1269
Journal of Cell Science 110, 1269-1278 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS1406
Tip localized Ca2+ pulses are coincident with peak pulsatile growth rates in
pollen tubes of Lilium longiflorum
Mark Messerli and Kenneth R. Robinson*
Department of Biological Sciences, West Lafayette, IN 47907-1392, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
It is known that locally elevated Ca2+ at the growing tips of
pollen tubes is necessary for pollen tube elongation. Here
we show that this localized Ca2+ is also temporally
regulated and is closely associated with pulsatile tip growth.
Lilium longiflorum pollen tubes were injected with the photoprotein, aequorin, and the Ca2+-dependent light output
was detected with a low noise photon-counting system. Ca2+
pulses with a mean period of 40 seconds were invariably
associated with growth. The pulses were sporadic and of
low amplitude for about the first 1.5 hours after germination. With subsequent growth, pulses increased in
amplitude and the period between pulses became
more regular. We have localized these Ca2+ pulses to the
INTRODUCTION
Pollen tube elongation is an essential process for the reproduction of flowering plants. After the pollen grain lands on the
stigma, it germinates a tube that grows down the style to the
ovary in order to transport the sperm from the grain to the
ovum. Tube elongation is a result of directed vesicle fusion,
which adds the necessary cell wall material and plasma
membrane components (Lancelle and Hepler, 1992; Larson,
1965; Rosen et al., 1964). Lilium longiflorum pollen grains,
typically about 90 µm in diameter, must grow tubes several
centimeters long in order to reach the ovary.
Pollen tube elongation is known to be a Ca2+-dependent
process. The presence of Ca2+ in the growth medium is
necessary for germination and growth (Brewbaker and Kwack,
1963). Tubes grown in 1% of normal Ca2+ elongate at half the
usual rate and tend to burst, while even lower Ca2+ concentrations reduce growth further and induce more bursting
(Weisenseel and Jaffe, 1976). Injection of tubes with the Ca2+
buffer, BAPTA, completely inhibits growth (Miller et al., 1992;
Pierson et al., 1994). La3+, an inorganic Ca2+ channel blocker,
has also been shown to stop pollen tube growth (Picton and
Steer, 1985).
It has long been thought that Ca2+ is spatially graded in
growing pollen tubes, with higher Ca2+ at the tip (Jaffe et al.,
1975). However, it was not until ratiometric fluorescent Ca2+
indicators, indo-1 and fura-2, were microinjected into pollen
tubes that such cytosolic gradients were demonstrated unequiv-
elongating end of the growing tube. The Ca2+ pulses are
asymmetrical, rising more slowly than they fall. We
estimate that the Ca2+ concentration at the peak of the
pulses reaches nearly 10 µM. The addition of 100 µM La3+,
a Ca2+ channel blocker, extinguished the pulses. An
analysis of growth of elongating tubes establishes that
extension is pulsatile, with a 42 second period between
pulses. Calcium imaging, using the fluorescent indicator,
Calcium Green dextran, shows that calcium pulses are
coincident with peak growth rates.
Key words: Ca2+ pulse, Aequorin, Calcium green, Tipgrowth, Pollen
tube
ocally (Miller et al., 1992; Rathore et al., 1991). Those experiments were done on Lilium longiflorum pollen tubes and it was
shown clearly that there is a steep, tip-directed gradient of Ca2+
localized to the distal 30 µm or so of the growing tube. Furthermore, it was shown by both groups that the elevated Ca2+
at the tip was absolutely correlated with growth; if the gradient
was disrupted by any means, growth ceased. Subsequent work
has shown that the extent of the region of elevated Ca2+ is
confined to 20 µm and that an influx of external Ca2+ is
required (Pierson et al., 1994). The existence of the Ca2+
gradient is not unique to lily pollen; similar gradients have been
found in Nicotiana sylvestris, Tradescantia virginiana and
Agapanthus umbellatus pollen tubes (Malhó et al., 1994;
Pierson et al., 1994).
