The Role of Aquaporins in Water Balance in Cheilanthes lanosa (Adiantaceae) Gametophytes

2012·11.3 MB·English

by  Hope L Diamond

The
Role Aquaporins Water
of Balance
in in
Cheilanthes lanosa (Adiantaceae) Gametophytes
Water and
osmoregulation has been a challenge since the beginning of
cellular life. The first protocells would have had to constantly control the
and
solutes water content of the cytoplasm to maintain Therefore, seems
life.
it
plausible that all bacterial, protozoan, animal, fungal, and plant would
cells
have and should contain highly conserved mechanisms move water
still to in
and out of cells (Chrispeels and Agre, 1994; Chrispeels and Maurel, 1994;
Maurel,
1997).
Water-specific channels for water balance, aquaporins, exist in every
kingdom and And, common
although
species. sense
calls for the existence of
such a practical mechanism, discovery of these channels was
difficult. Indirect
evidence demonstrated the rationale for such a simple mechanism
at least
twenty
years before their discovery. Philip (1958) reviewed the data on water
movement
and
across lipid bilayers, averred that the actual rate of water
movement much
more what
into cells is rapid than should occur across a lipid
were movement
bilayer. Yet, future botanists taught water
all that occurs
through osmosis across a plant plasma membrane. Finally, in 1984, aquaporins
were
concurrently discovered in bovine eyes (Gorin and
al, 1984)
erythrocytes (Macey, Agre Very
1984; et 1987). quickly, aquaporins were
al.,
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identified in rat kidney tubules, fungi, bacteria, and of course, plants
(reviewed Agre and
in et 1995; Chrispeels Agre,
al., 1994].
One
impediment to the discovery of aquaporins was Any
specificity. water-
specific channel would have exclude which
to ions, are smaller than water
molecules. Confusion
over potential structure helped delay the search
for a
water control mechanism. However, following the discovery of aquaporins, the
how
elucidation of the structure revealed an organism could move
effectively
water and exclude The membrane
ions. proteins are a constructed from
six
regions that form a spiral internal channel (Fotiadis et al, 2001]. Water, which
is angled, flows through the spiraled channels but smaller molecules, such
as
sodium and
potassium, cannot pass through the center (Fotiadis et ah, 2001).
Aquaporins
by
are controlled a phosphorylation switch (Johnson and
Chrispeels, Johansson and
1991;
et al., 1996, 1998], thus, cells are in control
of water movement. The channels may
are also bidirectional; water flow out of
Tyerman
cells or into cells (Steudle, 1992; For
et 1999]. plants, this a
al., is
perfect control mechanism. Cytoplasm solute control and aquaporin gating
combine
allow
to a functional cytoplasmic environment within a range of
conditions (Kaldenhoff Maurel
et al., 1993, 1995; et 1995; Maurel, 1997].
al.,
Studies on aquaporins have many
resolved complicated
questions regarding
plant functions. Aquaporins xylem and phloem and
exist in help prevent
cavitation (Voicu et al, 2009]. Aquaporins exist in root cortical and
endodermal and
help
cells generate or resolve pressure of water flow
(Vandeleur et 2005; Steudle and Peterson, Aquaporins guard
al., 1998]. in
cell tonoplasts respond to changes in blue light and abscisic acid and allow
water flow and
into out of the vacuole (Kaldenhoff Aquaporins
et al, 1993]. are
movement
also involved water and management
in during water (Suga
stress
et 2002].
al.,
Based on above we may
the findings, hypothesized aquaporins
that play a
key
role in the survival of fern gametophytes in xeric environments
as
mechanisms
water management. To we
for hypothesis, chose
test this a
common
North American
xerophytic fern, Cheilanthes lanosa (Michx.] D.C.
