Plant Physiol. (1998) 118: 305-313
A Model for Signal Transduction during Gamete Release in the
Fucoid Alga Pelvetia compressa1
Gareth Anthony Pearson2, * and
Susan Howard
Brawley3
Department of Plant Biology and Pathology, University of Maine,
Orono, Maine 04469-5722
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ABSTRACT |
Fucoid
algae release gametes into seawater following an inductive light period
(potentiation), and gamete expulsion from potentiated receptacles of
Pelvetia compressa began about 2 min after a
light-to-dark transition. Agitation of the medium reversed
potentiation, with an exponential time course completed in about 3 h. Light regulated two signaling pathways during potentiation and
gamete expulsion: a photosynthetic pathway and a
photosynthesis-independent pathway in which red light was active but
blue light was not. Uptake of K+ appears to have an
important role in potentiation, because a 50% inhibition of
potentiation occurred in the presence of the tetraethylammonium ion, a
K+-channel blocker. A central role of anion channels in the
maintenance of potentiation is suggested by the premature release of
gametes in the light when receptacles were incubated with inhibitors of slow-type anion channels. An inhibitor of tyrosine kinases, tyrphostin A63, also inhibited potentiation. A model for gamete release from P. compressa is presented that proposes that
illumination results in the accumulation of ions (e.g. K+)
throughout the cells of the receptacle during potentiation, which then
move into the extracellular matrix during gamete expulsion to generate
osmomechanical force, resulting in gamete release.
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INTRODUCTION |
Developmental and life history events in photosynthetic organisms
often involve complex responses to natural variations in light
intensity and quality. Light is processed in a variety of ways: either
through the photosynthetic apparatus (Durnford and Falkowski, 1997
, and
refs. therein) or through other photoreceptors such as the phytochrome
(Quail et al., 1995), cryptochrome (Ahmad and Cashmore, 1996), or
rhodopsin families (Robinson et al., 1998). In lower plants and algae,
light influences processes as diverse as cell differentiation (in
cyanobacteria [Campbell et al., 1993]), photopolarization of zygotes
(in fucoid algae [Robinson and Miller, 1997; Robinson et al., 1998]),
and control of branching (in mosses [Ermolayeva et al., 1996]).
Natural populations of fucoid algae release gametes into SW in the
light during periods of low water motion (Pearson and Brawley, 1996;
Serrão et al., 1996). Gamete release is photosynthesis dependent,
since blocking photosynthetic electron transport in the light with DCMU
prevents gamete release (Serrão et al., 1996). Low water motion
stimulates gamete release by limiting the inorganic carbon required for
photosynthesis (Pearson et al., 1998). We demonstrated this with
experiments in which addition of excess inorganic carbon to SW under
calm conditions blocked gamete release; conversely, gamete release
occurred in inorganic carbon-free SW independently of the hydrodynamic
conditions (Pearson et al., 1998). The chances of successful external
fertilization are increased by ensuring that gametes are released
during relatively calm periods, when dilution will be slow.
Some of the key environmental factors controlling gamete release are
known; however, we have little information about how these signals are
transduced into downstream events resulting in gamete expulsion.
Therefore, the aim of this study was to investigate the signaling
pathway. Since oogonia and antheridia are released by being forced
through pores in the subepidermal conceptacles of the reproductive
tissue (receptacles), our hypothesis is that environmental signals
ultimately result in ionic movements, leading to osmotic changes within
the receptacles that stimulate gamete expulsion.
Ionic fluxes are involved in several osmomechanical processes in lower
and higher plants. These include the K+- and
Cl
-driven swelling and shrinking of motor cells
that control leaf movements in several higher plants (Satter et
al., 1988; Lee, 1990; Antkowiak and Engelmann, 1995). In guard cells,
the best understood osmoregulatory system of higher plants,
light-dependent ionic movements drive turgor changes caused by fluxes
of K+ and the anions malate and
Cl (Assmann, 1993; Roelfsema and Prins, 1998).
Membrane depolarizations are often an early event in signal
transduction pathways involving ion channels, as in the
phytochrome-mediated, Ca2+-dependent
depolarizations involved in branching of the moss Physcomitrella patens (Ermolayeva et al., 1996) and in stomatal closure
(Schroeder and Keller, 1992; Schroeder et al., 1993).
Guard cell anion channels are currently thought to be a central control
mechanism in the signal transduction pathways for stomatal function,
allowing sustained plasma membrane depolarization (Schroeder and
Keller, 1992; Schroeder et al., 1993; Pei et al., 1997; for review, see
Schroeder, 1995). Down-regulation of S-type anion channels is
necessary during K+-driven stomatal opening,
whereas sustained plasma membrane depolarization resulting from the
opening of anion channels drives K+ efflux and
stomatal closure (Schwartz et al., 1995). Recent studies have
implicated phosphorylation and dephosphorylation events in the
regulation of inward and outward K+ currents
(Luan et al., 1993; Thiel and Blatt, 1994; Li et al., 1998) and anion
channels (Schmidt et al., 1995; Pei et al., 1997) in guard cells. This
suggested that it would be of interest to investigate the roles of
K+ and anion fluxes and of phosphorylation and
dephosphorylation in gamete release in fucoid algae. Furthermore, the
inorganic carbon sensitivity of gamete release in fucoids shows
intriguing functional parallels with the role of malate as a
CO2 sensor and modulator of guard cell
anion-channel activity (Hedrich and Marten, 1993; Hedrich et al.,
1994). Therefore, we also performed experiments to investigate the
effect of malate on gamete release.