Growth in lily pollen is pulsatile (Pierson et al., 1996),
mirroring the situation in fungal hyphae (López-Franco et al.,
1994). Pierson et al. (1996) also detected fluctuations in Ca2+,
but were unable to determine the periodicity due to limitations
on the rate at which ratiometric images could be gathered. We
too have detected Ca2+ fluctuations in lily pollen, using ratiometric fluorescent indicators, and have also failed to establish
their period. In addition to the problem of the rate of gathering
images, prolonged exposure to the ultraviolet excitation light
used with ratiometric Ca2+ indicators damaged the tubes and
interfered with growth. Consequently, we have elected to
sacrifice spatial information (which is already well established), by using aequorin, in order to achieve better temporal
resolution. We have also used the non-ratiometric fluorescent
1270 M. Messerli and K. R. Robinson
indicator, Calcium Green, in order to establish the phase relationship between growth pulses and calcium pulses.
The photoprotein, aequorin, emits light upon reacting with
Ca2+. Once this reaction has occurred the aequorin molecule is
irreversibly inactivated. The rate of light emission increases
with the 2.2 power of the Ca2+ concentration (Shimomura et
al., 1993), making aequorin an excellent indicator for spatially
or temporally localized increases in Ca2+. It can be microinjected into cells and is non-disturbing to biological processes
over periods of hours or even days (Miller et al., 1994). It is
available in a variety of semi-synthetic forms with differing
Ca2+ sensitivities (Shimomura et al., 1993). Its major drawback
is the low level of light emission, which makes imaging
difficult. However, total light emission from single cells can be
recorded by fairly simple means, with excellent temporal resolution (Cork et al., 1987; Keating et al., 1994). We have
employed this approach in order to detect Ca2+ fluctuations in
growing lily pollen tubes.
Although spatially-resolved Ca2+ dynamics can be determined with aequorin, using ultralow-noise imaging equipment,
a more accessible method was chosen. Scanning confocal fluorescence microscopy of injected Calcium Green dextran is
sufficient to show relative changes in Ca2+ concentration. As
the Calcium Green signal is not ratiometric, the fluorescence
will depend not only on the Ca2+ concentration but also on the
amount of indicator in the optical path. This factor can be controlled for by imaging a calcium-insensitive fluorescent
indicator; we used fluorescein dextran. Both Calcium Green
and fluorescein can be excited by the krypton/argon laser line
at 488 nm, which avoids UV irradiation and permits long-term
imaging of pollen tubes.
MATERIALS AND METHODS
Lilium longiflorum flowers were grown in a greenhouse and the pollen
was separated from the anthers, dried at room temperature for 1 week
and stored at −20°C. A small sample of pollen was hydrated in
modified Dickinson’s medium (in mM: 0.16 H3BO3, 0.127 Ca(NO3)2,
1.0 KNO3, 1.0 succinic acid, 292.0 sucrose), pH 5.5, which was mixed
with an equal volume of 2% low-temperature gelling agarose (FMC
Co., Rockland, ME), in modified Dickinson’s medium. The agarose
provided mechanical support during injection but also impeded the
rapid change of medium near the tubes. The pollen/agarose mixture
was then put into 35 mm ×10 mm Falcon culture dishes with No.1
coverglass bottoms, and placed at 4°C for 1 minute to gel the agarose
thus forming a gelled layer about 1 mm deep. A few milliliters of
modified Dickinson’s medium was added to immerse the gel and the
dish was then placed in a 25°C incubator. Pollen began germinating
in less than an hour under these conditions. Growth rate measurements
and Ca2+ measurements were done at room temperature, 21-22°C.
Aequorin
A high pressure injection system (Kiehart, 1982) was used to inject
pollen tubes with 1-2 pl of 10 mg/ml recombinant h-aequorin
(provided by Dr O. Shimomura at the Marine Biological Laboratory,
Woods Hole, MA). Briefly, mercury filled micropipettes were
connected to a threaded syringe filled with a fluorocarbon oil, Fluorinert (Sigma Chemical Co., St Louis, MO), via Fluorinert-filled
tubing. Positive pressure was applied to force the mercury to the
micropipette tip. The micropipette was then dipped into a small
volume of h-aequorin, which was drawn into the micropipette
followed by a small volume of dimethylpolysiloxane, DMPS (Sigma).
