cell types along the bean
hypocotyl. The high accumulation of PvLEA-18 in the embryo radicle
during the early stages of germination suggests that it may have a
protective role during this process.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
Bean (Phaseolus vulgaris L. cv Negro Jamapa; National
Institute for Forest and Agricultural Research, México) seeds
were surface-sterilized in 10% (v/v) sodium hypochlorite for 10 min, rinsed in running tap water for 2 h, sown on water-saturated paper towels, and germinated in the dark at 27°C ± 1°C and 100%
RH. After 5 d, we selected seedlings for uniform size and
transplanted them to vermiculite containing different amounts of water.
The control growth condition was 5 mL of water per gram of vermiculite (
w = 
0.074 MPa). The water-deficit
conditions corresponded to w = 0.35 MPa,
one-twelfth the amount of water relative to the control. We maintained
the vermiculite at a constant w throughout the
experiment. Seedlings grew in the dark at 27°C ± 1°C and 75% RH. The duration of the water deficit in the different treatments appears in the text and in the corresponding figure legends. Plant material was frozen immediately in liquid nitrogen after harvesting and
stored at 80°C until extraction.
w and s Measurements
We determined w and s
using the dew-point method with the C-52 sample chamber, as described
in the instruction manual from the manufacturer of the dew-point
microvoltimeter (model HR-33T, Wescor, Logan, UT). We calculated the
p values from the experimentally obtained
w and s values
according to the equation p = s w.
Protein Purification and Antibody Production
The open reading frame encoded by the gene Pvlea-18
(Colmenero-Flores et al., 1997) was expressed in Escherichia
coli using the pGEX3 expression vector. The PvLEA-18 protein fused
to GST was purified by affinity chromatography using
glutathione-agarose beads and a standard method (Ausubel et al., 1994).
Antibodies were raised in rabbits by conventional procedures using
purified preparations of the GST-PvLEA-18 fusion protein (Harlow and
Lane, 1988a). Antibodies that specifically recognized PvLEA-18 were purified by immunoaffinity using the antigen GST-PvLEA-18 covalently coupled to bromine cyanide-activated Sepharose-4B (Sigma). The coupled
resin was packed in a column and used for the immunoaffinity purification as described by Harlow and Lane (1988b).
Protein Extraction and Analysis in SDS-PAGE
Plant tissues were homogenized in a mortar in the presence of
liquid nitrogen and extracted in a solution containing 10% (w/v) TCA
and 0.3% (v/v) 2-mercaptoethanol prepared in 100% (v/v) acetone. After the sample was centrifuged, the pellet was washed three times
with cold 100% acetone (20°C). The resulting pellet was vacuum-dried and resuspended in PBS buffer for concentration
measurement before 2 volumes of 2× Laemmli buffer was added (Laemmli,
1970). We analyzed total proteins by electrophoresis in SDS-PAGE
gels after boiling the extracts for 3 min (Laemmli, 1970).
Western Analysis
Proteins separated by SDS-PAGE were transferred to
nitrocellulose according to the method of Towbin et al. (1979). We used a 1:1,500 dilution of the anti-PvLEA-18 antibody and a 1:10,000 dilution of goat anti-rabbit IgG conjugated to peroxidase. An ECL
chemiluminescence kit (Amersham) was used to detect the cross-reacting polypeptides.
Quantitation of PvLEA-18 in Plant Tissues
We extracted the proteins from fresh plant tissues by the
TCA/acetone method, as described above. Different dilutions of the total protein extract were separated in 12% SDS-PAGE and transferred to nitrocellulose. We included different known amounts of purified GST-PvLEA fusion protein as an internal standard. The protein bands
reacting with the anti-PvLEA-18 antibody were analyzed by western blot
and developed using the ECL kit. We quantified the chemiluminescent
signal in the cross-reacting bands with a one-dimensional analysis
program (Bio Image Products, Millipore). We constructed a standard
curve from the values obtained for the different amounts of the
purified GST-PvLEA-18 fusion protein, which was used as the
internal standard. This allowed an accurate estimation of the amount of
the PvLEA-18 protein in the plant extracts. The molar concentration of
the PvLEA-18 protein was determined according to the water content in
the sample tissues.
RNA Isolation and Northern Analysis
We followed the procedure described by de Vries et al. (1991) to
prepare total RNA. Northern blots were carried out by electrophoresis of 5 µg of total RNA on 1.5% (w/v) agarose gels containing 1.1% formaldehyde, according to the method of Sambrook et al. (1989), and
transferred onto nylon membranes (Hybond N+,
Amersham). The hybridization and washes took place at high stringency as described by Church and Gilbert (1984). Filters were exposed on
Kodak XAR film at 80°C using an intensifying screen. We quantified the mRNA levels using the one-dimensional analysis program.
