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Plant Physiol. (1998) 118: 483-492
Inhibition of the Gravitropic Response of Snapdragon Spikes by
the Calcium-Channel Blocker Lanthanum Chloride1
Haya Friedman,
Shimon Meir,
Ida Rosenberger,
Abraham H. Halevy,
Peter B. Kaufman, and
Sonia Philosoph-Hadas*
Department of Postharvest Science of Fresh Produce, Agricultural
Research Organization, The Volcani Center, Bet Dagan 50250, Israel
(H.F., S.M., I.R., S.P.-H.); The Kennedy-Leigh Centre for Horticultural
Research, Faculty of Agriculture, The Hebrew University of
Jerusalem, Rehovot, 76100, Israel (A.H.H.); and Department of
Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048
(P.B.K.)
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ABSTRACT |
The putative Ca2+-channel
blocker LaCl3 prevented the gravitropic bending of cut
snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301-310) and inhibited stem curvature to a greater extent
than vertical and horizontal stem elongation at the bending zone. This
might indicate that LaCl3, which modulates cytosolic Ca2+, does not influence general stem-growth processes but
may specifically affect other gravity-associated processes occurring at
the stem-bending zone. Two such specific gravity-dependent events were
found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells,
as seen in cross-sections, and establishment of an ethylene gradient
across the stem. Our results show that the lateral sedimentation of
chloroplasts associated with gravity sensing was prevented in
cross-sections taken from the bending zone of LaCl3-treated and subsequently gravistimulated spikes and that LaCl3
completely prevented the gravity-induced, asymmetric ethylene
production established across the stem-bending zone. These data
indicate that LaCl3 inhibits stem curvature of snapdragon
spikes by preventing several gravity-dependent processes. Therefore, we
propose that the gravitropic response of shoots could be mediated
through a Ca2+-dependent pathway involving modulation of
cytosolic Ca2+ at various stages.
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INTRODUCTION |
Various aboveground plant parts, such as coleoptiles, hypocotyls,
seedlings, grass-shoot pulvini, and flowering stems, respond to a
change in a gravity vector by differential growth that leads to upward
bending. Most of the work on negative gravitropism has been performed
with seedlings and specific graviresponsive organs such as coleoptiles,
epycotyls and grass-shoot pulvini (Kaufman et al., 1985 ; Migliaccio and
Galston, 1987; Gehring et al., 1990; Brock et al., 1992; Kim and
Kaufman, 1995). However, there have been several reports of
gravitropism of stem-like organs (Rorabaugh and Salisbury, 1989; Kiss
et al., 1997; Strudwick et al., 1997), vegetative stems (Meicenheimer
and Nackid, 1994), and flowering shoots (Kohji et al., 1979; Halevy and
Mayak, 1981; Woltering, 1991; Philosoph-Hadas et al., 1995, 1996;
Fukaki et al., 1996). In this respect, the graviresponding spikes of
snapdragon (Antirrhinum majus L.; Philosoph-Hadas et al.,
1996) provide an excellent model system for investigating the
gravitropic phenomenon in mature inflorescence stems.
The common underlying mechanisms of negative
gravitropism involve perception of the stimulus and its transduction
into physiological processes that lead to a differential growth
response (Salisbury, 1993). Gravity perception is considered to be
accomplished by a mass pressure on the lower cell membrane following
changes in organ orientation. This mass is believed to be either that
of specific plastids, usually amyloplasts or chloroplasts, which sediment only in cells of a specific developmental stage and location (the starch-statolith model; Sack, 1997; Vitha et al., 1998), or that
of the entire cell (the gravitational pressure model; Staves, 1997).
According to the prevailing Cholodny-Went theory, this gravity
perception leads to redistribution of auxin toward the lower side of
the gravireacting organ, thereby increasing its growth rate and
consequently the organ reorientation (Li et al., 1991; Trewavas, 1992;
Salisbury, 1993). However, it is clear now that the gravitropic
response is a complex, multistep event also influenced by factors such
as ethylene (Wheeler et al., 1986; Salisbury, 1993; Philosoph-Hadas et
al., 1996), responsiveness and sensitivity to auxin (Rorabaugh and
Salisbury, 1989; Kim and Kaufman, 1995), and
Ca2+ (Philosoph-Hadas et al., 1995, 1996;
Belyavskaya, 1996; Sinclair and Trewavas, 1997), which might act in
succession or in parallel.
In several shoot systems the gravitropic response has been reported to
be accompanied by significantly higher amounts of ethylene produced by
the lower half of a horizontally positioned stem (Clifford et al.,
1983; Kaufman et al., 1985; Wheeler et al., 1986; Woltering, 1991;
Philosoph-Hadas et al., 1996). However, in only a few cases could
inhibitors of ethylene synthesis or action block the gravitropic response (Wheeler and Salisbury, 1981; Wheeler et al., 1986;
Philosoph-Hadas et al., 1996). Therefore, the role of ethylene in
gravitropism is still controversial.