study because
Eat. for this inhabits a variety of substrates (Mickel,
it 1979],
though commonly among
found
other xerophytic species (Mohlenbrock,
it is
many
and
possesses
1959], of the features that other xerophytic ferns possess,
such
microphylly
as (Pickett, 1931; Hevly, 1963; Quirk and Chambers,
1981),
trichomes (Quirk and Chambers,
mycorrhizal and
1981), associations (Palmieri
on
Swatzell, 2004], cuticle the gametophyte (Lingle et al, 2004), and the ability
to regenerate after desiccation (Diamond et al, 2003). In addition, like many
other xerophytic gametophytes, lanosa gametophytes apogamous
C. are
(Steil,
The
1933, 1939; Hevly, 1963). gametophytes are also not heavily covered with
wax, and
are one-cell in thickness, appear generally unprotected from
their
surroundings. Thus, the lanosa gametophytes could
C. represent the
We
numerous
physiological state of xerophytic ferns. predicted that was
if
it
we
possible to identify aquaporins in this fern gametophyte, and could
if
poison these proteins with low levels of mercury, which blocks the protein
channel Kuwahara we
(Preston et al, 1993; et al, 1997), could effectively
DIAMOND ET AQUAPORINS CHEILANTHES LANOSA
AL.: IN
movement
control water out of desiccating This would
suggest
cells. at least a
potential role for aquaporins in xerophytic gametophyte water management.
Materials and Methods
To we
test our hypothesis, examined different developmental stages and in
microclimates
different for the Cbeilanthes lanosa gametophyte. Each
development and
stage microclimate condition will heretofore be designated
simply
as a "stage."
Plant Collection
Cbeilanthes lanosa sporophylls were collected in the fall of 2003 after the
from Makanda, cm
placed and
first frost Illinois, in glass 9 Petri dishes, stored
in the dark at 4°C. After several months, sporophylls were crushed using a
mortar and
pestle. Cheilantbes lanosa spores average 40 |im in diameter (Devi
and
et al., 1971] thus spores were separated from the plant material using a
mesh
65 )im brass Spores were C
sieve. stored at 4 in the dark.
Culture Conditions
Wet grown (WG) gametopbytes and protonemal were
callus (callus).—
S^pores
7%
surface sterilized in a (v/v) commercial bleach solution with 0.1%
(v/v)
Triton X-100 for 10 min. Spores were then rinsed ddHaO and sown on
in sterile a
mM mM mM
medium
modified tissue culture (TCM; 20 NH4NO3, 20 KNO3,
1.5
mM
MgS04-7H20, MnS04-H20,
1.0 30.0 ^iM ZnS04-7H20, jiM CuS04-5H20,
0.1
mM
mM mM
CaCl2-2H20,
3.0 5.0 [iM KI, 0.1 |iM CoCl2-6H20, 0.8 KH2PO4, 0.9
mM
mM
^M
H3BO3, Na2Mo04-2H20,
FeS04-7H20, Na2EDTA,
1.0
0.1 0.1
^M
0.23 )iM kinetin, 0.86 ^iM 2,4-D, 0.4 nicotinic acid, 0.3 jiM pyridoxine, 1.3 ^iM
mM
pH
cm
thiamine, 0.56 myo-inositol, 5.7; Smith, 1992) in 9 Petri dishes.
Spores were incubated at 25°C in 0.175 nmol-m"^-s^^ of continuous red
far light
nm)
(650-705 10
for
d.
Following germination and protonemal development, were
the plates
Some
were
separated. plates left in the far red light to enhance callus growth
and
the remaining plates were then exposed continuous white and
to light the
protonema began growth
planar into gametopbytes.
Dry grown (DG) gametopbytes.—Dry grown sown
gametopbytes were on
fine
grain white sand (Decor Sand, Activa Products USA)
Marshall, Texas,
Inc.,
WG
mL TCM
wetted with 20 of and incubated as the and callus cultures
DG
upon
described above. Thereafter, drying, gametopbytes wetted
erratically
with ddH20.
Antibody
Production
To
establish the molecular weight of the ensure
target protein, specificity of
binding of the anti-aquaporin antibody, and to detect the presence of
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aquaporin-like proteins in gametophytes, an antibody against maize PIPl
aquaporin was raised using homologous sequence from mapped
a previously
aquaporins maize (Chaumont
in et 2000). Rabbit anti-aquaporin antibody was
al.,
produced
by Biosource USA)
(Invitrogen; Biosource, Camarillo,
California,
against a conserved amino terminus sequence of PIPl maize aquaporin,
MEGKEEDVRVGANKFPERQPIGTSAQS
Chaumont
described by
as
et al. (2000).