Labeling of Probes
The complete Pvlea-18 cDNA and a 3
-noncoding fragment
of the PvleaIV-25 cDNA clone (Colmenero-Flores et al., 1997)
were labeled with a commercial random primer kit (DuPont-NEN) using
[
-32P]dCTP (3000 Ci mmol
1) (Amersham).
Immunocytochemistry
We dissected 6-d-old etiolated seedlings (either well watered or
subjected to water deficit for 72 h) and 36-h-soaked embryos into
4- to 8-mm sections. The sections were fixed overnight at room
temperature in a solution containing 3.7% (v/v) formaldehyde, 50%
(v/v) ethanol, and 5% (v/v) acetic acid. The 1st h of the fixation
procedure took place under a vacuum. We dehydrated the fixed tissues
and embedded them in paraffin using the method described by Van de Wiel
et al. (1990). Seven-micrometer-thick sections were rehydrated and
preincubated overnight at 4°C in blocking buffer (5% BSA in
PBS/0.1% Tween 20). We subsequently incubated the sections at room
temperature for 3 h in a solution containing the anti-PvLEA-18
affinity-purified antibody, diluted 1:50 in blocking buffer. After the
sections were washed three times for 10 min each time with blocking
buffer, we incubated them with the secondary antibody coupled to
alkaline phosphatase for 1 h at room temperature. The complex
developed in 100 mM Tris, pH 9.5, 100 mM NaCl,
50 mM MgCl2, 0.2 mg
mL1 nitroblue tetrazolium, and 0.2 mg
mL1 5-bromo-chloro-3-indolyl phosphate. After
the sections were dehydrated through an ethanol series and two changes
of xylene, we mounted them in Permount.
| |
RESULTS |
Identification of the PvLEA-18 Protein
The identification of the PvLEA-18 protein and the study of its
expression during development and in response to environmental stress
required the production of polyclonal antibodies. With this aim, we
purified the PvLEA-18 protein from E. coli as a GST-fusion protein. The Pvlea-18 cDNA (Colmenero-Flores et al., 1997)
was subcloned into the pGEX3 expression vector, giving rise to a
translational fusion of the GST gene with the bean Pvlea-18
cDNA. After we verified the correct reading frame of the chimera by
sequence analysis, the gene was overexpressed in E. coli and
we used glutathione-agarose beads to purify the GST-PvLEA-18 fusion
protein by affinity chromatography. Purified fusion protein elicited
antibodies in rabbits, as described in ``Materials and Methods''. We
carried out all of the experiments in this work with polyclonal
antibodies purified by affinity chromatography, coupling the
GST-PvLEA-18 fusion protein to Sepharose-4B.
To identify the product of the Pvlea-18 gene, we subjected
bean seedlings to water deficit and obtained total protein extracts from roots, the organs that present the highest transcript level (Colmenero-Flores et al., 1997). The western blot in Figure
1a shows that the antibodies cross-react
with a 14-kD polypeptide, which, like the Pvlea-18
transcript, accumulated in response to water-stress conditions. To test
the specificity of the detection reaction, the purified GST-PvLEA-18
fusion protein was added as a competitor when the immunoblot was
incubated with the purified anti-GST-PvLEA-18 antibody. The results
shown in Figure 1b indicate that an incubation with 5 µg of
GST-PvLEA-18 protein was sufficient to completely block the
cross-reaction with the 14-kD polypeptide. To discard the interference
of antibodies raised against the GST domain of the fusion protein
during the immunodetection, we performed a similar experiment using the
purified GST protein as the competitor.
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| Figure 1.
Characterization of the anti-PvLEA-18 antibody.
Bean seedlings grown in the dark for 5 d were transferred to
well-irrigated or water-stressed vermiculite and harvested after
24 h of treatment. Total protein extracts were purified from
roots, separated by SDS-PAGE, transferred to nitrocellulose, and
incubated with antisera as follows: a, Immunodetection of PvLEA-18 in
protein extracts obtained from control (C) or water-deficient (WD)
tissues using immune, anti-PvLEA-18, or preimmune antisera. b,
Competition of the antiserum by preincubation with different
concentrations of purified PvLEA-18-GST fusion protein: 0 ng (lane 1),
50 ng (lane 2), 500 ng (lane 3), and 5 µg (lane 4). c, Competition
analysis of the antiserum by addition of purified GST protein in
different amounts: 0 ng (lane 1), 5 µg (lane 2), and 50 µg (lane
3). As a control, 5 µg of purified PvLEA-18-GST fusion protein was
added in lane 4. Numbers at the left indicate the corresponding
molecular masses in kD.