Ca2+ ions have long been proposed to be essential
for gravitropic competence in plants (Pickard, 1985; Poovaiah et al.,
1987; Roux and Serlin, 1987; Trewavas, 1992; Bush, 1995; Belyavskaya, 1996; Sinclair and Trewavas, 1997). These ions were suggested to be
involved as a second messenger in all steps of the signal transduction
pathway leading to the gravitropic bending of higher plants: stimulus
perception (Pickard, 1985; Belyavskaya, 1996), auxin redistribution
(Migliaccio and Galston, 1987) or action (Saunders, 1990; Bush, 1995),
and differential cell growth (Brock et al., 1992; Jackson and
Hall, 1993). However, because of the difficulties in measuring
[Ca2+]cyt in intact plant
tissues, and because of the complexity of the gravitropic system
(Sinclair and Trewavas, 1997), evidence for the role of
[Ca2+]cyt in gravitropism
remains circumstantial. Only one example of what is apparently a direct
gravity-induced but only slightly sustained elevation of
[Ca2+]cyt in maize
coleoptiles has been reported so far (Gehring et al., 1990). This
finding was challenged recently by Legué et al. (1997), who
clearly demonstrated by direct measurements of [Ca2+]cyt that the
gravitropic response of Arabidopsis roots is not associated with
detectable changes in
[Ca2+]cyt.
Alternatively, the use of Ca2+ agonists and
antagonists has been extensively reported as providing indirect
evidence for the involvement of changes in
[Ca2+]cyt in the process
of gravistimulation (Poovaiah et al., 1987; Roux and Serlin, 1987;
Belyavskaya, 1996; Sinclair and Trewavas, 1997). Recently, we have
shown that several Ca2+ chelators (e.g. EGTA,
CDTA, and
1,2-bis[2-aminophenoxy]ethane-N,N,N ,N-tetraacetic acid)
and a Ca2+-channel blocker
(LaCl3) significantly inhibited the gravitropic response of snapdragon spikes (Philosoph-Hadas et al., 1996). These
agents had been similarly effective in inhibiting the gravitropic response of several other flowering stems capable of linear growth after harvest (Philosoph-Hadas et al., 1995).
The role of [Ca2+]cyt as
a second messenger requires the activity of Ca2+
channels between Ca2+ stores and the cytoplasm
that open upon signaling, thereby allowing the movement of
Ca2+ down its electrochemical gradient. In plant
and animal systems, La3+ ions inhibit various
Ca2+-dependent processes by interacting with
binding sites inside Ca2+ channels or by
stimulating Ca2+-ATPases, thereby preventing
elevation of [Ca2+]cyt
(Tester, 1990; Bush, 1995; Belyavskaya, 1996). Therefore, LaCl3 has been widely used to show
Ca2+ involvement in various physiological
responses (Knight et al., 1992; Jackson and Hall, 1993; Bush, 1995;
Polisensky and Braam, 1996; Rock and Quatrano, 1996), including
graviperception (Belyavskaya, 1996; Staves, 1997).
Since LaCl3 showed the most pronounced inhibitory
effect on the gravitropic response of snapdragon spikes
(Philosoph-Hadas et al., 1996), it was of interest to further
characterize its mode of action. For this purpose we examined whether
LaCl3 inhibits curvature through inhibition of
general stem-elongation processes and whether it affects other
gravity-induced processes such as amyloplast sedimentation, which is
associated with gravity perception, and differential ethylene
production, which is associated with the physiological response. Our
results indicate that LaCl3 significantly and
specifically affects these gravitropism-associated events and therefore
may provide additional evidence for the possible second-messenger role
of [Ca2+]cyt in the
gravitropic bending process of shoots.
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MATERIALS AND METHODS |
Plant Material and Treatments
Experiments were performed with several cultivars (Maryland White,
Maryland Appleblossom, and Potomac Royal) of snapdragon (Antirrhinum majus L., F1 hybrid),
purchased from the Pan-American Seed Company (West Chicago, IL).
Freshly cut spikes bearing four to six open florets were obtained from
local commercial growers. All treatments were performed as previously
described (Philosoph-Hadas et al., 1996) in a standard,
controlled-environment room maintained at 20°C with 60% to 70% RH
and 24 h of light at an intensity of 14 µmol
m 2 s1 provided by
cool-white fluorescent tubes. To enable their straightening after
harvest and transport, spikes were held vertically overnight with the
cut ends of the stems in distilled water. Spikes were subsequently
trimmed to a length of 60 to 70 cm, and 10 to 30 flower stalks were
placed in a 2-L plastic cylinder containing 400 mL of distilled water
or a solution of LaCl3 (Sigma) at concentrations between 10 and 30 mM (pH 6.0-7.0) for 20 to 24 h.