The DMPS oil cap in the micropipette prevented the aequorin from
being exposed to Ca2+ before it could be injected into the tube. Tubes
growing parallel to the coverslip plane and with lengths between 300
and 500 µm were selected for injection. Micropipettes were pulled
from 1 mm OD Microcaps glass (Drummond Scientific, Broomall,
PA) to approximately 1 µm OD and were silanized before use. For
silanization, micropipettes were dried at 200°C for 1 hour, treated with
N,N-dimethyltrimethylsilylamine
(Fluka
Chemical
Co.,
Ronkonkoma, NY) for 1 hour at 200°C in an enclosed chamber and
then cooled before use.
Photomultiplier system
Photon counting was performed with a system previously described
(Cork et al., 1987; Keating et al., 1994). The equipment comprised an
R464s photomultiplier tube (Hamamatsu, Bridgewater, NJ) and a
Pacific Precision Instruments (Concord, CA) amplifier/discriminator.
The photon-generated pulses were sent to a PC-LPM-16 data acquisition card (National Instruments, Austin, TX) installed in an IBMPC/XT, which recorded the number of pulses in each one second
interval. The data were then converted to ASCII format and imported
into Axum 5.0 (Mathsoft Inc., Cambridge, MA). Data were then
averaged over 5 second intervals before graphing, except in Fig. 2
where the 1 second, unaveraged data were used. The average period
of the Ca2+ pulses is given as the mean ± s.e.m.
The coverslip-bottomed Petri dish was placed directly on the photomultiplier’s end-window photocathode, the photon-sensing
component of the system, and a light-tight cover was placed over the
dish. The position of the tube on the coverslip was marked to ensure
that the whole tube and grain were centered on the photocathode.
Likewise, when the tip of the tube was placed just off the photocathode, similar markings were made. Marks were made adjacent to the
grain, opposite the side of germination (within 100 µm). When the
tube had grown for a few hours, about 1.5 mm long, two marks were
made approximately two-thirds down the tube, one on each side of
the tube. During recording these marks were placed off the photocathode, so that the two marks along with the distal third of the tube
were off the photocathode while the proximal two-thirds of the tube
and the grain were maintained on the photocathode.
In order to change the medium or reposition the Petri dish, the high
voltage to the photomultiplier tube was turned off and the cover was
removed. The dish was left in place on the photocathode while the
medium was changed. After the treatment had taken place, the cover
was replaced and the tube was powered up. This total procedure took
approximately 2 minutes.
Growth records
Time-lapse bright field images of elongating pollen tubes were taken
on an inverted microscope (Nikon Diaphot TMD) with a Newvicon
camera and microchannel plate image intensifier (Video Scope International, Ltd, Sterling, VA). Collection and analysis of data were
performed on a Sun Workstation using software from G. W.
Hannaway and Associates (Boulder, CO). Each image was an average
of 16 video frames taken over a period of 0.5 seconds. An image was
collected every 5 seconds. Tubes could be imaged for about 3 minutes
before the faster-growing tubes grew out of the field of view. Successive pollen images were then viewed on the monitor and a line representing the path of elongation was drawn through the leading edge
of the tubes, using the ‘vector’ function of the image processing
software. Individual images were then viewed with the line overlay
and the pixel position of the line on the pollen tube’s leading edge of
growth was recorded. Growth rates were calculated from the differences between the tube’s leading edge pixel position of successive
images, along with a known 5 second time interval and known spatial
parameters. Average growth rates and the average period of the growth
pulses are given as the mean ± s.e.m.
Fluorescence imaging
Pollen tubes were injected 2 hours after germination with 1 pl of either
Ca2+ pulses and pollen tube elongation 1271
2.5 mM Calcium Green dextran (Mr 10×103) or 2.5 mM fluorescein
dextran (Mr 3×103), (Molecular Probes, Eugene, OR) in 100 mM KCl
and imaged with an MRC 1024 laser scanning confocal imaging
system (Bio-Rad, Hercules, CA) on an inverted Nikon microscope
through a ×40, 1.3 NA Nikon objective. Both dyes were excited at
488 nm with the emission light viewed through a 522 nm filter with
a bandpass of 25 nm. Laser intensity was set to 1% of maximum and
the diaphragm was fully opened to give a brighter signal. The resulting
confocal scanning section was approximately 10 µm thick. Under
these conditions, photodamage and photobleaching were minimized,
and pollen tubes could be studied for hours without apparent harm.
Pollen tubes that were growing parallel to the bottom of the chamber
were chosen for study. An average of 5 time-lapse recordings were
taken for each tube. Each recording consisted of 30-45 images taken
at 5 second intervals; each image took less than a second to collect.