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The results in Figure 1c show that even 50 µg of GST was unable to
block the detection of the 14-kD protein, indicating that the detected
protein was not related to GST. Therefore, we can conclude that the
antibodies specifically recognized the PvLEA-18 protein. As indicated
above, the immunodetected signal corresponding to the PvLEA-18 protein
showed an apparent molecular mass of 14 kD, which did not correspond to
the 9 kD inferred from the protein deduced from the cDNA sequence. This
inconsistency could be explained by an anomalous migration in SDS-PAGE
given the PvLEA-18 amino acid composition. This possibility is
supported by the fact that the Pvlea-18 cDNA expressed in
Saccharomyces cerevisiae and E. coli also
produced a 14-kD protein that was immunodetected with the anti-PvLEA-18
antibodies (data not shown).
Accumulation of the Pvlea-18 Transcript and Protein
during Embryogenesis and Germination
We reported that the Pvlea-18 transcript accumulated in
response to water-deficit conditions and ABA treatment and that it was
stored in the dry bean seed. These data, together with the amino acid
composition of the deduced polypeptide, suggested that the
Pvlea-18 gene encoded a LEA-like protein (Colmenero-Flores et al., 1997). To define whether the PvLEA-18 protein was a bona fide
LEA protein, we analyzed the pattern of Pvlea-18 transcript and protein accumulation during embryogenesis. Figure
2 shows that the PvLEA-18 protein and its
transcript accumulated during late embryogenesis (after 20 d
postanthesis; Fig. 2). Embryos 20 d postanthesis initiated the
desiccation process and at 26 d postanthesis had reached maturity
and their lowest water content. By quantitation of the immunoreactive
proteins, we estimated that the concentration of the PvLEA-18 protein
was approximately 10 µM at this developmental
stage. These accumulation patterns allowed us to unambiguously classify
this protein as a bean LEA protein.
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| Figure 2.
Analysis of the accumulation of the
Pvlea-18 transcript and protein during embryogenesis. a,
Pvlea-18 transcript accumulation as determined by
northern analysis of RNA extracts obtained from bean embryos at
different days postanthesis (dpa): lane 1, 5 dpa; lane 2, 15 dpa; lane
3, 20 dpa; lane 4, 22 dpa; lane 5, 24 dpa; and lane 6, 26 dpa. Five
micrograms of total RNA was electrophoresed, blotted on nylon
membranes, and hybridized against the indicated probes. Hybridization
against a 28S-rRNA probe was used as an RNA-loading control. b,
Accumulation of the PvLEA-18 protein was analyzed by western blot of
total protein extracts obtained from the same samples described in a.
The molecular mass of PvLEA-18 is indicated on the right in
kilodaltons. Proteins were separated by SDS-PAGE and transferred to
nitrocellulose membranes before incubation with the immunopurified
anti-PvLEA-18 antiserum.
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We previously determined that the Pvlea-18 transcript
decreased gradually during the germination process: the transcript
levels remained the same as those in the dry seed during the first
12 h of imbibition and then decreased after 24 h, and no
transcript was observed after 48 h of imbibition (Fig.
3a; Colmenero-Flores et al., 1997). When
the analysis was extended during seedling establishment in the dark and
under optimal irrigation, a reinduction of the transcript occurred
after 72 h in the hypocotyl/radicle system (Fig. 3a) but not in
the cotyledons or plumules (Fig. 3b).
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| Figure 3.
Analysis of the accumulation of the
Pvlea-18 transcript during germination. Bean seeds were
germinated in the dark and seedlings were harvested at different times
after imbibition (0, 2, 3, and 5 d postimbibition). Five
micrograms of total RNA was purified from different seed or seedling
organs, blotted onto nylon membranes, and hybridized as indicated.
Hybridization against a 28S-rRNA probe was used as an RNA-loading
control. a, Northern-blot analysis of RNA extracts obtained from the
hypocotyl-root system. b, Northern-blot analysis of total RNA extracts
obtained from cotyledons and plumules.
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Analysis of the accumulation of the Pvlea-18 transcript in
the growing and mature regions of the hypocotyls and roots showed that
a transient reinduction took place at d 3 of imbibition in the
hypocotyl basal region (nongrowing) and in roots, whereas we detected a
reinduction only 1 d later in the hypocotyl-growing regions (Fig.
4, a and b). In the latter case, we
observed that the Pvlea-18 transcript levels progressively
increased, even 8 d after imbibition (data not shown). To
correlate the transcript levels with those of the protein, we performed
western blots using anti-PvLEA-18 and protein extracts obtained from
the same samples described above (Fig. 4). In contrast to the
transcript accumulation pattern, the PvLEA-18 protein levels remained
without a major change even after 2 d of imbibition (Fig. 4c),
when the transcript was no longer detectable (Fig. 4b). We observed a
similar phenomenon in the hypocotyl-growing regions after d 3 of
imbibition and again in cotyledons in which the PvLEA-18 protein was
still present even though its transcript was not detectable (Figs. 4, b
and c, and 5), which suggested that there
was a low turnover of the protein. We did not observe this
characteristic in the hypocotyl mature region or in the root at
these germination stages when the accumulation patterns for the
transcript and the protein are similar (Fig. 4, b and c).