This duration of pulsing treatment was determined in preliminary
experiments.
After the spikes were pulsed, they were divided into bunches of five
stalks each and transferred to 1-L plastic cylinders containing 400 mL
of preservative solution (TOG-6, Assia Reizel Ltd., Ramat Gan, Israel)
containing 5 mg mL1 chlorine complexed with
sodium dichloro-isocyanureate to prevent microorganism contamination.
Gravitropic stimulation was provided by tilting the cylinders
horizontally at an angle of 30° to the table surface to avoid
drifting of the liquid inside the cylinder. In this position stems
could be kept with their cut ends inside the solution and still be
oriented initially at an angle of 180°, which represents noncurved
stems (Fig. 1A). The curvature angle (Fig. 1A) of 10 to 15 spikes was measured at hourly intervals with a
protractor to monitor the kinetics of stem bending. The stem thickness
at the bending zone of all of the snapdragon cultivars assayed ranged
between 4 and 10 mm. The bending rate of all cultivars was similar and
was not dependent on stem diameter (H. Friedman, S. Meir, I. Rosenberger, A.H. Halevy, and S. Philosoph-Hadas, unpublished data).
Experiments were repeated three to five times with similar results, and
data from individual representative experiments are presented.
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| Figure 1.
Schematic drawing illustrating the various
experimental settings used for snapdragon spikes. A, Gravitropic
stimulation of a snapdragon spike from a noncurved (180°) to a curved
(145°) position and the angle () measured as the curvature of the
stem. B, The numbers I through IV were given to each 5-cm segment
starting from the stem apex to 20 cm below for determination of the
elongation rate in vertical or horizontal spikes. C, Longitudinally
halved stem sections, excised from the stem-bending zone during
gravistimulation and used for ethylene measurements.
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Determination of Stem Elongation
Elongation measurements were performed with spikes of the cvs
Maryland White and Maryland Appleblossom. For stem-elongation measurements, leaves and florets were removed from the whole spike except for the top 5-cm apical section, which contains only buds. These
bare spikes responded to gravistimulation in a manner similar to that
of floret-bearing stalks (data not shown). Four sections at 5-cm
intervals, designated I to IV counting from the apical part of the
inflorescence stem, were marked on the bare stalk with a thin marker
(Fig. 1B). The marker did not cause any obvious damage to stem cells,
as judged from microscopic analysis and curvature measurements. The
marked stems were then pulsed for 22 h with various
LaCl3 concentrations (10-30 mM) and
placed either vertically or horizontally in the preservative solution
for an additional 24 h, after which time the elongation of each
stem section was determined with a ruler. The elongation rate was
calculated separately for each 5-cm stem section.
Staining of Starch-Containing Chloroplasts (Amyloplasts)
Snapdragon spikes were pulsed with the lowest concentration of
LaCl3 that caused maximal inhibition of curvature
(10-20 mM LaCl3 depending on spike
diameter). Following pulsing, spikes were either kept vertical or
gravistimulated for an additional 6 or 10 h (to obtain curvatures
of 150° or 90°, respectively). Hand-cut cross-sections were
prepared from the middle part of the stem-bending zone (section II)
taken from LaCl3-treated or nontreated spikes at
the indicated intervals during gravistimulation. The sections were
stained with iodine solution (10% KI/5% I2 in distilled water), as described previously (Song et al., 1988) and
immediately examined with a light microscope (Vanox, Olympus), and
photographs were taken with Kodak 100-ASA film.
Ethylene Measurements
For measurements of ethylene-production rates during the
gravitropic bending of snapdragon spikes, 5-cm stem segments were excised from the bending zone of treated and untreated spikes (Fig. 1C)
at various intervals following gravistimulation, as described
previously (Philosoph-Hadas et al., 1996). After leaves and florets
were removed, the longitudinally halved stem sections (Fig. 1C) were
weighed and individually placed in 25-mL Erlenmeyer flasks sealed with
rubber serum caps for 1 h at 20°C. The upper and lower halves of
horizontally placed stems were maintained in their original positions
during ethylene measurement. The ethylene concentration in each flask
was analyzed by withdrawing a 2-mL gas sample with a hypodermic syringe
and injecting it into a gas chromatograph (Varian, Palo Alto, CA)
equipped with an activated-alumina column and a flame-ionization
detector.