There are two distinct phases in gamete release in the fucoid alga
Pelvetia compressa (J. Agardh) De Toni (formerly P. fastigiata, Silva, 1996). First, receptacles become competent to
release gametes following culture for 
4 h under calm conditions in
the light. This is referred to as potentiation in this report. Second,
gamete expulsion is a rapid process (minutes) that is triggered by
transferring receptacles to darkness (Jaffe, 1954). Gamete expulsion
does not occur normally during potentiation under laboratory conditions unless old receptacles are used. The temporal and functional separation of potentiation and gamete expulsion in P. compressa makes
it a useful model in which to study the mechanistic basis underlying these processes. On the basis of our results, we suggest that (a) light
signals during potentiation are processed via two separate pathways:
one sensed via photosynthetic electron transport and the other
photosynthesis independent and possibly red-light dependent, (b)
K+ uptake plays a role in potentiation and gamete
expulsion may involve changes in anion-channel activities, and (c)
phosphorylation events involving Tyr kinase(s) are involved in the
signaling pathway for potentiation.
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MATERIALS AND METHODS |
Reproductive branches of the intertidal brown alga Pelvetia
compressa (J. Agardh) De Toni were collected at Pigeon Point, California, and shipped overnight between layers of moistened paper in
Styrofoam boxes that contained cool packs. For the experiments reported
here, material was stored at 5°C in a cold room and used within
10 d and normally within 1 week. To minimize artifacts associated
with storage, receptacles (50-100) were preincubated in 2-L flasks
containing 1 L of SW for 6 h in the light (150-200 µmol photons
m2 s1) with water
motion provided by an orbital shaker (150 rpm, Lab-Line Instruments,
Inc., Melrose Park, IL) prior to experiments, unless otherwise stated.
Slightly different periods of potentiation were used in different
experiments as a result of small seasonal effects in responses of
tissue. Gamete release in experiments was quantified by counting the
number of eggs present in the medium following a 30-min transfer to
darkness (unless otherwise stated) with a dissecting microscope, and is
expressed as a function of the fresh weight of receptacle tissue after
release.
Time Course of Gamete Expulsion in Darkness
The time course of gamete expulsion in darkness (two receptacles
per replicate in 15 mL of ASW, n = 5) was determined
following potentiation under calm conditions in the light for 6 h.
Following potentiation, receptacles were placed in darkness for 30 s, 1 min, 5 min, or 30 min, and then irradiated for an additional
period of 30 min (to allow completion of any gamete expulsion under
way) before quantitation of release.
Time Course of Stimulation and Inhibition of Potentiation Related
to Hydrodynamic Conditions
To study the time course of potentiation in the light, which is
known to be inhibited by high water motion in this and other species of
fucoid algae (Serrão et al., 1996; Pearson et al., 1998), two to
three receptacles (approximately 0.5-1.0 g fresh weight) per replicate
(n = 5) were incubated in 25 mL of filtered SW in
125-mL flasks. Receptacles in flasks were incubated at 15°C ± 1°C in the light (250 µmol photons m2
s1) under either agitated (150 rpm, Lab-Line
Instruments, orbital shaker) or calm conditions for 8 or 13 h.
Other treatments (n = 5) included agitation first for
8 h and then 1, 2, 3, 4, or 5 h of light under calm
conditions. A second experiment was performed under the same culture
conditions as described above, except that receptacles were placed
under calm conditions for 6 h in the light and then agitated for
1, 2, 3, 4, or 5 h in the light. Positive and negative controls
were incubated under calm or agitated conditions for 6 or 11 h in
the light. In both experiments, receptacles were used directly from
storage in darkness at 5°C without pretreatment (see above), and
gamete release was determined under a dissecting microscope by counting
the number of eggs released after transfer to darkness (30 min).
Inhibition of Photosynthetic Electron Transport
The effects of inhibiting photosynthesis in the light on gamete
expulsion following potentiation were investigated by adding an
inhibitor of PSII electron transport, DCMU, to receptacles after 7 h in light (two receptacles per replicate in 4 mL of ASW, n = 5). DCMU was added from a 100 mM stock
in 95% ethanol to a final concentration of 10 µM.
Control treatments were incubated in ASW or ASW plus 0.01% ethanol.
Gamete release was determined after 30 min in the light with no dark
transfer.
Malate Sensitivity
The effect of malate on gamete release in P. compressa
was studied by addition of L-(
)malate and
D-(+)malate (as sodium salts, Sigma) during potentiation.
Malate (up to 50 mM) was added to receptacles (two per
replicate in 15 mL of ASW, n = 5) at the beginning of a
6-h potentiation period, and gamete release was quantified following a
30-min dark transfer.
Effects of Light Quality on Potentiation and Gamete Release
To investigate the effects of light quality on gamete release
independently of photosynthetic rate, the photosynthesis versus irradiance responses of receptacles to white, red, and blue light were
determined in preliminary experiments using oxygen electrodes (data not
shown). Red and blue wavelengths were provided using 50-mm-diameter
dichroic color separation glass filters (Edmund Scientific Co.,
Barrington, NJ). In subsequent experiments to investigate the effects
of light quality on potentiation and gamete expulsion, photon flux
densities were selected that gave equal rates of
O2 production in different treatments. Relatively
low fluences of blue light were obtainable with the fluorescent light source used, and photon flux densities were lower than those used in
other experiments (white light = 50 µmol photons
m2 s1; red light = 35 µmol photons m2
s1; and blue light = 70 µmol photons
m2 s1). Receptacles
(three per replicate) were potentiated for 8 h in 60-mm Petri
dishes wrapped in aluminum foil containing 15 mL of ASW
(n = 6). The transparent lids of the dishes were either unaltered (white light) or replaced with the appropriate filter (red or
blue light). Following potentiation, receptacles were incubated for an
additional 30 min in darkness or in white light, red light, or
blue light. Gamete release was assayed as described previously.
Inhibitors of Ion Channels
An inhibitor of K+ channels (Taylor and
Brownlee, 1993), TEA-chloride (100 mM), was added either at
the start of the potentiation period (i.e. for 5 h) or by transfer
of receptacles to ASW plus TEA+ for 60 min or for
5 min prior to dark treatment. The appropriate controls for both
osmotic effects (100 mM NaCl) and for transfers of
receptacles between different solutions were used to allow the
comparison of inhibitor and noninhibitor treatment effects.
Several compounds reported to block S-type anion channels in both
animals and plants were tested for their effects on gamete release.