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| Figure 4.
Analysis of the accumulation of
Pvlea-18 transcript and protein in roots and in
different regions of the hypocotyl during seedling establishment. a,
Schematic description of the hypocotyl regions used. E1 and E2,
Hypocotyl-growing regions (E2 shows the highest elongation rate); M,
nongrowing or mature zone. b, Northern-blot analysis of the
Pvlea-18 transcript. Bean seeds were germinated in the
dark and seedlings were harvested at different times (0, 2, 3, 4, and
5 d after imbibition). Five micrograms of total RNA was purified
from different seed or seedling organs and from the hypocotyl regions
indicated above, blotted on nylon membranes, and hybridized.
Hybridization against a 28S-rRNA probe was used as an RNA-loading
control. c, Western-blot analysis of the PvLEA-18 protein accumulation
from total protein extracts obtained from the same samples as described
in b. Numbers at the right indicate the corresponding molecular masses
in kilodaltons. Proteins were separated by SDS-PAGE and transferred to
nitrocellulose membranes before incubation with the immunopurified
anti-PvLEA-18 antiserum.
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| Figure 5.
Analysis of the Pvlea-18 transcript
and protein accumulation in cotyledons during seedling establishment.
a, Northern-blot analysis of the Pvlea-18 transcript.
Bean seeds were germinated in the dark and seedlings were harvested
after different times (0, 1, 2, 4, and 6 d postimbibition). Five
micrograms of total RNA was purified from the seed or seedling
cotyledons, blotted onto nylon membranes, and hybridized as indicated.
Hybridization against a 28S-rRNA probe was used as an RNA-loading
control. b, Western-blot analysis of the PvLEA-18 protein accumulation
from total protein extracts obtained from the same samples described in
a. Numbers at the right indicate the corresponding molecular masses in
kilodaltons. Proteins were separated by SDS-PAGE and transferred onto
nitrocellulose membranes before incubation with the immunopurified
anti-PvLEA-18 antiserum.
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Distribution of the Pvlea-18 Transcript and Protein in
the Different Hypocotyl-Growing Regions from Well-Irrigated and
Water-Stressed Bean Seedlings
The Pvlea-18 gene not only responded to water-stress
situations but was also reinduced in the hypocotyl during seedling
establishment. Because it is known that water status changes along the
stem region (Nonami and Boyer, 1993), we investigated in more detail
the water status of the different bean hypocotyl regions from
well-irrigated and water-stressed seedlings. For this, we divided the
bean hypocotyls into discrete regions: E1 and E2 corresponding to the
elongating regions (E2 was the section with the highest elongation
rate) and M, the most basal or mature region (Fig. 4a). The water
status of these regions was determined as described in ``Materials and Methods''. The results shown in Table I
indicate that the most negative values of p and s were found in the most apical regions of
well-irrigated and water-stressed seedlings. However, as expected, the
values for both potentials were lower in water-stressed than in
well-irrigated seedlings. Furthermore, the most apical region showed
the highest turgor pressure, which did not change after the
water-deficit treatment. In contrast, turgor in the mature region
showed an 8-fold decrease in water-stressed plants (Table I).
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Table I.
Water status of the hypocotyl regions
The data in this table are expressed as the means ± SD (shown in parentheses) from 10 measurements of three
independent experiments, according to Student's t
distribution analysis with 95% confidence intervals.
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We investigated the accumulation pattern of the Pvlea-18
transcript in the expanding hypocotyl regions described above. In agreement with the results in the previous section, Figure
6 shows that the Pvlea-18 mRNA
was present in the hypocotyl-elongating regions (E1 and E2) but not in
the nongrowing region (M) of nonstressed bean seedlings (Fig. 6a).
These results also showed that the highest accumulation of the
transcript occurred in the region with the highest elongation rate
(E2). In contrast, the Pvlea4-25 mRNA, corresponding to a
member of a typical LEA family, was not detected in any region from the
nonstressed seedlings (Fig. 6c). When seedlings grew under water-stress
conditions, both Pvlea-18 and PvleaIV-25 transcripts accumulated in all regions and had the highest levels in
the mature zone, the area with the lowest turgor values.
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| Figure 6.
Pvlea-18 transcript and protein accumulation
patterns in different hypocotyl regions from seedlings grown under
well-irrigated and water-stress conditions. E1 and E2,
Hypocotyl-growing regions; M, nongrowing or mature zone; R, root. a,
Northern-blot analysis of the Pvlea-18 transcript.