The limitation on the number of images collected in each recording
was the growth of the pollen tube tip out of the field of view. Images
were displayed with a 256 pixel intensity gray-scale and were
exported and analyzed using MetaMorph imaging software (West
Chester, PA).
Growth rates were determined from the fluorescent images by
drawing a line 20 pixels thick over the long axis of the image of the
growing tube. Each set of 20 pixels was averaged and a single value
was returned to the middle point on the line thus generating a profile
of the fluorescence under the line. The coordinates of each point on
the line as well as the averaged pixel intensity of that point was saved
to a spreadsheet. The leading edge of growth was determined to be
the leading edge pixel value with approximately half the peak
intensity value of the line profile (see Fig. 8A, arrows). The pixel coordinates of this point on the line were recorded for each image in the
recording. The vector difference between coordinates along with a
known time interval and distance calibrated coordinate plane were
used to calculate growth rates. The growth rate between each time
point was averaged with its two nearest neighbors to reduce noise
before graphing.
Tip localized fluorescence was determined by drawing a circle/oval
approximately 10 µm in diameter and placing it on top of the images
of the tubes within a micron of the tip and front edges of the tubes.
The average pixel intensity of the region beneath the circle/oval was
recorded in this way for each of the images in a recording. The average
pixel intensity under the region varied by 1-2 pixel values when
moving the region a few pixels left/right or up/down. The average tip
localized fluorescence for each image was averaged with its two
nearest neighbors before determining the relative fluorescence. The
relative fluorescence was calculated by dividing each of the averaged
pixel values per time point by the lowest averaged pixel value. This
allowed comparison of changing fluorescent intensities despite
differing gain settings between recordings.
RESULTS
Calcium pulses
We have monitored intracellular free Ca2+ in 12 elongating
pollen tubes of Lilium longiflorum. Fig. 1 shows a complete
recording of an aequorin-injected pollen tube along with three
15 minute segments of the recording. Fig. 1A shows a 6.25
hour recording of the Ca2+ in the pollen tube and grain
beginning 30 minutes after germination. Pulses appeared
superimposed on a baseline Ca2+ signal that changed only
slowly with time. The sharp drop at about 180 minutes
occurred when the growing tip of the tube was positioned off
the photocathode for 10 minutes while the rest of the tube along
with the grain remained on the photocathode (see below). The
baseline of the recording decreased during this procedure but
still remained above instrumental background, and the Ca2+
pulses ceased. The pulses returned when the whole tube was
returned to the photocathode. The final pulse occurred when
Fig. 1. (A-D) Recordings of
aequorin luminescence from a
growing lily pollen tube and grain.
(A) Full length recording of a
growing lily pollen tube and grain
beginning about 30 minutes after
germination. (B) A segment of the
first 15 minutes of the recording in
A with an expanded time scale.
(C) A 15 minute segment of the
recording in A starting about 3
hours after germination. (D) The
final 15 minutes of the recording
in A showing aequorin burnout
and the resulting instrumental
background signal.
Aequorin luminescence (photons/s)
A
B
150
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0
0
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300
400
0
10
15
375
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385
D
C
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5
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0
370
Time (minutes)
1272 M. Messerli and K. R. Robinson
Calcium pulse isolation
As discussed above, it is known that the elevated calcium in
elongating tubes is localized to the distal 20 µm of the tube. In
order to confirm that the recorded Ca2+ pulses we have detected
were at the elongating end of the growing tube, we repositioned
the tube so that the distal third of the tube was located off the
photocathode of the photomultiplier tube while the proximal
two-thirds of the tube along with the grain was still on the photocathode. Fig. 3 shows recordings from two different tubes
Aequorin luminescence
(photons/s)
A
110
100
90
80
70
60
50
40
30
20
-10
-5
0
5
10
Time (seconds)
B
2.5
Relative [Ca2+ ]
the membrane was permeablized with the detergent, Triton X100, allowing Ca2+ influx from the medium and causing
complete inactivation, ‘burnout’, of the aequorin sample (Fig.
1A,D). The remaining signal subsequently is the instrumentgenerated background.