9-AC was prepared as a 0.5 N stock solution in ethanol:DMSO (95:5 [v/v]) and used at a final concentration of 1 mM in
ASW. Probenicid was used at a final concentration of 2 mM,
from a 1 N stock in 1 N NaOH. Niflumic acid was
prepared as a 200 mM stock in ethanol and used at a final
concentration of 1 mM. Control treatments were done in ASW
and in ASW with the appropriate solvent concentration (9-AC control,
0.02% [v/v] 95:5 ethanol:DMSO; probenicid control, 0.02%
[v/v] 1 N NaOH; and niflumic acid control, 0.5% [v/v]
ethanol). Experiments were carried out in 35- × 10-mm Petri dishes
containing 4 mL of Tris-buffered ASW (pH 7.8). Each dish (n = 5) contained two receptacles. Anion-channel
blockers were added at the beginning of the 7-h potentiation period,
after which time receptacles were removed to new dishes containing
appropriate inhibitor or control solutions for a 30-min dark period;
the number of gametes expelled was thus quantified independently for
both the light (potentiation) and the dark phases. The results were analyzed by a two-factor analysis of variance (SYSTAT, version 5.2.1, SPSS, Inc., Chicago, IL) and significant differences
between means (
= 0.05) were identified using Tukey's test.
In similar experiments, the anion-exchange inhibitor
4,4
-diisothiocyanatostilbene-2,2-disulfonic acid was added at
concentrations of 200 µM and 1 mM (two
receptacles per treatment in 4 mL of ASW, n = 5). After
5 h of potentiation, gamete release was quantified following a
30-min transfer to darkness. Gamete release in the light was not
observed in these experiments and was not quantified separately.
Inhibitors of Protein Phosphorylation
The involvement of protein phosphorylation in gamete release was
investigated in several experiments with the use of drugs known to be
active against protein kinases in other systems. Staurosporine, a
broad-range inhibitor of Ser/Thr protein kinases, was added to a
concentration of 10 µg mL1, from a 10 mg
mL1 stock solution in DMSO. Tyrphostins A25 and
A63, specific inhibitors of protein Tyr kinases (Yaish et al., 1988),
were added at 50 to 200 µM from stock solutions in DMSO,
either at the beginning of the potentiation period or for 30 min prior
to the dark transfer period. Controls for the effects of solvents
(0.1% DMSO), in addition to ASW controls, were used in these
experiments.
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RESULTS |
Time Course of Gamete Expulsion and of Stimulation and Inhibition
of Potentiation by Hydrodynamic Conditions
A quantitative analysis of the temporal effect of darkness on
potentiated receptacles under calm conditions (Jaffe, 1954) showed that
a sustained period of darkness was required to stimulate gamete
expulsion (Fig. 1). Receptacles returned
to light after periods of 
2 min of darkness released few gametangia.
A period of darkness between 2 and 5 min was sufficient to saturate
gamete expulsion (Fig. 1). The response of potentiated receptacles to darkness was gradual, as demonstrated by the data for 1, 2, and 5 min
of darkness (Fig. 1).
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| Figure 1.
Gamete expulsion from potentiated receptacles
following transfer to darkness for periods between 0 and 30 min.
Potentiation was for 6 h in the light. Following dark transfers,
receptacles were returned to the light for an additional 30 min to
allow for the completion of gamete release. Results are means ± SE (n = 5). Fwt, Fresh weight.
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The potentiation of gamete release in P. compressa was
inhibited by high water motion (agitation) relative to calm conditions in laboratory experiments (Fig. 2).
Gamete release from receptacles was maximized by incubation for 6 h under calm conditions; no further increase was observed by extending
the potentiation period to 11 h (Fig. 2A). However, incremental
increases in the duration of agitation (1-5 h) following a 6-h period
of calm resulted in an exponential decline in gamete release. Following
an 11-h potentiation period, 48,720 ± 5,937 eggs
g1 fresh weight were released under calm
conditions, but only 306 ± 161 eggs g1
fresh weight were released when 6 h of calm was followed by 5 h of agitation (Fig. 2A). Agitated controls in both experiments (Fig.
2) indicated that gamete release was reduced progressively by periods
of agitation for 6 to 13 h. Several properties are evident using
receptacles in calm:shaken and shaken:calm treatments: (a) an extended
period of agitation is required to fully reverse potentiation, (b)
potentiated (calm) receptacles are very sensitive; only 1 to 2 h
of agitation greatly reduced the number of gametes released (Fig. 2),
and (c) the rapid, exponential kinetics of the reversal of potentiation
by agitation (Fig. 2A) compared with the slower increases in
potentiation after agitation (Fig. 2B) suggest that these two
processes might not be simply the inverse of one another. Following
8 h of agitation, >1 h of calm incubation was necessary to
observe increases in gamete release (Fig. 2B), which then increased
with the duration of the calm interval.
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| Figure 2.
The effects on potentiation of shaking receptacles
following calm conditions (A,  ) and calm following shaking
conditions (B,  ). The numbers of gametes released were determined
following a 30-min dark transfer after completion of each experimental
treatment. Results are shown relative to calm controls ( ) and shaken
controls ( ). Values are means ± SE
(n = 5). Fwt, Fresh weight.
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Inhibition of Photosynthetic Electron Transport
When added to potentiated receptacles in the light, DCMU caused a
massive expulsion of gametes, mimicking a transfer to darkness (Table
I). The time course of the response
(about 30 min for completion following addition of the inhibitor) was
slower than that seen in response to darkness (Fig. 1). But given the
time necessary for DCMU to reach its site of action in the
chloroplasts, these results are consistent with a role for
photosynthesis in signaling the rapid response of gamete expulsion in
P. compressa, in addition to the requirement for
photosynthesis during potentiation shown in a previous report
(Serrão et al., 1996).
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Table I.
DCMU triggers gamete expulsion in the light from
potentiated receptacles
DCMU (10 µM) or ethanol (0.01%) was added following
7 h of potentiation of receptacles in the light, and the number of
gametes released was determined 30 min after addition. Control (ASW)
and treated receptacles remained in the light for a total of 7.5 h. Values are means ± SE (n = 5).