Five-day-old bean seedlings grown in the dark were transferred to
well-irrigated (C) or water-deficient (WD) vermiculite and harvested
after 24 h of treatment. Five micrograms of total RNA was purified
from the indicated seedling regions, blotted onto nylon membranes, and
hybridized against the indicated probes. Hybridization against a
28S-rRNA probe was used as an RNA-loading control. b, PvLEA-18 protein
accumulation pattern obtained by western analysis of total protein
extracts purified from the same samples described in a. The molecular
mass of PvLEA-18 is indicated on the right in kilodaltons. Proteins
were separated by SDS-PAGE and transferred to nitrocellulose membranes
before incubation with the immunopurified anti-PvLEA-18 antiserum. c,
Northern-blot analysis of the PvleaIV-25 transcript by
blotting total RNA obtained from the samples described in a. Five
micrograms of total RNA was blotted onto nylon membranes and hybridized
against the indicated probes. Hybridization against an 28S-rRNA probe
was used as an RNA-loading control.
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The analysis of the PvLEA-18 protein levels along the different
hypocotyl regions showed a similar accumulation pattern, i.e. the
protein accumulated in the growing but not in the mature regions of
well-irrigated plants. However, in contrast to its transcript, which
showed a higher accumulation in the E2 than in the E1 region, the
accumulation of the protein was similar in both sections (Fig. 6b).
Additionally, in water-stressed seedlings, the protein showed different
levels along the hypocotyl, presenting the highest accumulation in the
most apical region and the lowest in the basal zone (Fig. 6b), where we
had detected the highest accumulation of transcript (Fig. 6a). Roots
from water-stressed seedlings and the mature hypocotyl region had the
lowest protein-to-transcript ratio (Fig. 6, a and b).
Distribution of the PvLEA-18 Protein in Different Tissues of
Bean Seedlings
Given the differential accumulation of the PvLEA-18 protein during
normal development and stress conditions, we decided to investigate its
tissue distribution. The immunolocalization in Figure
7 shows that, in agreement with the
results obtained from the immunoblot-detection experiments, the
PvLEA-18 protein responded to water deficit and preferentially
accumulated in the most apical regions of hypocotyls (Fig. 7, a-h).
Additionally, the PvLEA-18 protein was present in all hypocotyl cell
types, although we did detect a preferential accumulation in the
vascular cylinder and epidermal tissues. This distribution was more
remarkable in the mature or nongrowing regions from stressed seedlings
because we detected less accumulation of the protein in the parenchymal
tissues, pith, and cortex (Fig. 7, g, h, and i, respectively). The
immunolocalization experiments also showed that the PvLEA-18 protein
was present in a high concentration in the nuclei of most cell types
(Fig. 8). Electron microscopic analysis
of mature embryo tissues supported these results by showing the
presence of the protein in cytoplasm and nuclei (data not shown).
Another characteristic of PvLEA-18 protein distribution was its
accumulation in protoxylem cells present in young, growing tissues
(Fig. 8, a and d) and in mature tissues (Fig. 8, e and f).
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| Figure 7.
Immunohistochemical detection of the PvLEA-18
protein in transverse sections of different hypocotyl regions from
well-watered and water-stressed seedlings using immunopurified
anti-PvLEA-18 antibody. Bean seedlings grown in the dark for 5 d
were transferred to well-irrigated or water-stressed vermiculite and
harvested after 36 h of treatment. Five-millimeter fragments
corresponding to the E1 (a-c), E2 (d-f), and mature (g-i) hypocotyl
regions were embedded in paraffin for subsequent immunohistochemical
analysis of microtome sections. Sections from well-irrigated seedlings
are shown in a, d, and g; water-stressed sections are shown in b, e,
and h; and water-stressed sections incubated with preimmune antiserum
are shown in c, f, and i. c, Cortex; e, epidermis; p, pith; vc,
vascular cylinder.
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| Figure 8.
Immunohistochemical detection of PvLEA-18 in
protoxylem cells and nuclei from root and hypocotyl tissues using
immunopurified anti-PvLEA-18 antibody. Tissues were dissected and
soaked in paraffin as described in Figure 7. a and c, Sections
from the hypocotyl-elongating region E1 obtained from water-stressed
seedlings corresponding to ×2 and ×5 magnifications of Figure 7b,
respectively. b, Section from the hypocotyl mature region obtained from
water-stressed seedlings corresponding to a ×2 magnification of Figure
7h. d, Section from the emerging hypocotyl obtained from well-irrigated
seedlings after 36 h of imbibition. e, Section from a root
obtained from well-irrigated seedlings grown as described in Figure
7. f, Detail of a protoxylem cell from the mature region of the
hypocotyl of a 6-d-old well-irrigated seedling. Arrows indicate
protoxylem cells and arrowheads indicate nuclei. c, Cortex; e,
epidermis; p, pith; vc, vascular cylinder; x, xylem.