Low intensity, irregularly spaced pulses were observed as
soon as recording started (Fig. 1B), less than 10 minutes after
injection. Pulses reached higher intensities and became more
regular about 1.5 hours after germination. Fig. 1C shows a
segment of the recording at 3 hours after germination. The
baseline of this section is around 20 photons/second with
averaged pulses being between 0.5 and 6.5 times higher. Pulses
occur with 10-15 second breaks between them. It can be seen
that not all of the Ca2+ pulses are of the same intensity; there
is a second population of pulses that is two to three times
higher than surrounding pulses. We could not determine any
regular pattern for these higher pulses among the different
tubes, but these occasional higher pulses did occur in a
majority of the recordings, particularly in longer tubes. The
pulse frequency changed only slightly over the course of a
recording. The segment of recording in Fig. 1C displays pulses
with a period of 47 seconds while the average period for the
total record during regular pulsing was 44 seconds. A similar
pattern of Ca2+ pulses was observed in all 12 tubes that were
studied. The mean period of the pulses was 40±3 seconds with
a range of 29 to 57 seconds. During regular period pulsing, the
periods showed no consistent pattern of change. Tubes
continued with higher pulses until they either burst and stopped
growing or until the experiment was ended by either blocking
Ca2+ influx with lanthanum or by permeabilizing the cell
membrane and burning out the aequorin with Triton X-100.
Toward the end of the recording, pulse intensity and frequency
became more sporadic (Fig. 1D). As recording continues and
the aequorin is being used up, we expected a slight decline in
the baseline counts and the pulse intensity. However the sudden
drop at 190 minutes (Fig. 1A) is too large to be accounted for
by aequorin exhaustion and must represent a decline in Ca2+.
In order to extract information about the shape of the pulses,
we have averaged a total of 80 pulses from two different
recordings, using the 1-second raw data. Pulses with peaks
between 70 and 150 photons/second, during regular period
pulsing, were chosen for the study. A graph of this averaging
is shown in Fig. 2A. It is clear that the rising component (solid)
of the pulses initially is higher than the falling component
(dash), indicating an increased average free [Ca2+] during the
rising component. Fig. 2B shows the calculated relative [Ca2+]
of the two components. The mirror image of the falling
component was drawn on top of the rising component to facilitate comparison. The asymmetry is evident in many of the
individual pulses as well, especially when viewed on an
expanded time scale (data not shown).
2.0
rising component
1.5
falling component
1.0
-10
-5
0
5
10
Time (seconds)
Fig. 2. (A) An averaging of a total of 80 Ca2+ pulses from 2 different
tubes. Pulses were selected whose peaks were between 70 and 150
photons/second. The peaks of the pulses were aligned and the data
were averaged second by second for 15 seconds on either side of the
peak. The solid line represents the rising component while the
dashed line represents the falling component. (B) The relative [Ca2+]
levels of the rising (solid) and falling (dashed) components. The
falling component is shown as the mirror image of itself in order to
facilitate comparison. To calculate the relative [Ca2+] from the curve
shown in A, the instrumental background along with the signal from
the grain and the proximal 2/3 of the tube (5 photons/second) were
subtracted from the average value of each time point. The ratio of
these points with the adjusted baseline (15 photons/second) and the
2.2 power dependence of light on Ca2+ were used to calculate the
resulting [Ca2+] values.
before and after the tip of each tube was moved off the photocathode. There are breaks in the recordings of about 2 minutes
when the photomultiplier tube was turned off in order to reposition the pollen tube. Both Fig. 3A and Fig. 3B show a loss
of the Ca2+ pulses as well as an overall drop below the baseline
of the tip recording. The instrumental background signal was
still lower than the signal where Ca2+ was only being measured
from the grain and proximal two-thirds of the tube.
Source of calcium pulses
We wanted to identify the source of Ca2+ that produced the
pulses. Our first approach was to chelate the extracellular Ca2+
by adding 1 mM BAPTA to the medium of growing tubes. We
found that the tubes burst before adequate measurements could
be made. As an alternative, we added LaCl3 to a final concentration of 100 µM. Fig. 4 shows two such recordings. The La3+
caused these two tubes to swell at the tip before growth
stopped. The arrows on the figures indicate the beginning of
the recording after the La3+ was added to the medium. The
Ca2+ pulses and pollen tube elongation 1273
A
250
Aequorin luminescence (photons/s)
200
150
100
50
0
135
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B
100
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40
30
20
10
0
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Time (minutes)
Aequorin luminescence (photons/s)
pulses stopped within 10 minutes of adding La3+. Most of this
delay can be accounted for by the time required for La3+ to
diffuse through the 1 mm of agarose. In both recordings, there
is a rise and fall of pulses just before pulses are extinguished.