ELISA
(enzyme-linked immunosorbent
assay)
In order to test for the presence of aquaporin-like proteins, an enzyme-linked
immunosorbent was
assay performed wide
to detect a range of aquaporin
WG
mg
concentrations in various gametophyte stages. Approximately 20 of
mM
gametophyte was homogenized
material 200
in of coating buffer
\xL (15
mM
pH
NasHCOa. NaHCOg,
35 mortar and The sample was
9.5) in a pestle.
rpm
then centrifuged at 10,000 for 10 min. The supernatant was retained and
rpm
centrifuged again at 10,000 for 10 min. The sample was then diluted in
1 ml of coating buffer. The concentration was approximately of material
3 ^g
for every 50 of coating buffer. The antigen-coating buffer solution was then
fiL
pipetted in 50 increments into a 96-well polystyrene microtiter The
plate.
was
plate covered and allowed to incubate overnight 4°C. The following
at
morning, the antigen solution was removed by inverting the plate and washing
mM mM
three times with Tris-Tween washing (TTW;
buffer 10 150 NaCl,
Tris,
pH
0.1%
Tween-20, 7.2). In all the washing steps, the wells were completely
3%
(-350
filled ^L). Plates were blocked with (w/v) low-fat milk powder in
TTW
for 30 min. The wells were then washed three times with TTW. Once the
TTW
was
removed, 100
of rabbit anti-aquaporin Biosource,
|iL (1:500;
USA) TTW,
Camarillo, California, preimmune serum
in rabbit
(1:500;
TTW
USA) TTW,
Biosource, Camarillo,
California, in or only (secondary
antibody only control) was placed into the wells and allowed incubate
to for
TTW
60 min. The wells were washed three times with TTW. The was removed
and
50 nL of goat anti-rabbit horseradish peroxidase Sigma-
Aldrich,
(1:500;
St.
TTW
USA) TTW,
Louis, Missouri,
in or only (primary antibody only
control)
was added and allowed to incubate for 60 min. The wells were again washed
TTW.
three times with The chromogenic enzyme was
reaction then
initiated
by mg
addition of freshly prepared solution of 1.5 o-phenylenediamine and
M
30% mL
H2O2
2 |iL of (v/v) dissolved in 2 of citrate-phosphate buffer
(0.2
M
pH
Na2HP04,
0.1 citric acid, 5.0) at 50 |iL per well. H2O2 was added
immediately The enzyme was
prior to use. reaction allowed incubate
to for
M
min and was
15 the reaction halted by the addition of 50 ^L of 0.5 H2SO4
nm
per well. The plate was then read 492 on a Beckman DU640B
at
spectrophotometer (Beckman
USA).
Coulter, Fullerton, California,
Immunoblotting
To
establish the molecular weight of the protein and ensure
target to
specificity of binding of the anti-aquaporin antibody, immunoblotting was
DIAMOND
ET AQUAPORINS CHEILANTHES LANDSA
AL.: IN
DG
WG,
performed. Approximately 100 of Cheilanthes lanosa callus, or plant
mM mM mM
was
material ground in homogenization buffer [10 KCl, 1 MgCla, 50
mM
mM
HEPES, BSA pH
300 sorbitol, 0.1% (w/v) and EDTA, Samples
1 to
7.2].
were mixed 2X Laemmli 4%
sample [Sambrook
1:1 buffer 1989; (w/v) SDS,
et
al.,
10% 20%
mercaptoethanol, 0.002% Bromphenol
(v/v) (w/v) Blue, (v/v) glycerol,
mM
pH
120 incubated 10 min 90 rpm
Tris, 6.8], for at C, centrifuged at 10,000 for
10 min. Total protein concentration was determined using the Bradford Assay
12%
(Stoschek, 1990). Samples were loaded onto a (w/v) polyacrylamide
gel at
and ^g/mL
35 per well 20 Samples were
concentration. electrophoresed
jil at
mA
A
16 constant current for approximately 30 min. broad range marker
(Kaleidoscope; BioRad, Hercules, USA) was used
California, for reference.
mA
Proteins were then transferred at 45 constant current for 20 min onto a
membrane
nitrocellulose ^im pore Following the membranes
(0.2 size). transfer,
mM mM mM
TTBS
were
rinsed three times in [137 NaCl, 2.7 KCl, 24.8 0.2%
Tris,
Tween 3%
and blocked in (w/v) bovine serum albumin (BSA) TTBS.