|
|
The immunolocalization results in Figure
9 show that in nonstressed seedlings the
PvLEA-18 protein was abundantly and homogeneously distributed in the
radicle during the early stages of germination (36 h of imbibition)
(Fig. 9a). In contrast, the PvLEA-18 protein was barely detectable in
roots from the older, well-irrigated seedlings (6 d postimbibition;
Fig. 9b), in agreement with the results obtained from western-blot
analysis (Fig. 4c). As in the mature region of water-stressed
hypocotyls, the PvLEA-18 protein in roots responded to water deficit,
showing a higher accumulation in the epidermis and vascular cylinder
(Fig. 9c). Emerging secondary roots had a high accumulation and a
uniform distribution of PvLEA-18 (Fig. 8a). The same characteristics
can be seen at the tip of water-stressed secondary roots (Fig. 8d) as
can be seen at their basal region: as in the hypocotyl, the protein
accumulated preferentially at the epidermal and vascular tissues. The
magnification in Figure 8f shows that, although the PvLEA-18 protein
was present in the root tip, it could not be detected in the root cap
cells.
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| Figure 9.
Immunohistochemical detection of PvLEA-18
protein in root tissues from well-watered and water-stressed seedlings.
Tissues were dissected and soaked in paraffin as described in Figure
7. a, Section from a radicle obtained from germinating seedlings
grown under well-irrigated conditions. Seeds were germinated in the
dark and harvested 36 h after imbibition. b, Section from a root
obtained from well-irrigated 6-d-old seedlings. Root sections were
obtained from the same seedlings transplanted to well-irrigated
vermiculite described in Figure 7. c, Section from a root obtained
from water-stressed 6-d-old seedlings. Root sections were obtained from
the same seedlings transplanted to water-limited vermiculite described
in Figure 7. d, Longitudinal section of a lateral root from a
water-stressed seedling. e, Longitudinal section of a lateral root from
a water-stressed seedling corresponding to the section described in d
incubated with preimmune antiserum. f, Detail of the secondary root tip
corresponding to a ×2 magnification of d. Arrows indicate protoxylem
cells and arrowheads indicate nuclei. c, Cortex; e, epidermis; lr,
lateral root; n, nucleus; p, pith; rc, root cap; rm, root meristem; vc,
vascular cylinder.
|
|
The PvLEA-18 Protein Is a Member of a Novel Family of LEA Proteins
Previous analysis of the deduced amino acid sequence of
the PvLEA-18 protein did not show significant homology with known LEA
proteins (Colmenero-Flores et al., 1997). We investigated the
possibility that the PvLEA-18 protein is a member of a new family of
LEA proteins by looking for homologous proteins in other plant species,
using the anti-PvLEA-18 antibodies for detection. As shown in Figure
10, related proteins were identified in
total protein extracts obtained from seeds of tobacco, tomato, soybean, pea, maize, and Arabidopsis. The cross-reacting proteins from the
different plant species varied in their molecular masses, from higher
than 97 kD to close to 14 kD (Fig. 10, lanes 4-10). Characteristically, for several LEA protein families described, polypeptide sizes were variable among members of the same family (Close
et al., 1993; Houde et al., 1995; Ingram and Bartels, 1996). Other
bean cultivars also had high-molecular-mass PvLEA-18-related proteins
(Fig. 10).
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| Figure 10.
Detection of PvLEA-18-related proteins in
different plant species by western analysis. Protein extracts obtained
from the seeds of the different species described below were separated
by SDS-PAGE and transferred to nitrocellulose before incubation with
the immunopurified anti-PvLEA-18 antiserum. PvLEA-18 cross-reacting
proteins in dry seeds from: lane 1, cv Cacahuate of bean; lane 2, cv
Flor de Mayo of bean; lane 3, cv Negro jamapa of bean; lane 4, tobacco;
lane 5, maize; lane 6, Arabidopsis; lane 8, tomato; lane 9, soybean;
and lane 10, pea. For S. lepidophylla, the protein
extract was obtained from vegetative tissues of desiccated whole plants
(lane 7). Numbers at both sides indicate the corresponding molecular
masses in kilodaltons.
|
|
We obtained similar results from the immunoblot analysis of total
protein extracts from desiccated tissue of a representative of an
ancestral vascular plant, Selaginella lepidophylla
(Lycopodiophyta) (Fig. 10, lane 7). In some cases, such as in maize and
soybean, we detected an 18-kD protein when seedlings were subjected to water stress (data not shown). The existence of similar proteins is
supported by the presence of a homologous open reading frame (40%
identity and 46% similarity) in the Arabidopsis genome (accession no.
U78721/ATU78721), as well as a similarly expressed sequence tag gene
(accession no. AA395541) from Arabidopsis (34% identity and 44%
similarity) (Fig. 11a). This homology
was significant in the central Gly-rich domain. Furthermore, these
proteins presented very similar hydropathic patterns (Fig. 11b). Taken
together, these data suggested that the PvLEA-18 protein is a
representative member of a novel family of LEA proteins.
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| Figure 11.