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Effects of Light Quality
Receptacles potentiated in white light expelled gametes normally
when incubated in darkness for 30 min (Fig.
3). As expected, gamete expulsion was
very low when white-light-potentiated receptacles were transferred to
white or red light, but a transfer from white to blue light triggered
gamete expulsion that exceeded that induced by darkness (although in
another experiment, it was of a similar magnitude). Moreover, although
potentiation in red light was effective (similar to white light) and
resulted in a large expulsion of gametes after transfer to darkness,
very few gametes were expelled when potentiation was carried out in
blue light (Fig. 3). Thus, blue light was insufficient to potentiate
gamete release, despite providing photosynthetically active irradiance,
and had a similar effect to darkness in triggering gamete expulsion
following normal potentiation in white light.
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| Figure 3.
The effects of light quality on potentiation and
gamete expulsion. Receptacles were potentiated for 8 h in red
light (RL), blue light (BL), or white light (WL). Receptacles were then
transferred to darkness (D) for 30 min and gamete release was assayed.
For white-light-potentiated receptacles additional treatments were
given by transferring to red or blue light for 30 min, while controls
were kept in white light, and gamete release was assayed. Results are
means ± SE (n = 6). Fwt, Fresh
weight.
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Effects of Malate and Inhibitors of Ion Channels
When L-()malate (50 mM) was present in
ASW during potentiation, gamete release was reduced significantly
relative to controls in ASW or to treatments with the biologically
inactive stereoisomer (Table II,
F2,12 = 4.56; P = 0.034). We found that the
effective concentration of L-malate necessary to inhibit
potentiation varied from experiment to experiment; 50 mM
was always effective, but in some experiments a significant response
was observed with concentrations as low as 5 mM.
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Table II.
The effects of D- and
L-malate on potentiation
Malate (50 mM) was added at the start of potentiation in
ASW (6 h), and gamete release from receptacles was assayed following 30 min of darkness. Values are means ± SE
(n = 5).
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The K+-channel blocker TEA+
(100 mM) inhibited potentiation, thereby reducing gamete
release by approximately 50% (Table
III), suggesting that
K+ movements are involved in achieving
potentiation in receptacles. In contrast, gamete expulsion was
unaffected when TEA+ was added to fully
potentiated receptacles for periods of up to 1 h before placing
these receptacles (still in TEA+) in darkness
(data not shown).
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Table III.
Inhibition of potentiation by the
K+-channel blocker TEA+
Receptacles were potentiated for 6 h in ASW (518 mmol/kg
Cl), ASW plus NaCl (618 mmol/kg Cl), or ASW
plus 100 mM TEA+. Gamete release was assayed
following 30 min of darkness. Values are means ± SE
(n = 5).
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Gamete release was significantly greater in the light relative to
controls in the presence of all three anion-channel inhibitors tested
(Fig. 4; two-factor analysis of variance,
F6,56 = 39.12; P <0.0001). The most effective
inhibitor at the concentrations used was 9-AC, which resulted in levels
of gamete expulsion in the light that were similar to levels of
dark-induced expulsion in controls (Fig. 4). The presence of
solvents (DMSO or ethanol) or carrier (NaOH) at concentrations
equal to those used in inhibitor treatments had no significant effects
on gamete release relative to controls; in every case very few gametes
were expelled in the light, almost all expulsion occurring rapidly
following the transfer of receptacles to darkness. Cumulative gamete
release (light plus dark periods) did not differ significantly between
treatments (analysis of variance, F6,28 = 1.58;
P = 0.190). Therefore, the biological significance of the observed
reduction in gamete expulsion during the dark phase in the presence of
inhibitors might simply have been a consequence of depletion of the
gamete pool due to release in the light (i.e. the magnitude of release
in the dark was not independent of release in the light in these
treatments). These results suggest that anion channels play a central
role in the normal functioning and regulation of gamete expulsion in P. compressa.
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| Figure 4.
The effects of anion-channel blockers on gamete
release. Receptacles were incubated for 7 h in the light in 9-AC
(1 mM), niflumic acid (1 mM), or probenicid (2 mM). Gametes released were counted at the end of the light
period and after an additional 30 min of darkness. Hatched bars
represent treatments in which inhibitor was present, and black bars
represent ASW controls or ASW plus solvent controls. Values are
means ± SE (n = 5). Fwt, Fresh
weight.
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Effects of Inhibitors of Protein Phosphorylation
Gamete release was almost completely blocked when specific
inhibitors of Tyr kinases were added to ASW at the beginning of potentiation (Table IV), whereas
staurosporine (10 µg/mL), a Ser/Thr kinase inhibitor, had no effect
(Table IV). Tyrphostin A63 (200 µM) reduced gamete
release from 19,107 ± 2,200 eggs g1 fresh
weight in controls (ASW) to 733 ± 409 eggs
g1 fresh weight (n = 5). A
second tyrphostin, A25 (200 µM), had no significant
effect on gamete release, indicating that inhibition was not a
consequence of any general cytotoxicity of these compounds or of the
concentration used. Neither tyrphostin had any effect on gamete
expulsion when added after potentiation, for up to 30 min before
receptacles were transferred to darkness (data not shown). Thus, we
conclude that protein Tyr phosphorylation is essential for potentiation
but appears not to be involved in the rapid response to darkness that
triggers gamete expulsion.
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Table IV.
Effects of protein kinase inhibitors on
potentiation
Inhibitors of Tyr kinases, tyrphostin A25 and A63 (200 µM), and, in a separate experiment, the Ser/Thr kinase
inhibitor staurosporine (10 µg/mL) were added at the beginning of
potentiation (6 h), and gamete release was determined following a
30-min transfer to darkness. Values are means ± SE
(n = 5).