100
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70
60
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40
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0
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Fig. 3. (A,B) Aequorin luminescence
recordings from two different tubes. The
first and last segment of the recording is the
signal from the entire tube and grain while
the middle segment of the recording
(separated by breaks) is the signal from the
grain and the proximal 2/3 of the tube.
This may be the Ca2+ signal that causes the tubes to swell at
their tips before growth stops. The latter portion of these
recordings shows the signal after aequorin burnout. These
recordings show that active aequorin is still present in the tube
A
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B
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Time (minutes)
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Fig. 4. (A,B) Recordings of aequorin
luminescence in two pollen tubes before and
after the addition of 100 µM La3+. Arrows
indicate the beginning of the recording after
addition of La3+. In both records, Triton X100 was added near the end of the recording
in order to burn up the remaining aequorin.
1274 M. Messerli and K. R. Robinson
Fig. 5. (A-D) Growth records of
growing pollen tubes at two
different times after
germination: (A,B) 30 minutes
after germination, (C,D) 3 hours
after germination. Pixel
coordinates of the leading edge
of growing pollen tubes were
recorded from successive bright
field images. The difference
between successive, calibrated
coordinates at 5 second time
intervals was used to calculate
growth rates. Final values were
averaged with the two nearest
neighbors.
Lily Pollen Tube Growth Rates (µm/s)
A
B
0.35
0.35
0.32
0.32
0.29
0.29
0.26
0.26
0.23
0.23
0.20
0.20
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0.17
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0.14
0.11
0.11
0.08
0.08
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D
C
0.35
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0.23
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0.14
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100
and grain after the pulses and growth have stopped. As La3+ is
a more potent aequorin activator than Ca2+ (Blinks et al., 1982)
the recordings show that significant amounts of La3+ were not
entering the cell. If La3+ were entering the tube, the pulses just
before the tube stopped growing would be expected to be
higher than preceding pulses.
Growth rates
It has been shown that pollen tube growth is pulsatile (Pierson
et al., 1996). In order to investigate the temporal relationship
between pulsatile tip growth and the calcium pulses reported
here, we have measured growth rates with sufficient resolution
to detect growth pulses. Fig. 5 shows growth records from
tubes at 0.5 and 3 hours after germination. Although these
growth records are from different tubes than the ones used for
Ca2+ measurements, they can be compared to the Ca2+ pulse
records in Fig. 1B and C which were also taken at 0.5 and 3
hours after germination, respectively. Fig. 5A and B show two
growth records about 0.5 hours after germination. Growth rates
fluctuated between 0.2 and 0.1 µm/second for the two tubes,
with an average growth rate of 0.17±0.01 µm/second (n=6). No
clearly defined period can be obtained for these early recordings. However, the growth records taken at 3 hours after germination clearly show periodic growth (Fig. 5C,D). Growth
rates fluctuated between 0.34 and 0.08 µm/second. The mean
period of growth pulses for these tubes was 42±2 seconds (n=8)
with a range of 38 to 55 seconds. The average growth rates for
these longer tubes was 0.18±0.01 µm/second.
Fluorescence imaging
We have determined the phase relationship between the tip
localized Ca2+ pulses and pulsatile growth by imaging Calcium
150
0
50
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150
200
Time (seconds)
Green dextran in elongating pollen tubes between 2.5 and 4
hours after germination. Growth rates and calcium pulses were
measured from the same fluorescent images. Data collected
from two recordings of a single tube are shown in Fig. 6A. The
temporal resolution for these data is 5 seconds and the relative
Calcium Green fluorescence data is plotted on the 5 second
interval starting at 0 seconds. The growth rate data, however,
were calculated as the distance a tube grew between images,
so these data have been plotted at the time point half-way
between successive images, starting at 2.5 seconds and incremented every 5 seconds.