Putative PvLEA-18 homologous proteins from
Arabidopsis. a, Alignment of the amino acid sequence predicted from the
bean Pvlea-18 cDNA with the amino acid sequence deduced
from Arabidopsis genomic (Atlea-18GEN) and partial cDNA
sequences (Atlea-18EST). b, Comparison of the
corresponding hydropathy profiles.
|
|
| |
DISCUSSION |
In a previous report we described a novel stress gene
from common bean whose transcript accumulated in dry seeds and in
different organs of plants subjected to water-deficit conditions or ABA treatment. Although this gene and its deduced protein did not show
significant similarity with any known genes or proteins in the
database, analysis of the protein sequence and amino acid composition
revealed structural characteristics similar to those of the LEA
proteins, such as high hydrophilicity, high content of Gly and
charged amino acids, and high percentages of randomly coiled moieties.
Given the characteristics of the putative protein and the accumulation
pattern of its transcript, we named the corresponding gene
"Pvlea-18" (Colmenero-Flores et al., 1997). In this work we show that the PvLEA-18 protein is a bona fide LEA protein, because,
in addition to its response to dehydration and ABA treatment, it also
accumulated in the seed during the final stage of embryogenesis. Also,
as was the case for other lea genes (for review, see Dure, 1993b; Ingram and Bartels, 1996), the Pvlea-18 transcript
accumulated to high levels in dry seed and decreased to undetectable
levels during the germination process (Figs. 3 and 4).
Analysis of the protein-accumulation pattern revealed that, although
the transcript was not detected after 2 d of imbibition, the
protein maintained levels comparable to those present in the dry seed.
A similar phenomenon occurred in the emerging hypocotyls 3 d
postimbibition (Fig. 4) and in cotyledons, in which the transcript was
undetectable after 2 d of seed imbibition, although the protein was present even after 6 d (Fig. 5). These observations suggest the existence of posttranscriptional mechanisms favoring differential turnover rates of the protein and/or the transcript in these regions during seedling development (see below).
Although LEA proteins have been associated with dehydration, seed
development, and adverse environmental conditions, we show in this work
that the PvLEA-18 protein and its transcript also accumulated during
the growth of etiolated bean seedlings under conditions of optimal
irrigation. In etiolated seedlings with no expanded leaves,
evapotranspiration in the apical zone was not a major driving force for
water transport. However, a w gradient must be
generated along the hypocotyl to ensure water flux (Meyer and Boyer,
1972). It has been proposed that this w
gradient was generated by the accumulation of osmolytes in the apical
zones, which is also important for the maintenance of cell growth in growing tissues (Meyer and Boyer, 1972, 1981; Creelman et al., 1990;
Nonami and Boyer, 1993).
As indicated above, the Pvlea-18 transcript decreased to
undetectable levels during the germination process; however, it was transiently reinduced in the radicle, the earliest emerging organ, and
hours later in the emerging hypocotyls, where we detected a progressive
increase in the transcript levels (Fig. 4). The possibility that this
transcript reinduction was related to the elongation process under
optimal growth conditions was investigated by looking at the
transcript-accumulation pattern along the different hypocotyl-growing
regions. The data described in ``Results'' and shown in Figures 4 and
6 indicate that the Pvlea-18 transcript accumulated in those
hypocotyl regions that were in active elongation (E1 and E2).
Furthermore, the fastest-elongating zone, E2, coincided with the
highest Pvlea-18 transcript accumulation. A similar
accumulation pattern was observed for the protein, although the
protein-to-transcript ratio in the most apical region (E1), which
exhibited the lowest s and
w, was higher than that in the region below
(E2). As indicated above, a similar situation (a high
protein-to-transcript ratio) was observed in emerging hypocotyls (Fig.
4) and in cotyledons (Fig. 5) during seedling establishment. It is
interesting that the lowest osmotic potentials were detected in
cotyledons (data not shown). These results led us to the hypothesis
that the low turnover of the PvLEA-18 protein is associated with a
stabilization mechanism that is favored in tissues with a high
concentration of osmolytes.
We observed a different transcript accumulation pattern in seedlings
grown under water-deficit conditions (Fig. 6). The Pvlea-18 transcript accumulated in all hypocotyl regions, presenting its highest
levels in the most basal or mature region, the one with less turgor
under water stress, and where no transcript was detected in
well-irrigated seedlings. The high transcript accumulation in the
low-turgor regions has also been observed for other genes whose
expression is induced under drought conditions (J.M. Colmenero-Flores, unpublished results). Even though the PvLEA-18 protein also accumulated in all hypocotyl regions under water-deficit conditions, it showed higher levels in the most apical regions, again suggesting a
correlation with the osmotic status. The fact that transcript
accumulation occurred in the growing regions of well-irrigated and
water-stressed seedlings suggests that the Pvlea-18 gene is
modulated by at least two different control mechanisms, one that is
mediated by ABA during water stress (Colmenero-Flores et al., 1997) and
another possibly mediated by hormones involved in growth induction.