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DISCUSSION |
A model that summarizes our present understanding of potentiation
and gamete expulsion is presented in Figure
5. Our data indicate that photoreception
and subsequent signal transduction occur via two pathways. One of these
requires photosynthesis (Serrão et al., 1996; see ``Results''),
and to become fully potentiated, receptacles must experience inorganic
carbon limitation during at least a portion of the potentiation period
(see figure 9 in Pearson et al., 1998). Fucoid algae have a
carbon-concentrating mechanism that provides relatively high
[CO2] to the chloroplasts (Surif and Raven,
1989; Raven and Osmond, 1992), and they continue to evolve
O2 in inorganic carbon-free SW (Surif and Raven,
1989). This suggests that internal stores of carbon might be utilized under carbon-limiting conditions (e.g. via the decarboxylation of
organic acids), which could drive the uptake of
K+ as a counterion. Gamete expulsion in the dark
from receptacles treated with TEA+ during
potentiation was only one-half that of controls; thus, part of the
requirement for potentiation is K+ uptake.
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| Figure 5.
A model for the signaling pathways controlling
light-dependent potentiation and gamete expulsion in fucoid algae. The
top part of the figure shows a diagrammatic section through a
receptacle illustrating the general organization of tissues and
extracellular matrix (ECM) in receptacles of P. compressa. The black blocks on the left of the model indicate
those parts of the pathway involved in potentiation and gamete
expulsion, respectively. The brackets on the right indicate the
probable sites within the receptacle for the events indicated.
Stimulation of pathway components is shown by open arrows, and
inhibitors by blocked lines. The photosynthesis-dependent pathway
activates under low inorganic carbon (IC) conditions.
Potentiation is prevented by increasing [IC] and also by blocking
PSII electron transport with DCMU (Serrão et al., 1996).
Potentiation under red light (RL) or white light (WL) is blocked in
blue light (BL), independently of photosynthetic rate. Protein Tyr
kinase (PTK) activity is required during potentiation. Unconfirmed
components of the pathway are shown by a "?," and possible
pathways are shown by broken arrows. Potentiation ultimately
results in movements of K+ and anions, and the
extracellular matrix provides the osmomechanical forces necessary to
cause gamete release from conceptacles. PS, Photosynthesis.
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Light Regulation of Gamete Release
The epidermal cells of the receptacle are likely to be key sites
for potentiation (Fig. 5) because chlorophyll autofluorescence is found
here primarily (S.H. Brawley, unpublished data), and the plasma
membrane of the epidermal cells is deeply invaginated, suggesting the
importance of these cells in secretion and/or absorption (McCully,
1968a). Ions taken up during potentiation, however, are likely to be
distributed throughout the receptacle during potentiation because the
cells are coupled by distinctive cytoplasmic junctions (Fritsch,
1945; McCully, 1968b; Moss, 1983; G.A. Pearson and S.H.
Brawley, unpublished data). Taken together, these observations suggest that the receptacle is a reproductive organ that functions as
an ionic syncytium.
Gamete expulsion is complete within about 30 min after the application
of DCMU to receptacles in the light (see ``Results''). This is dramatic, although somewhat slower than the time course demonstrated here for darkness. Hypotheses (Fig. 5) arising from the
observed effects of darkness/DCMU include: (a) photosynthetically
supplied ATP may be required by ion pumps involved in maintenance of
potentiation (Fig. 5) and/or (b) redox signaling (Campbell et al.,
1993; Danon and Mayfield, 1994; Escoubas et al., 1995) may be important
in the transition from potentiation to gamete release. Other signals could arise under high light or carbon limitation as a result of
oxidative stress and/or the thioredoxin pathway (Danon and Mayfield,
1994; Allen, 1995; Bohnert et al., 1995; Ingram and Bartels, 1996).
A second photosynthesis-independent pathway must have been present,
because potentiation occurred in red or white light, but not in blue
light, independently of photosynthetic rate. Since potentiation occurs
normally in white light, it appears that blue light is not inhibitory.
Blue light was as effective as darkness in triggering gamete expulsion
from potentiated receptacles. Investigations of the action spectra for
the response will reveal whether this was simply due to removal of red
light or to a blue-light effect. Blue light does have substantial
effects on some processes in fucoids, including photopolarization of
zygotes (Hurd, 1920; Robinson and Miller, 1997), which may occur via
rhodopsin, since retinal has been identified in P. compressa
zygotes (Robinson et al., 1998). The ubiquitous red-far-red
photoreceptors of higher plants, the phytochromes (Quail et al., 1995;
Chamovitz and Deng, 1996), are candidates for the red-light
photoreceptor of fucoid receptacles. Phytochrome has not been
identified in fucoids, but genes with homology to phytochrome are
widely distributed (prokaryotes: Schneider-Poetsch et al., 1991; Kehoe
and Grossman, 1996; Hughes et al., 1997; green algae: Kidd and
Lagarias, 1990; Winlands and Wagner, 1996; lower plants: Thümmler
et al., 1992; Pratt, 1995; Ermolayeva et al., 1996; ascomycete fungi
and slime moulds: Griffith et al., 1994; Starostzik and Marwan, 1995).
Given the identification of retinal in P. compressa, it is
also of interest that animals obtain selective spectral information,
including from red light, with retinal in combination with different
opsin proteins (Bowmaker, 1991).
Role of Protein Phosphorylation
Successful potentiation requires Tyr kinase activity. Gamete
release was reduced to 13% of controls when tyrphostin A63 was present
during the potentiation period. Gamete release was unaffected, however,
when the drug was added 30 min prior to dark transfer, leading to the
(tentative) conclusion that a protein Tyr kinase is not involved in the
direct triggering of gamete expulsion in darkness. Tyr phosphorylation
has been found as part of a number of processes that may be relevant to
our model (e.g. in stress-response pathways involving mitogen-activated
protein kinase [Shinozaki and Yamaguchi-Shinozaki, 1997];
modulation of K+ channels [Holmes et al.,
1996]; regulation of some Rubisco [Aggarwal et al., 1993];
phytochrome-mediated signaling [Bowler et al., 1994; Sommer et al.,
1996]). It should be noted, however, that translation from 70S and 80S
ribosomes was not required for potentiation in this study (data not
shown); thus, the functional consequences of light signaling and/or
protein phosphorylation are probably not expressed through changes in
gene regulation and transcription. Inward-rectifying
K+ channels in guard cells are sensitive to
TEA+ (Blatt, 1992), with regulation through
calmodulin-dependent protein phosphatase 2B (calcineurin); however, we
have not detected any significant change in gamete release when
inhibitors of Ser/Thr kinases (staurosporine, H-7, ML-7) or the
phosphatase inhibitor okadaic acid were present during
potentiation, and inconsistent results have been obtained in
experiments with the calmodulin inhibitors W7 and W5 (data not shown).