The growth rate and Calcium Green relative fluorescence
data both appear oscillatory with similar periods and closely
related phasing. The phase relationship was studied by
comparing the time difference between the minimum and
maximum extreme points of growth pulses and tip localized
Calcium Green fluorescence. We found that the Calcium Green
fluorescence extremes lagged the growth rate extremes by
3.8±0.9 seconds (136 extremes from 4 tubes). This is below
our temporal resolution of 5 seconds, so the Ca2+ pulses, within
our limits of resolution, are coincident with growth pulses.
In order to determine that the Calcium Green fluorescence
changes were not simply due to the changing shape of the pollen
tube tip, we imaged fluorescein dextran as a control. Fig. 6B
shows data from two recordings of a single pollen tube. Growth
rates were still oscillatory but the fluorescein signal showed
only small differences in intensity, compared to the Calcium
Green signal. The small oscillations in the fluorescein intensity
were out of phase with the growth pulses. We compared the
growth maximum and minimum extremes with the nearest fluorescein minimum and maximum extremes, respectively, and
found that they differ only by 3.9±0.9 seconds (74 extremes for
Ca2+ pulses and pollen tube elongation 1275
Growth Rate
Calcium Green Fluorescence
A
0.32
1.24
Growth Rates (µm/s)
0.29
1.20
0.26
1.16
0.23
1.12
0.20
1.08
0.17
1.04
0.14
1.00
0.11
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Relative Calcium Green Fluorescence
0.35
0.96
Time (seconds)
B
Growth Rate
Fluorescein Fluorescence
0.35
0.32
0.29
Growth Rates (µm/s)
1.20
0.26
1.16
0.23
1.12
0.20
1.08
0.17
1.04
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50
Time (seconds)
3 tubes). Not all of the growth extremes were accompanied by
a change in fluorescein fluorescence. Those extremes were
excluded from the preceding analysis. Growth extremes are a
half-cycle out of phase with fluorescein oscillations. We have
noticed periodic distortions in the growing tips. When tubes
grew out rapidly, the tip was narrower and when the growth
slowed, the tube thickened. These periodic distortions leading
to a changing level of fluorescein in the optical path may explain
the weak fluorescein oscillations. These same factors will apply
to the Calcium Green data; the shape-induced oscillations are
100
150
0.96
Relative Fluorescein Fluorescence
1.24
Fig. 6. Simultaneous growth and tip
fluorescence of a Calcium Green
dextran injected pollen tube (A) and
a fluorescein dextran injected tube
(B). Two recordings for each dye are
plotted and have their own time
scales. The growth rate and relative
fluorescence scales, plotted on the yaxis, are identical for both dyes.
(A) Tip localized Calcium Green
fluorescence oscillates with large
intensity changes and is in phase
with pulsatile tip growth. (B) Tip
fluorescein fluorescence oscillates
with weak intensity changes and is
out of phase with pulsatile tip
growth.
out of phase with the Ca2+ oscillations. Correcting the Calcium
Green results for the shape-induced changes in fluorescence
would increase the amplitude of the Ca2+ oscillations. Alternatively, the fluorescein fluorescence oscillations may represent
weak pH oscillations as fluorescein’s emission decreases with
decreasing pH.
Fig. 7 shows the images that were used to generate the final
growth pulse/calcium pulse shown in Fig. 6A (arrow). The
middle image (#2) was used to generate the value at the peak
of the Calcium Green fluorescence curve while the images
1276 M. Messerli and K. R. Robinson
for the fluorescein injected tube. The #2 profile for fluorescein
is the profile for the first of the two images used to calculate
the peak growth rate for the pulse labeled in Fig. 6B (arrow).
Profiles #1 and #3 are the profiles of the images taken 20
seconds before and after the #2 profile. A reference line was
drawn at a pixel intensity of 200. The peak fluorescence was
located a few microns further from the tip for fluorescein than
for Calcium Green and the fluorescein intensity changes only
slightly for the different images both among different tubes and
within the same tube (n=3). Uninjected tubes were not detected
by the imaging system even at maximum gain, indicating that
autofluorescence was insignificant.