(v/v)], in
Membranes were then rinsed three times min each TTBS. Membranes
for 5 in
were then incubated overnight 4'C preimmune serum
at in either (BioSource,
USA;
Camarillo, California, 1:500 in TTBS), rabbit anti-PIPl aquaporin
USA; TTBS
(BioSource, Camarillo, Cahfornia, 1:500 in TTBS), or only (secondary
antibody only Membranes were min TTBS.
control). again rinsed 3 times for 5 in
membranes
Following the were incubated
rinse, in goat anti-rabbit alkaline
TTBS;
phosphatase (1:500 in Sigma-Aldrich, Louis, Missouri, USA), with the
St.
exception of the primary antibody only which was incubated TTBS
control, in
each h Membranes were min
only, for 1 at 25°C. rinsed three times for 5 each in
mL
TTBS. The membrane was
then colorized with a fresh mixture of 20 of
mM mM mM
phosphate MgC^, pH
alkaline buffer (100 NaCl, 5 100 Tris,
9.5),
132 [iL of nitro blue tetrazohum (NBT) stock, and 66 ^L of 5-Bromo-4-chloro-3'-
indolyphosphate P-Toluidine (Sambrook
(BCIP) stock et 1989). After agitation
al.,
for 5 min, the membrane was briefly rinsed with ddHaO and the reaction was
mM
stopped with 20 ethylene diamine acid (EDTA).
tetraacetic
Osmolality
Stress
In order to test for aquaporin function and potential control of function
in
gametophyte gametophytes were exposed
stages, to desiccating levels of
osmolytes with and without mercury exposure. Although the preferred
test for
mRNA
Xenopus
function of aquaporins expression of in oocytes (which do
is
mRNA
not express aquaporins), PIPl does not express well in oocytes (Preston
1992). Therefore, another standard, the immersion samples
of
al., in
mM;
mercuric chloride low Macey, was Wet grown
at levels 1984) used.
(1
(WG) gametophytes, callus and dry grown (DG) gametophytes were exposed
to
One
2 separate treatments. treatment consisted of time increments of NaCl,
GaGl2, or sucrose and was used to show gametophyte response long term
to
environmental Gametophytes were
increasing stress. pretreated in 200 well
|iL
mM
ddHzO ddH^O
slides in 100 of or plus HgCl2 20 min. Gametophyte
1 for
images were captured At and subsequently every min
digitally. 5 afterward,
to,
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1
mM
50 increments of the respective solute were added
until the solution in the
mM
well slide reached 500 osmolality. Images of gametophytes were captured
The
second was show
after h. treatment used gametophyte
1 to response
to
immediate
stress. This treatment began with a 20 min pretreatment in a 200
mM
well
slide in 100 isotonic solute solution (NaCl,
CaCls, or sucrose) or
mM
mM
100
isotonic solution plus HgCl^. Gametophyte images were
1
mM
captured digitally. At 400 of solute in 100 of ddHaO was added
to, |iL to
mM
the well so that the solution in the well reached 500 osmolality. Images of
the gametophytes were
captured
after 1 h.
volume was
Cell determined using six diameter measurements. Average
radii were used to extrapolate the sphere volume and Only
surface hourly
area.
were
rates of efflux calculated. This because diffusion out of these was
is cells
slow enough
to allow the necessary resolution demonstrate
to loss of function
of water channels under mercury
poisoning. Well were
slides incubated in a
moisture chamber
in the interim times between increments prevent water
to
from The
loss the wells. efflux of water out of each of 100 from each
cells
was
treatment measured
using Law:
Pick's
Flux=
-PS[(Osmo-Osmi)/D]
Where =
-P permeability (negative because
against the concentration
gradient),
= =
S Osmo =
surface area, osmolality outside of cell, Osmi osmolality inside
D =
and
of cell, distance (modified from Qui
et 2000).
al.,
Data Capture and Image
Analysis
For volume and
cell frichome position, gametophyte images were captured
on an Olympus SZ40 (Olympus
digitally America, Center
Valley, Pennsylvania,
USA) SPOT
with Advanced
software
3.2 (Diagnostic Instruments,
Sterling
Heights, Michigan, USA). Measurements were made using SPOT Advanced
3.2
Measurements
software. of 100 were conducted each To
cells for treatment. assess
,
the influence of freatment, medium, and
fern stage timing, a three-way Analysis of
(ANOVA; - =
Variance P
0.05; n 3600) with interaction was undertaken using
SAS
the General Linear Model Procedure (SAS 1999-2000; SAS, Gary, North
A
USA).