Anion-Channel Regulation and Gamete Expulsion
Experiments using anion-channel blockers suggest that S-type anion
channels play a role in controlling gamete expulsion. Several anion-channel antagonists caused gametes to be released prematurely in
the light during the normal potentiation phase. However,
4,4-diisothiocyanatostilbene-2,2-disulfonic acid, a potent
inhibitor of rapid-type anion channels (Marten et al., 1993), had no
effect on gamete release under the same conditions. The control of
anion efflux through S-type anion channels in guard cells is central to
the process of turgor regulation and thus stomatal opening (Schroeder
and Keller, 1992; Schroeder et al., 1993; Schroeder, 1995; Schwartz et
al., 1995; Pei et al., 1997). These channels can remain open over a
wide range of membrane potentials and allow for sustained membrane
depolarization, upon which turgor loss and stomatal closing depend. The
activation state of these channels is also important in the control of
stomatal opening, which is enhanced in the presence of anion-channel
blockers (Schwartz et al., 1995). We propose that anion-channel
activity is necessary to maintain the potentiated state, because of the effects of inhibitors in causing gamete expulsion in the light; however, further studies are needed to clarify their regulation and
role (e.g. in the maintenance of turgor). The inhibitory effect of
malate on potentiation that we observed might be due to disruption of
turgor by an activation of anion efflux channels (Hedrich and Marten,
1993; Hedrich et al., 1994).
A Model for Signal Transduction Leading to Gamete
Release
Our view of the potentiated receptacle is one in which light and
inorganic carbon-limited uptake of K+ has
occurred. Underlying the epidermal and cortical cells of the
receptacles is a thick layer consisting of medullary filaments in a
copious extracellular matrix of alginic acid and fucoidan (McCully,
1968b). We hypothesize that a rapid release of ions into the
extracellular matrix occurs when potentiated receptacles are
transferred to darkness, such that this extracellular gel swells
rapidly to provide the physical force required for gamete expulsion
(Fig. 5). As shown above, TEA+ inhibits gamete
release by partially blocking potentiation. The failure of
TEA+ to inhibit gamete expulsion following
potentiation in the absence of the inhibitor might seem contrary to our
model, but the inhibitor may not penetrate to sites deep within the
receptacle and, or the channels through which K+
efflux occurs may be TEA+ insensitive (for
reviews, see Cook, 1990; Hedrich and Dietrich, 1996). Preliminary
studies of the spatial distribution of ions within receptacles during
potentiation and during gamete expulsion with x-ray microanalysis are
supportive of the proposed K+ flux into the
extracellular matrix (Speransky et al., 1998).
The exquisite control mechanisms of potentiation and gamete expulsion
in P. compressa in response to water motion ([inorganic carbon]) and light signals may have arisen as adaptations allowing increased fertilization success in intertidal habitats. Potentiation builds gradually under calm conditions but declines exponentially if
water motion increases (see ``Results''), conditions under which
gamete dilution would be expected to decrease fertilization success. Rapid light-to-dark transitions, which trigger gamete expulsion in the
laboratory, are unlikely to happen in nature; however, sharp reductions
in light intensity and changes in quality (reduction in the relative
amount of red light) may occur when algae are immersed by the incoming
tide. Questions concerning the potentiation status of receptacles of
P. compressa in natural populations, and thus the timing of
natural gamete release (Johnson and Brawley, 1998), might be best
addressed using a physiological marker, e.g. the spatial distribution
of important ions at different phases of the tidal cycle (Speransky et
al., 1998). Our model for signal transduction during gamete release
suggests the presence of elements common to other osmoregulatory model
systems (guard cells and sensitive plants) and offers a basis for
further investigation.
| |
FOOTNOTES |
1
This work was supported by National Science
Foundation award OCE 92 16981 to S.H.B.
2
Present address: Unidade de Ciências e
Technologias dos Recursos Aquaticos, Campus de Gambelas, Universidade
do Algarve, 8000 Faro, Portugal.
3
Present address: School of Marine Sciences,
University of Maine, Orono, ME 04469-5722.
*
Corresponding author; e-mail gpearson{at}ualg.pt; fax
351-89-818353.
Received May 20, 1998;
accepted June 10, 1998.
| |
ABBREVIATIONS |
Abbreviations:
9-AC, anthracene-9-carboxylic acid.
ASW, artificial seawater.
S-type, slow-type.
SW, seawater.
TEA+, tetraethylammonium ion.
| |
ACKNOWLEDGMENTS |
The authors would like to thank Jon Ashen for providing regular
shipments of P. compressa. We appreciate the help and
support of Dr. Ester Serrão during both the experimental work and
the preparation of the manuscript. Comments by two anonymous reviewers improved an earlier version of the manuscript.
| |
LITERATURE CITED |
Aggarwal KK,
Saluja D,
Sachar RC
(1993)
Phosphorylation of Rubisco in Cicer arietinum: non-phosphoprotein nature of Rubisco in Nicotiana tabacum.
Phytochemistry
34:
329-335
[CrossRef]
Ahmad M,
Cashmore AR
(1996)
Seeing blue: the discovery of cryptochrome.
Plant Mol Biol
30:
851-861
[CrossRef][ISI][Medline]
Allen RD
(1995)
Dissection of oxidative stress tolerance using transgenic plants.