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RESULTS |
Effect of LaCl3 on the Gravitropic Response of
Snapdragon Spikes
cv Maryland White spikes were pulsed with 20 mM
LaCl3 and the effects on the bending kinetics are
illustrated in Figure 2. The results show
that LaCl3 delayed the onset of upward bending of
this cultivar by 3 h and reduced the bending rate from 7° to 2° h1. A similar curvature inhibition pattern
was found for the other snapdragon cultivars after pulsing with
LaCl3 concentrations between 10 and 40 mM, without any detectable damage (H. Friedman, S. Meir, I. Rosenberger, A.H. Halevy, and S. Philosoph-Hadas, unpublished data). It
should be noted that spikes (7-10 mm thick) pulsed with 20 mM LaCl3 for 24 h absorbed an
average amount of 0.9 mg LaCl3 g1 fresh weight, which was effective in
blocking their gravitropic response (Fig. 2). Therefore, for effective
curvature inhibition of thinner spikes (4-6 mm thick), this amount of
LaCl3 was obtained by shorter pulsing time or
lower LaCl3 concentrations.
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| Figure 2.
Effect on the kinetics of the gravitropic response
after pulsing cv Maryland White spikes with LaCl3.
Flowering stems were pulsed with 20 mM LaCl3
for 22 h and placed horizontally. Each point and bar represent an
average angle of curvature ± SE of 15 spikes.
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Effect of LaCl3 on Stem-Elongation Rate
To examine the possibility that LaCl3
inhibits the gravitropic response by inhibiting stem elongation, we
studied the effect of LaCl3 on the elongation
rate of vertically and horizontally positioned spikes of two snapdragon
cultivars; detailed results of cv Maryland White are presented in
Figure 3. The elongation rates of four
different sequential stem sections (I-IV; Fig. 1B) were determined for
vertical and horizontal spikes after pulsing with various
LaCl3 concentrations. The elongation rates of the three top sections (I-III) of vertical spikes were in the same range
(60-90 µm h1), whereas that of section IV
was much lower (Fig. 3A). Unlike the elongation profile of vertical
stems, section II of horizontally positioned spikes, i.e. the
stem-bending zone, showed the highest elongation rate (almost twice as
high as that of horizontal sections I and III or of vertical sections
I-III; Fig. 3A). This indicates that gravistimulation induces stem
elongation, particularly in the bending zone.
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| Figure 3.
Elongation profiles of vertical and horizontal
stem sections of cv Maryland White spikes in the absence (A) or
presence of 10 mM (B), 20 mM (C), or 30 mM (D) LaCl3. Spikes were pulsed for 22 h
with the various LaCl3 concentrations, and elongation rates
of each of the four designated stem sections (I-IV) were determined
24 h later. Each column and bar represent an average elongation
rate ± SE of 10 stem replicates. Numbers in
parentheses represent the average angle of curvature ± SE of 10 horizontally positioned spikes.
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Pulsing spikes with 10 mM LaCl3
either promoted or did not significantly affect the elongation rates of
the various sections of vertical and horizontal spikes (Fig. 3B), nor
did this LaCl3 concentration change the curvature
of horizontal spikes (Fig. 3B). On the other hand, higher
LaCl3 concentrations, which markedly delayed stem
curvature, significantly inhibited the elongation rate of all stem
sections in both horizontal and vertical spikes (Fig. 3, C and D). The
highest degree of inhibition was obtained in section II of vertical
stems, whereas sections II and III of horizontal stems were equally
inhibited by LaCl3 (Table
I), indicating that
LaCl3 does not interfere with growth of all stem
sections but, rather, affects a specific region in both vertical and
horizontal stems. It is noteworthy that, in general, the elongation
rate of section II of horizontal spikes was higher than that of the corresponding section of vertical spikes at all
LaCl3 concentrations examined, even at 30 mM, a concentration that completely eliminated the bending
response.
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Table I.
Effect of two LaCl3 concentrations on
the percent inhibition of the elongation rates of the three top stem
sections in vertical and horizontal cv Maryland White spikes
Data were obtained from Figure 3 and are presented as percentages of
control.
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When the relative extents of inhibition by LaCl3
of stem curvature and elongation rate of section II of cv Maryland
White spikes are analyzed, it can be seen that these two processes are not similarly inhibited. Thus, 20 and 30 mM
LaCl3 inhibited the angle of spike curvature by
80% and 100%, respectively, although these concentrations inhibited
the elongation rates of vertical and horizontal shoots similarly, but
to a lesser extent, by only 33% to 35% and 54% to 60%, respectively
(Fig. 3, C and D; Table I). The differential inhibition pattern of
these two processes was even more pronounced in cv Maryland
Appleblossom. Figure 4 shows the relative
changes in the angle of curvature and stem-elongation rates of section
II of vertical and horizontal spikes of this snapdragon cultivar after
pulsing with various LaCl3 concentrations.
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| Figure 4.