Carolina, Tukey's Studentized was then performed
determine
test
to
significant differences between media, with a primary focus on the comparison
of
ddH20
HgCl2 and To
graph
prefreataients. individual box were
treatments,
plots
developed JMP
using Statistical Discovery Software (SAS Prograimning,
Serial
©
No. GV0KZ9JJ07, SAS, USA)
2007; Gary, North Carolina, and modified using
PaintShopPro
6.02 (Jasc Software, Ottawa, Ontario, Canada).
ELISA and
Immunoblotting
To
establish the potential presence of an aquaporin-like protein
in
germinating and ELISA
spores gametophytes, was performed on and
callus
DIAMOND
ET AQUAPORINS
CHEILANTHES LANDSA
AL.: IN
WG
DG
immunoblotting was
performed on and
gametophytes. ELISA,
In
primary antibody only, secondary antibody only, and the preimmune serum
controls were negative The
(Fig. lA). positive control, application of
anti-
aquaporin antibody applied radish homogenate,
to root resulted in a strong
cross-reaction of root total protein with the anti-aquaporin antibody. The
immunoblotting procedure produced band when
a single the anti-aquaporin
antibody was
applied the mature gametophyte sample
to Primary
(Fig. IB).
antibody only, secondary antibody and preimmune serum
only, were
controls
The
negative. positive control, application of anti-aquaporin antibody applied
radish homogenate,
to root resulted in a strong cross-reaction of root
total
protein with the anti-aquaporin antibody.
Increment
Solute Treatments
—
NaCl and To
measure
CaCls. the response
of different stages of gametophytes
hyperpolarizing which
to solutes, destabilize plasma membranes and
often
alter
membrane
protein function, samples were exposed
incremental
to increases of
When
NaCl and
ddHaO
CaCls- preincubated and
in then exposed NaCl
to
increments, callus quickly plasmolyzed to the extent that protoplasm was
fluid
almost entirely extracted and chloroplasts clumped with and
tightly nuclei other
contents when
cell (Fig 2A1. Callus cells visibly plasmolyzed exposed slow
to
increments NaCl Mean
in concentrations. volume
of
cell loss in treatments in
cm^
which NaCl was added in slow increments was 6.65-10"^ ± 10.85-10"^.
Following an incubation
in HgCls, callus flux levels significantly dropped
mM
appear up
(Fig. 2B). Cells fully intact to 500 NaCl. In addition, variation in
the response was greatly reduced. Mean of volume NaCl
cell flux in treatments
with a HgCla incubation prior the incremented NaCl was
to treatment
WG
cm
±
0.21-10 0.47-10"^. when
^ Plasmolysis also occurred gametophytes
were exposed slow NaCl Mean
to increases in (Fig. 2C). of cell volume loss in
cm^
which
treatments in NaCl was added in slow increments was 4.86-10"^ ±
WG
6.93-10"'^.
flux dropped
levels significantly after the introduction of HgCla
(Fig. 2D). Cells appear uncompromised. Standard deviations and variation was
WG
Mean
similar for both treatments. of cell volume flux in NaCl treatments with
HgCl2
incubation prior to the incremented NaCl treatment was 0.55-10^^ cm"^ ±
DG
3.23-10 gametophytes
appeared
^. maintain NaCl
to cell viability in
up
increments 500 NaCl Some
to available volume
(Figs, firom author).
cell loss
was
visible in older cells toward the center of the gametophyte. Mean
of
cell
volume 10~^
loss following a slow increase in NaCl was 3.96- cm"^ ± 10"''.