Plant Physiol
107:
1049-1054
[CrossRef][ISI][Medline]
Antkowiak B,
Engelmann W
(1995)
Oscillations of apoplasmic K+ and H+ activities in Desmodium motorium (Houtt.) Merril. pulvini in relation to the membrane potential of motor cells and leaflet movements.
Planta
196:
350-356
Assmann SM
(1993)
Signal transduction in guard cells.
Annu Rev Cell Biol
9:
345-375
[CrossRef][ISI]
Blatt MR
(1992)
K+ channels of stomatal guard cells.
J Gen Physiol
99:
615-644
[Abstract/Free Full Text]
Bohnert HJ,
Nelson DE,
Jensen RG
(1995)
Adaptations to environmental stresses.
Plant Cell
7:
1099-1111
[CrossRef][ISI][Medline]
Bowler C,
Yamagata H,
Neuhaus G,
Chua N-H
(1994)
Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms.
Genes Dev
8:
2188-2202
[Abstract/Free Full Text]
Bowmaker JK
(1991)
The evolution of vertebrate visual pigments and photoreceptors.
In
JR Cronly-Dillon,
RL Gregory,
eds, Vision and Visual Dysfunction, Vol 2: Evolution of the Eye and Visual Systems.
Macmillan, London, pp 63-65
Campbell D,
Houmard J,
Tandeau de Marsac N
(1993)
Electron transport regulates cellular differentiation in the filamentous cyanobacterium Calothrix.
Plant Cell
5:
451-463
[Abstract]
Chamovitz DA,
Deng X-W
(1996)
Light signaling in plants.
Crit Rev Plant Sci
15:
455-478
Cook NS (1990) Potassium Channels: Structure, Classification,
Function and Therapeutic Potential. E Horwood, Chichester, UK
Danon A,
Mayfield SP
(1994)
Light-regulated translation of chloroplast messenger RNAs through redox potential.
Science
266:
1717-1719
[Abstract/Free Full Text]
Durnford DG,
Falkowski PG
(1997)
Chloroplast redox regulation of nuclear gene transcription during photoacclimation.
Photosynth Res
53:
229-241
[CrossRef]
Ermolayeva E,
Hohmeyer H,
Johannes E,
Sanders D
(1996)
Calcium-dependent membrane depolarization activated by phytochrome in the moss Physcomitrella patens.
Planta
199:
352-358
[ISI]
Escoubas J-M,
Lomas M,
LaRoche J,
Falkowski PG
(1995)
Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.
Proc Natl Acad Sci USA
92:
10237-10241
[Abstract/Free Full Text]
Fritsch FE (1945) The Structure and Reproduction of the Algae, Vol
2. Cambridge University Press, Cambridge, UK, pp 366-368
Griffith GW,
Jenkins GI,
Milner-White EJ,
Clutterbuck AJ
(1994)
Homology at the amino acid level between plant phytochromes and a regulator of asexual sporulation in Emericella (=Aspergillus) nidulans.
Photochem Photobiol
59:
252-256
[Medline]
Hedrich R,
Dietrich P
(1996)
Plant K+ channels: similarity and diversity.
Bot Acta
109:
94-101
Hedrich R,
Marten I
(1993)
Malate-induced feedback regulation of plasma membrane anion channels could provide a CO2 sensor to guard cells.
EMBO J
12:
897-901
[ISI][Medline]
Hedrich R,
Marten I,
Lohse G,
Dietrich P,
Winter H,
Lohaus G,
Heldt H-W
(1994)
Malate-sensitive anion channels enable guard cells to sense changes in the ambient CO2 concentration.
Plant J
6:
741-748
[CrossRef][ISI]
Holmes TC,
Fadool DA,
Ren R,
Levitan IB
(1996)
Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
Science
274:
2089-2091
[Abstract/Free Full Text]
Hughes J,
Lamparter T,
Mittman F,
Hartmann E,
Gartner W,
Wilde A,
Borner T
(1997)
A prokaryotic phytochrome.
Nature
386:
663
[CrossRef][Medline]
Hurd AM
(1920)
Effect of unilateral monochromatic light and group orientation on the polarity of germinating Fucus spores.
Bot Gaz
70:
25-50
[CrossRef]
Ingram J,
Bartels D
(1996)
The molecular basis of dehydration tolerance in plants.
Annu Rev Plant Physiol Plant Mol Biol
47:
377-403
[CrossRef][ISI][Medline]
Jaffe LF
(1954)
Stimulation of the discharge of gametangia from a brown alga by a change from light to darkness.
Nature
174:
743
Johnson LE, Brawley SH (1998) Dispersal and recruitment of a
canopy-forming intertidal alga, Pelvetia compressa
(Phaeophyceae). Oecologia (in press)
Kehoe DM,
Grossman AR
(1996)
Similarity of a chromatic adaptation sensor to phytochrome and ethylene receptors.
Science
273:
1409-1412
[Abstract]
Kidd DG,
Lagarias JC
(1990)
Phytochrome from the green alga Mesotaenium caldariorum: purification and preliminary characterization.
J Biol Chem
265:
7029-7035
[Abstract/Free Full Text]
Lee Y (1990) Ion movements that control pulvinar curvature in
nyctinastic legumes. In RL Satter, HL Gorton, TC
Vogelmann, eds, The Pulvinus: Motor Organ of Leaf Movement. American
Society of Plant Physiologists, Rockville, MD, pp 130-141
Li J,
Lee Y-RJ,
Assmann SM
(1998)
Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel.
Plant Physiol
116:
785-795
[Abstract/Free Full Text]
Luan S,
Li W,
Rusnak F,
Assmann SM,
Schreiber SL
(1993)
Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells.
Proc Natl Acad Sci USA
90:
2202-2206
[Abstract/Free Full Text]
Marten I,
Busch H,
Raschke K,
Hedrich R
(1993)
Modulation and block of the plasma membrane anion channel of guard cells by stilbene derivatives.
Eur Biophys J
21:
403-408
McCully ME
(1968a)
Histological studies on the genus Fucus. III. Fine structure and possible functions of the epidermal cells of the vegetative thallus.