Effect of three LaCl3 concentrations
on the relative changes in curvature angles of gravistimulated spikes
and in elongation rates of the stem-bending zone section II of cv
Maryland Appleblossom spikes incubated for 24 h in either the
vertical or the horizontal orientation. Results are expressed as
percent inhibition or promotion, based on control values and calculated
from the means of 15 spikes.
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The results show that 10 mM LaCl3
enhanced the elongation rates of vertical and horizontal stems in this
cultivar but did not affect the angle of curvature (Fig. 4), as it did
for cv Maryland White (Fig. 3B). However, 20 and 30 mM
LaCl3 reduced the angle of curvature of cv
Maryland Appleblossom spikes by 40% and 90%, respectively. This
significant inhibition was obtained even though 20 mM
LaCl3 promoted vertical growth and inhibited
horizontal growth by only 20%, and 30 mM
LaCl3 inhibited elongation of vertical and
horizontal shoots by only 20% and 60%, respectively (Fig. 4). These
results indicate that (a) in all cases LaCl3
inhibited horizontal stem elongation more than it inhibited vertical
elongation and (b) the extent of reduction of the angle of curvature by
LaCl3 was in all cases almost double the extent
of inhibition for the elongation of both vertical and horizontal
spikes. It seems, therefore, that LaCl3 affects
bending much more than it affects vertical or horizontal stem
elongation.
Effect of LaCl3 on Sedimentation of Chloroplasts in
Stems during Gravistimulation
It has been suggested that amyloplast/chloroplast sedimentation is
responsible for gravity stimulus perception (Sack, 1997). Therefore,
the effect of LaCl3 on this gravity-dependent
event was studied in cells of gravistimulated snapdragon spikes. Figure 5 illustrates the chloroplast
distribution as seen in stem cross-sections prepared from the bending
zones of vertically and horizontally positioned cv Potomac Royal
spikes. In vertical spikes the chloroplasts usually sediment in the
direction of the gravitational field in the lower part of the cortex
stem cells (Brock et al., 1989). This typical pattern of chloroplast
position appeared as a random distribution in stem cells seen in
cross-sections of vertical spikes (Fig. 5, A and B). On the other hand,
in gravistimulated spikes that had fully curved to 90°, the
cross-sections were characterized by a polar position of chloroplasts
at the bottoms of the cells (Fig. 5, C and D). It is noteworthy that
this gravity-induced orientation of chloroplasts occurred only in the
cortex cells close to the vascular system (Fig. 5D).
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| Figure 5.
Light micrographs of chloroplast distribution in
cortical cells of the stem-bending zone as seen in cross-sections
prepared from vertical (A and B) or gravistimulated (90°; C and D) cv
Potomac Royal spikes. Cross-sections of the stem-bending zone taken
from vertical or gravistimulation spikes were stained with
KI/I2 solution and examined under the microscope. c,
Cortex; v, vascular cylinder. Bars = 25 µm.
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A similar sedimentation pattern was obtained in spikes curved only to
150° (Fig. 6A). Pulsing spikes with 10 mM LaCl3, which completely inhibited
the gravitropic curvature of cv Potomac Royal, prevented the
typical chloroplast sedimentation obtained in untreated spikes (Fig. 6,
B and C). However, LaCl3 did not affect the
chloroplast distribution in either cortex or pith cells of vertical
shoots, as seen in stem cross-sections (Fig. 6D). Therefore, their
chloroplast distribution was very similar to that of untreated vertical
shoots (Fig. 5, A and B). A similar pattern of chloroplast
sedimentation in the absence or presence of LaCl3
was also obtained in cross-sections of the other snapdragon cultivars
assayed in this work (data not shown). Therefore, it seems that
LaCl3 prevents the gravity-induced sedimentation
of chloroplasts in snapdragon stem cells.
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| Figure 6.
Effect of LaCl3 on chloroplast
distribution in cortical (c) and pith (p) cells of the stem-bending
zone as seen in cross-sections prepared from gravistimulated (A-C) or
vertical (D) cv Potomac Royal spikes. Spikes were pulsed with either
distilled water or 10 mM LaCl3 for 20 h
and gravistimulated or held vertically for an additional 6 h.
Cross-sections of the stem-bending zones were stained and examined as
detailed in Figure 5. The arrows point to individual cells in which the
chloroplast distribution is clearly evident. A, Control gravistimulated
spikes (150°); B and C, LaCl3-treated and gravistimulated
spikes (180°); D, LaCl3-treated, vertical spikes. v,
Vascular cylinder; c, cortex; p, pith. Bars = 25 µm.