0.40-
Mean
volume
of cell loss in NaCl increment treatment following
a pre-incubation
DG
HgCl2 was 0.80-10"'' cm"^ ± 0.1510"^.
in
flux levels significantly dropped
the introduction
after of HgCla-
These were
results mirrored in the CaClz increment treatments
(Fig. Callus
3).
when
cells visibly plasmolyze exposed to slow increments in CaCl2 concentrations
Mean
volume
(Fig 3A). of cell loss in treatments in which CaCl2 was added slow
in
increments was 12.18-10~'' cm""* ± 6.59-10"'^. Flux volume
in callus
cell
levels
dropped
significantly following pre-incubation in HgCls (Fig 3B). Mean of
cell

DIAMOND
ET AQUAPORINS
AL.: IN CHEILANTHES LANDSA
volume CaC^
loss following treatment with
a pre-incubation HgCl2 was
in
cm
±
2.20-10 ^ ' 2.80-10-^. The heatment with
pre-incubation HgCl2
in also
much
exhibited amount
a smaller
of variation than the treatment without
HgCla.
WG
Heavy
when
plasmolysis occurred
gametophytes were
exposed slow
to
mM
up
increments of CaCls 500 Mean
to volume
(Fig. 3C]. of cell loss in heataients
in which CaCla was added in slow increments was ± WG
e-QS-lO""* cm~'^ 6.59-10"^.
flux levels significantly dropped after the inhoduction of HgCl2 Mean
(Fig. 3D). of
volume CaCh
cell loss in increment heatments were preceded by
pre-incubation
in
HgCl2 was 1.95-10 cm ' ± 1.23-10-^ DG
gametophytes
from
(Figs, available
author) plasmolyzed
higher concenhations mM). Mean
at of CaCla (up 500
to of
cell
volume
loss after increasing increments of CaCla was cm"^ ± 2.38-10"*.
S.ZS-IO"'*
DG
volume
were when
cell flux levels lower
significantly gametophytes were
pre-
incubated in HgCl2. There was
also a substantial increase
in variation in the response
with Mean
pre-incubation in HgCla. of cell volume loss in gametophytes preheated
with HgCl2 was 2.65-10"* cm~^ ±
1.05-10"*.
Sucrose.— was
Sucrose used
gametophyte
to test response pure osmolyte
to a
that does not depolarize membranes
or hyperpolarize
Callus
(Fig. cells
4).
were
desiccated in a treatment of slow sucrose increments up
to a total of
mM
Mean
500 sucrose. of volume was 9.45-10"* cm^ ±
cell loss 8.86-10"*.
Callus flux levels significantly dropped
in treatments included
that a pre-
incubation HgCl2 Mean
in
(Fig. 4B). of callus volume was
cell loss
cm
0.09-10 * ± 0.51-10"*.
^ was when
In addition, variation reduced
greatly
WG
protonemal was
callus pre-incubated
in HgCls. gametophytes
in a
mM
which
treatment
in sucrose increased in increments up 500
is to exhibited
some volume
Mean
cell loss in older
cells (Fig. 4C). of cell loss in sucrose
cm^ WG
increment was 9.6210"*
treatment ±
1.64-10"*.
flux levels
dropped
significantly in sucrose increment treatments were
that pre-incubated
Mean
HgCl2
in (Fig. 4D). of cell volume loss in these treatments with HgCla
was
pre-incubation 0.48-10 * cm"' ± 3.2010"*.
Variation response
in
when
decreased markedly gametophytes were DG
pre-incubated
in HgCla-
gametophytes
(Figs, available from author) experienced some volume
cell loss
in treatments involving increments in sucrose molarity (up 500 mM). Mean
to
DG
volume
of cell loss in sucrose increment treatments was 10.80-10"* cm"^ ±
DG
54.15-10 volume
cell loss significantly dropped in sucrose increment
.
treatments included
that a pre-incubation
in HgCls. Variation in gametophyte
response was
also less in treatments that included HgCl2
pre-incubation.
Mean
volume
of
cell loss in treatments with HgClz was
pre-incubation
±
0.95-10 * cm"' 2.45-10"*.
ELISA
plate. (B) Positive Radish Root Control. Each immunoblot and ELISA
contained
radish
a root
radish root reliably produced a strong Immunoblotting
signal. (C) procedures resulted In a sinele
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