In both cases the stimuli could be the low
s. The Pvlea-18 expression pattern
resembles that of some plant aquaporin genes that are preferentially
expressed in zones of cell division and elongation, as well as in the
epidermis and youngest portions of the xylem (Yamada et al., 1995;
Barrieu et al., 1998; Chaumont et al., 1998).
The PvLEA-18 protein-immunolocalization experiments clearly showed that
many cell types are able to accumulate the protein in their cytoplasm,
even in well-watered seedlings. The highest accumulation of the protein
occurred in cells from the epidermis and the vascular cylinder (Figs. 7
and 8), tissues that may have exhibited more negative
w because they were more exposed to the changing environment (Nonami and Boyer, 1983; Davies, 1986). The accumulation of the PvLEA-18 protein in the apical regions of elongating roots (radicle and lateral roots) from either stressed or
nonstressed seedlings could also be related to the low
s present in the emerging roots (Creelman et
al., 1990). The absence of the protein in the root cap cells indicates
that its accumulation was in response to specific regulatory factors.
We also observed a high accumulation of the PvLEA-18 protein in
immature xylem cells in hypocotyls and roots (Fig. 8). Although we do
not know the role of this protein during xylogenesis, its accumulation may be related to the osmotic status of this cell type. It has been
proposed that high concentrations of sugars, auxins, and cytokinins are
needed to induce xylem development (Aloni, 1987). In addition, a high
concentration of potassium ions has been detected during xylogenesis
(McCully, 1994), suggesting low s
during the development of this cell type.
The presence in Arabidopsis of genes encoding putative
PvLEA-18-homologous proteins, together with the detection of
PvLEA-18-immunorelated polypeptides in protein extracts from different
plant species, such as Arabidopsis, maize, tomato, tobacco, pea,
soybean, and the primitive vascular plant S. lepidophylla,
indicates that PvLEA-18 is a member of a novel LEA protein family. Even
though the putative proteins deduced from the expressed sequence tag
and genomic sequences in Arabidopsis have low molecular masses,
polypeptides of this size were not detected in the western-blot
experiments. Because the protein extracts analyzed were obtained only
from seeds, we cannot discard the presence of the expected
low-molecular-mass proteins in tissues from different developmental
stages or growth conditions. More experiments are needed to clarify
this observation.
The fact that the PvLEA-18 protein belongs to a different LEA protein
group than the ones described indicates that it may carry out a
different function. Although the Pvlea-18 expression pattern
and protein localization during water-deficit conditions were very
similar to other lea genes reported (Mundy and Chua, 1988;
Close et al., 1993; Godoy et al., 1994; Houde et al., 1995; Ingram and
Bartles, 1996; Niogret et al., 1996), no other LEA protein has been
found during seedling establishment and growth under optimal irrigation
conditions. These characteristics seem to be particular for the
Pvlea-18 gene and protein. The possibility that other
proteins from the PvLEA-18 group present a similar behavior awaits
testing.
Finally, the presence of PvLEA-18 in cells with high osmolyte
accumulation (embryos, germinating embryonic axes, hypocotyl growing
tissues, root tips, cotyledons, and water-stressed tissues) may be
related to the capability of this loosely structured hydrophilic protein to capture water and/or to a protective function in conditions of low w. The ubiquitous presence of PvLEA-18
among many cell types and in different cell compartments, such as the
cytoplasm and the nucleus, suggests a nonspecific protective role.
Experiments are being carried out to elucidate the function of PvLEA-18
and the factors involved in the regulation of its expression.
| |
FOOTNOTES |
1
This work was partially supported by grants from
the Consejo Nacional de Ciencia y Tecnología, México (no.
0131P-N), and from the Dirección General de Asuntos del Personal
Académico, Universidad Nacional Autónoma de México (no.
IN204496 to A.A.C.).
2
Present address: Max Planck Institut für
Züchtungsforschung, Carl von Lineé-Weg 10, D 50829 Köln, Germany.
*
Corresponding author; e-mail crobles{at}ibt.unam.mx; fax
52-73-13-9988.
Received September 15, 1998;
accepted February 2, 1999.
| |
ABBREVIATIONS |
Abbreviations:
GST, glutathione S-transferase.
p, pressure potential.
s, osmotic
potential.
w, water potential.
| |
ACKNOWLEDGMENTS |
We thank G. Cassab and S. Gilmore for critical reading of the
manuscript. We also thank P.C. Zambryski for her support during the
establishment of the microscopic techniques, R.M. Solórzano for
technical assistance, P. Gaytán and E. López for the
synthesis of oligonucleotides, and E. Mata for animal care during the
antibody production. We are also grateful to N. Capote for her constant support.
| |
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Top
Abstract
Introduction