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Effect of LaCl3 on Ethylene-Production Rates during the
Gravitropic Response
To further elucidate the mode of action of
LaCl3 on physiological processes occurring during
the gravitropic response, we examined its effect on ethylene-production
rates of longitudinally halved stem sections taken from the
spike-bending zone during gravistimulation (Fig. 1C). Figure
7A shows that the ethylene-production rates of lower stem halves were relatively low (2 nL
g1 h1) for the first
2 h of gravistimulation, but it then started to increase, reaching
a maximum of 13 nL g1
h1 after 8 h and declining slightly
thereafter. However, except for an initial increase to 5 nL
g1 h1 during the first
2 h, ethylene-production rates of upper stem halves remained low,
in the range of 2 to 3 nL g1
h1, during 25 h of gravistimulation (Fig.
7A). Consequently, the ratio between the ethylene-production rates of
lower and upper stem halves, which was approximately unity during the
initial 4 h of gravistimulation, eventually reached values of
3 to 4 (Fig. 7C). This ethylene gradient coincided with the initiation
of stem curvature and persisted during the progress of the bending
response (Fig. 7D). Thus, the initial increase in ethylene-production
rates in the lower stem halves (Fig. 7A) appeared together with bending initiation (Fig. 7D), and the maximal ethylene gradient (Fig. 7C)
appeared when a curvature angle of 130° was attained (Fig. 7D).
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| Figure 7.
Effect of pulsing cv Maryland White spikes with
LaCl3. Shown are the effects on the kinetics of ethylene
evolution (A and B) from longitudinally halved stem sections, cut from
their bending zone, on the ethylene-production ratio (C) and on the
kinetics of spike curvature (D) during 25 h of gravistimulation.
Spikes were pulsed with 20 mM LaCl3 for 24 h and placed horizontally for an additional 25 h. Stem sections (5 cm long) were excised from the bending zone at the indicated intervals,
cut longitudinally into halves, and incubated for 1 h in sealed
vials for ethylene accumulation. Points and bars are the averages ± SE of seven replicates. LH, Lower halves; UH, upper
halves.
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Pulsing spikes with 20 mM LaCl3 prior
to gravistimulation modified this pattern of ethylene-production rate
(Fig. 7B); it markedly increased ethylene-production rates of both
upper and lower stem halves (Fig. 7B) and prevented the development of
the ethylene gradient between these two stem sections (Fig. 7C). In parallel, the bending response of LaCl3-treated
spikes was completely inhibited and their angle of curvature remained
at 180° during 25 h of gravistimulation (Fig. 7D).
| |
DISCUSSION |
In our previous study (Philosoph-Hadas et al., 1996) we showed
that the putative plasma membrane Ca2+-channel
blocker LaCl3 significantly inhibited the
gravitropic response of snapdragon. The present study demonstrates that
the inhibitory effect of LaCl3 on gravitropic
bending may result from its inhibitory effect on various
gravity-associated processes. This effect was found in several
different snapdragon cultivars, with slight variations, indicating that
the phenomenon may be widespread. Until now,
LaCl3 has rarely been used to block the gravitropic response in plants: apart from inhibiting gravity-induced cytoplasmic streaming in characean algae (Staves, 1997),
La3+ has been shown to suppress
gravisensitivity only in maize coleoptiles and roots (for review, see
Belyavskaya, 1996) and in pea roots (Belyavskaya, 1992).
Analysis of the elongation profiles of various stem sections of
snapdragon revealed that, despite the elongation potential of all three
top sections (sections I-III) of vertical spikes, only section II
exhibited a significantly increased elongation rate following
gravistimulation (Fig. 3A). This higher elongation rate of section II
seems to contribute to the upward bending of gravistimulated spikes and
could be attributed to the ability of cells in this particular region
to perceive and/or respond to the gravity stimulus. Therefore, like the
root cap (Sack, 1997), stems seem to have specific sites for gravity
perception and response.
The elongation rate of section II of both vertical and horizontal
spikes was also the most responsive to LaCl3
inhibition (Fig. 3, C and D; Table I). Therefore,
LaCl3 interferes mainly with the elongation
process, occurring specifically in this section of both vertical and
horizontal spikes. Moreover, the following observations suggest that
LaCl3 also interferes with several other distinct
processes that lead to the asymmetric stem elongation induced by
gravity, rather than inhibiting bending merely by blocking general stem
elongation. LaCl3 inhibited the gravitropic
response by reducing the bending rate and by extending the lag period
until the visible response was apparent (Fig. 2), indicating that
LaCl3 might interfere with initial processes
occurring during this lag period. Also, LaCl3
inhibited the elongation rate of horizontal spikes to a greater extent
than it inhibited the elongation rate of vertical spikes (Figs. 3 and
4; Table I), indicating that LaCl3 interferes
with gravity-induced elongation processes. And finally,
LaCl3 was more efficient at inhibiting the
bending response than at inhibiting the elongation rate (Figs. 3 and
4). This was particularly pronounced in the cv Maryland Appleblossom
(Fig. 4), in which LaCl3 could specifically
inhibit both bending and elongation rates of horizontal stems in spite
of its promotive effect on vertical spike elongation.