J Cell Sci
3:
1-16
[Abstract/Free Full Text]
McCully ME
(1968b)
Histological studies on the genus Fucus. II. Histology of the reproductive tissues.
Protoplasma
66:
205-230
[CrossRef]
Moss BL
(1983)
Sieve elements in the Fucales.
New Phytol
93:
433-437
Pearson GA,
Brawley SH
(1996)
Reproductive ecology of Fucus distichus (Phaeophyceae): an intertidal alga with successful external fertilization.
Mar Ecol Prog Ser
143:
211-223
Pearson GA,
Serrão EA,
Brawley SH
(1998)
Control of gamete release in fucoid algae: sensing hydrodynamic conditions via carbon acquisition.
Ecology
79:
1725-1739
[CrossRef]
Pei Z-M,
Kuchitzu K,
Ward JM,
Schwartz M,
Schroeder JI
(1997)
Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.
Plant Cell
9:
409-423
[Abstract]
Pratt LH
(1995)
Phytochromes
differential properties, expression patterns and molecular evolution.
Photochem Photobiol
61:
10-21
Quail PH,
Boylan MT,
Parks BM,
Short TW,
Xu Y,
Wagner D
(1995)
Phytochromes: Photosensory perception and signal transduction.
Science
268:
675-680
[Abstract/Free Full Text]
Raven JA,
Osmond CB
(1992)
Inorganic C acquisition processes and their ecological significance in inter- and sub-tidal macroalgae of North Carolina.
Funct Ecol
6:
41-47
Robinson KR,
Lorenzi R,
Ceccarelli N,
Gualtieri P
(1998)
Retinal identification in Pelvetia fastigiata.
Biochem Biophys Res Commun
243:
776-778
[CrossRef][ISI][Medline]
Robinson KR,
Miller BJ
(1997)
The coupling of cyclic GMP and photopolarization of Pelvetia zygotes.
Dev Biol
187:
125-130
[CrossRef][ISI][Medline]
Roelfsema MRG,
Prins HBA
(1998)
The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification.
Planta
205:
100-112
[CrossRef][ISI][Medline]
Satter RL,
Morse MJ,
Lee Y,
Crain RC,
Coté GG,
Moran N
(1988)
Light- and clock-controlled leaflet movements in Samanea saman: a physiological, biophysical and biochemical analysis.
Bot Acta
101:
205-213
[ISI]
Schmidt C,
Schelle I,
Liao Y-J,
Schroeder JI
(1995)
Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events.
Proc Natl Acad Sci USA
92:
9535-9539
[Abstract/Free Full Text]
Schneider-Poetsch HAW,
Braun B,
Marx S,
Schaumburg A
(1991)
Phytochromes and bacterial sensor proteins are related by structural and functional homologies. Hypothesis on phytochrome-mediated signal-transduction.
FEBS Lett
281:
245-249
[CrossRef][ISI][Medline]
Schroeder JI
(1995)
Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions.
Plant Mol Biol
28:
353-361
[CrossRef][ISI][Medline]
Schroeder JI,
Keller BU
(1992)
Two types of anion channel currents in guard cells with distinct voltage regulation.
Proc Natl Acad Sci USA
89:
5025-5029
[Abstract/Free Full Text]
Schroeder JI,
Schmidt C,
Sheaffer J
(1993)
Identification of high-affinity slow anion channel blockers and evidence for stomatal regulation by slow anion channels in guard cells.
Plant Cell
5:
1831-1841
[Abstract]
Schwartz A,
Ilan N,
Schwarz M,
Scheaffer J,
Assmann SM,
Schroeder JI
(1995)
Anion-channel blockers inhibit S-type anion channels and abscisic acid responses in guard cells.
Plant Physiol
109:
651-658
[Abstract]
Serrão EA,
Pearson GA,
Kautsky L,
Brawley SH
(1996)
Successful external fertilization in turbulent environments.
Proc Natl Acad Sci USA
93:
5286-5290
[Abstract/Free Full Text]
Shinozaki K,
Yamaguchi-Shinozaki K
(1997)
Gene expression and signal transduction in water-stress response.
Plant Physiol
115:
327-334
[CrossRef][ISI][Medline]
Silva PC
(1996)
California seaweeds collected by the Malaspina expedition, especially Pelvetia (Fucales, Phaeophyceae).
Madroño
43:
345-354
Sommer D,
Wells TA,
Song P-S
(1996)
A possible tyrosine phosphorylation of phytochrome.
FEBS Lett
393:
161-166
[Medline]
Speransky VV, Brawley SH, McCully ME (1998) Ion fluxes during
potentiation and gamete release in the fucoid alga Pelvetia
compressa (J. Agardh) De Toni (abstract). J Phycol
34(suppl): 56
Starostzik C,
Marwan W
(1995)
A photoreceptor with characteristics of phytochrome triggers sporulation in the true slime mould Physarum polycephalum.
FEBS Lett
370:
146-148
[CrossRef][Medline]
Surif MB,
Raven JA
(1989)
Exogenous inorganic carbon source for photosynthesis in seawater by members of the Fucales and Laminariales (Phaeophyta): ecological and taxonomic implications.
Oecologia
78:
97-105
Taylor A,
Brownlee C
(1993)
Calcium and potassium currents in the Fucus egg.
Planta
139:
109-119
Thiel G,
Blatt MR
(1994)
Phosphatase antagonist okadaic acid inhibits steady-state K+ currents in guard cells of Vicia faba.
Plant J
5:
727-733
[CrossRef]
Thümmler F,
Algarra P,
Fobo GM
(1992)
Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase.
Plant Mol Biol
20:
1003-1017
[CrossRef][ISI][Medline]
Winlands A,
Wagner G
(1996)
Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position.
Plant Mol Biol
32:
589-597
[CrossRef][ISI][Medline]
Yaish P,
Gazit A,
Gilon C,
Levitski A
(1988)
Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors.
Science
242:
933-935
[Abstract/Free Full Text]
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Abstract
Introduction