Additional support for the view that LaCl3
affects specific processes induced by gravistimulation is provided by
studies of amyloplast/chloroplast sedimentation, which is believed to
be one of the susceptors that trigger gravitropic sensing in roots and
shoots of higher plants (Song et al., 1988; Sack, 1997; Vitha et al.,
1998). As was previously found for columella cells of roots or for
endodermis of stem seedlings (Sack, 1997), the bending zone of the
snapdragon stem was found to be very rich in starch-containing chloroplasts (Figs. 5 and 6D). This might contribute to the high responsiveness of this spike to gravity. Although the chloroplasts were
present in the cortex and pith, their gravity-induced sedimentation was
restricted to specified stem zones in the inner cortex and around the
vascular system in the stele (Figs. 5, C and D, and 6A).
The chloroplast-distribution pattern in cross-sections of vertical
(Fig. 5, A and B) and horizontal (Fig. 5, C and D) snapdragon spikes
was similar to that found in cross-sections of barley (Brock et al.,
1989) and oat (Brock and Kaufman, 1990) pulvini taken from the
corresponding shoot orientations. This provides additional correlative
evidence for their important role in the gravitropic sensing of shoots.
To our knowledge, our observations show for the first time that
LaCl3 treatment prevented the chloroplast sedimentation in gravistimulated spikes (Fig. 6, B and C), which indicates that LaCl3 could inhibit bending by
preventing the gravity-induced sedimentation of the chloroplast
gravisensors. Similarly, LaCl3 has previously
been demonstrated to disturb the polar distribution of amyloplasts in
statocytes of pea roots (Belyavskaya, 1992). Since
LaCl3 is known to inhibit
Ca2+ fluxes across the plasma membrane, these
results imply that the gravity-induced sedimentation of chloroplasts
could be mediated by changes in
[Ca2+]cyt. Alternatively
or additionally, since LaCl3 can penetrate the
cell, the possibility that LaCl3 may directly
disturb the sedimentation process cannot be excluded.
Another gravity-related process that was influenced by
LaCl3 was the development of an ethylene gradient
across the stem (Fig. 7, A-C). It is possible that this gradient is
necessary for curvature development, since the timing of its formation
was correlated with the initiation of curvature development (Fig. 7, C
and D), as reported previously (Philosoph-Hadas et al., 1996). Similar correlative evidence for the formation of curvature and the development of gravity-induced ethylene gradients across the stem, with more ethylene in the lower half, has been reported previously in various other systems (Clifford et al., 1983; Wheeler et al., 1986; Woltering, 1991). The relevance of this ethylene gradient to differential cell
elongation and, hence, to the gravitropic bending of flowering stems
was previously hypothesized as possibly reflecting an asymmetric auxin
distribution across the stem (Philosoph-Hadas et al., 1996). Additionally, the direct contribution of such an asymmetric ethylene production to differential stem elongation cannot be excluded. This
possibility is indicated by the close correlation found in the present
study between the remarkably increased rates following gravistimulation
of elongation in the stem-bending zone (Fig. 3A) and of the ethylene
production in the lower half of the stem-bending zone (Fig. 7A). In
this respect, the recent findings of Smalle et al. (1997) showing that
ethylene can induce marked hypocotyl elongation in light-grown
Arabidopsis may provide further support for this idea. However, such an
ethylene-induced elongation seems to be dependent on ethylene levels in
the tissue, because when endogenous ethylene-production rates reached
very high values (40-45 nL g1
h1) in the presence of 20 mM
LaCl3 (Fig. 7B), horizontal stem elongation was
suppressed (Fig. 3C).
LaCl3 prevented ethylene gradient development by
increasing the ethylene-production rates of both the upper and lower
halves of horizontal spike stems (Fig. 7, A and B). This may be because LaCl3 induces expression of genes involved in
ethylene production, as shown recently for mRNA levels of several other
genes (Polisensky and Braam, 1996; Rock and Quatrano, 1996). On the
other hand, CDTA, a Ca2+ chelator that also
inhibited gravitropic bending, prevented the development of such an
ethylene gradient in snapdragon shoots by reducing the
ethylene-production rates in the lower stem section (Philosoph-Hadas et
al., 1996). This indicates that, although these two
Ca2+ antagonists affected ethylene production in
an opposite manner, they both resulted in elimination of the ethylene
gradient across the stem and in a parallel blocking of spike curvature.
It seems, therefore, that the development of such an ethylene gradient
could be an important prerequisite for development of stem curvature. In addition, the opposite effect of these two
Ca2+ antagonists on ethylene production indicates
that ethylene is induced directly by LaCl3
application (Fig. 7, A and B) rather than by modulation of
[Ca2+]cyt.
LaCl3 can exert its inhibitory effect on various
gravity-induced and possibly Ca2+-dependent
processes by several modes of action. At the cellular level,
La3+ ions can prevent increases in
[Ca2+]cyt through a
direct inhibitory effect on Ca2+ channels and a
stimulatory effect on several Ca2+-ATPases,
thereby interfering with free Ca2+ availability
(Bush, 1995; Belyavskaya, 1996). It was demonstrated by using
aequorin-containing transgenic plants that LaCl3
directly prevented the
[Ca2+]cyt increase
induced by cold treatment (Knight et al., 1992; Polisensky and Braam,
1996). Thus, it seems that the putative elevation of
[Ca2+]cyt following
gravistimulation may require extracellular Ca2+
and may derive from an influx through Ca2+
channels. This possibility is reinforced by our findings showing that,
in addition to LaCl3, snapdragon bending was also
inhibited by Ca2+ chelators such as CDTA or
1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid
(Philosoph-Hadas et al., 1995, 1996), which are known to reduce
Ca2+ fluxes across the plasma membrane.
Nevertheless, it is still possible that, unlike the
Ca2+ chelators, La3+ ions
have more complex effects in addition to antagonizing
Ca2+ (Polisensky and Braam, 1996).
It has been reported that LaCl3 can inhibit other
intracellular ion channels (Tester, 1990), can directly induce mRNA
levels of several genes (Polisensky and Braam, 1996; Rock and Quatrano, 1996), can bind proteins and various ligands in biological molecules, and can often mimic Ca2+ action inside the cell
(Belyavskaya, 1996). These direct effects of
LaCl3 may be attributed to the fact that it can
be taken up by plant cells, whereas the Ca2+
chelator CDTA remains in the extracellular space (Bush, 1995; Polisensky and Braam, 1996). In addition, unlike the findings of
Gehring et al. (1990) in oat coleoptiles, Legué et al. (1997) recently directly demonstrated that the gravitropic response of Arabidopsis roots is not associated with detectable changes in [Ca2+]cyt. Therefore,
although these contradictory results could be ascribed to the different
plant organs used in these studies and in this study (coleoptiles and
stems versus roots), regarding the recent observations in roots and the
direct effects of LaCl3, it is still possible that the
inhibitory effect of LaCl3 might be unrelated to
[Ca2+]cyt changes.
In summary, the present study shows that the putative
Ca2+-channel blocker LaCl3
inhibits the negative-gravitropic curvature of snapdragon spikes by
only partially inhibiting the stem-elongation rate. Its more pronounced
inhibitory effect on the bending response seems to be exerted through
prevention of several gravity-induced processes, including
starch-containing chloroplast sedimentation and formation of an
ethylene gradient across the stem.
The exact sequence of these gravity-induced events is not yet clear.
One possibility is that the change in stem orientation leads to changes
in [Ca2+]cyt, which may
induce sedimentation of chloroplasts in the stem-bending zone. This
sedimentation process may elicit, through interaction with cell
membranes, additional transient changes in
[Ca2+]cyt, which may
affect membranous IAA receptors, altering IAA transport (Gross and
Sauter, 1988) and leading to the asymmetric production of ethylene
across the stem, as well as to differential stem growth. Accordingly,
LaCl3 might prevent this cascade of events by
blocking Ca2+ channels, preventing
[Ca2+]cyt elevation, and
thereby disturbing chloroplast sedimentation and subsequent
gravity-associated processes. In addition, LaCl3 might prevent bending through direct disturbance of chloroplast sedimentation, direct induction of ethylene biosynthesis genes, and/or
inhibition of IAA transport. A careful analysis is now being performed
to examine the effects of LaCl3 on the
differential stem-growth process and on endogenous IAA distribution in
the bending zone of snapdragon spikes and may shed light on this
hypothesis.
| |
FOOTNOTES |
1
This work was supported by grant no. IS-2434-94
from the United States-Israel Binational Agricultural Research and
Development Fund, by grant no. 95/26 from the Joint Dutch-Israeli
Agricultural Research Program, and by the Pearlstein Family Fund for
Research in Ornamental Horticulture at the Hebrew University (to
A.H.H.). This study is no. 407 in the Agricultural Research
Organization (Volcani Center, Bet Dagan, Israel) series.
*
Corresponding author; e-mail vtsoniap{at}volcani.agri.gov.il; fax
972-3-968-3622.
Received April 13, 1998;
accepted June 11, 1998.
| |
ABBREVIATIONS |
Abbreviations:
[Ca2+]cyt, [Ca2+] of the cytosol.
CDTA, trans-1,2-
cyclohexane dinitro-N,N,N,N-tetraacetic acid.
| |
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Top
Abstract
Introduction