1
Calcium Green
Fluorescence
DISCUSSION
2
3
0
10
µm
Fig. 7. Time-lapse images of a Calcium Green dextran-injected
pollen tube. Images are 10 seconds apart and were used to generate
the indicated pulse shown in Fig. 6A (arrow). Each of these images
was filtered with a low-pass filter and the pixel intensities below 20
were masked to reduce background noise. The tip localized
fluorescence intensity rises between the first image and the middle
image, and then falls during the last two images. Growth between the
first two images and the last two images is lower than the growth
between the second and third and between the third and fourth
images.
above were taken 10 and 20 seconds before and the images
below were taken 10 and 20 seconds afterward. The profiles of
the labeled images in Fig. 7 are shown in Fig. 8A. These
profiles not only show the growth of the tube’s leading edge
but give an idea of the relative fluorescence down the tube. A
reference line was drawn at a pixel intensity of 175 to facilitate comparison with the tip localized fluorescence. Profile #2,
the peak of the calcium pulse, clearly shows a more intense fluorescence in the tip than images taken 20 seconds before (#1)
and 20 seconds afterward (#3). Fig. 8B shows similar profiles
The data presented here establish that the cytosolic Ca2+ in
growing Lilium longiflorum pollen tubes is pulsatile, with large
pulses of increased Ca2+ superimposed on a steady background.
During the early stages of growth, the pulses are sporadic, but
as the tubes elongate, the pulses become quite regular. We find
that pollen tube growth is similarly pulsatile, with the growth
pulses being irregular initially but becoming large and regular
in longer tubes. Both growth pulses and Ca2+ pulses continue
until growth ceases, typically several hours after germination;
thus pulsing is the dominant mode of growth. If our in vitro
results can be extrapolated to the in vivo situation, then pulsatile
growth accompanied by Ca2+ pulses would be the mode of
growth during nearly all of the tube’s trek from the stigma to
the ovary. Our data further establish that the changes in Ca2+
are in phase with the changes in growth rate; that is, the time
of fastest growth corresponds to the time of highest Ca2+ and
slowest growth occurs when Ca2+ is lowest.
The method that we have used for detecting Ca2+, photon
counting of aequorin luminescence by means of a lens-less
system, allows us little spatial resolution of the dynamic
changes in Ca2+ that occur during growth. Nevertheless, we are
confident that the Ca2+ pulses that we detect occur at the
growing tip of the pollen tube. The ratiometric fluorescence
images of calcium distribution indicate that the only regions of
elevated Ca2+ in growing lily pollen tubes are at the tip (Miller
et al., 1992; Pierson et al., 1996; Rathore et al., 1991). The
limited spatial resolution that we were able to attain, by positioning the distal 1/3 of the tube off the photocathode, showed
that the Ca2+ signal from the remainder of the tube and the
grain was unchanging and small. A major advantage of the
aequorin method is the 2.2 power dependence of light output
on Ca2+ concentration. This non-linearity greatly amplifies
increases in Ca2+, which in turn allows for better temporal resolution.
The analysis of the shape of the pulses shows that they are
asymmetric about the peak, with the rising component being
slower than the falling component. Initially, Ca2+ rises in a
nearly linear manner, until it becomes about 1.5 times the basal
level. It then increases in a non-linear manner, perhaps exponentially, suggesting a regenerative process. The falling phase
is also non-linear, with an identical time course to the nonlinear part of the rise in Ca2+.
The periodicity and amplitudes of the Ca2+ pulses reflect the
variations in the rate of pollen tube elongation. In shorter tubes,
the Ca2+ pulses are of low amplitude and sporadically distrib-
Ca2+ pulses and pollen tube elongation 1277
Pixel Intensity
Fig. 8. Fluorescence profiles
A
B
Calcium Green
Fluorescein
250
250
1
from images taken from a
1
200
200
Calcium Green dextraninjected tube (A) and a
150
150
fluorescein dextran-injected
100
100
tube (B). The profiles are from
the images used to generate the
50
50
arrow-labeled pulses in Fig.
0
0
6A,B. The #2 profiles represent
0
10
20
30
40
50
0
10
20
30
40
50
60
the peak of the growth pulses
250
250
while the #1 and #3 profiles
2
2
represent images taken 20
200
200
seconds before and 20 seconds
150
150
after the image used to
generate profile #2. Arrows
100
100
indicate the leading edge of
50
50
growth. (A) A reference line
0
0
has been drawn at pixel
0
10
20
30
40
50
0
10
20
30
40
50
60
intensity 175. The peak
intensities start at the front of
250
250
3
the tube and are changing, with
3
200
200
the middle profile having the
highest intensity. (B) The peak
150
150
intensities are located a few
100
100
microns behind the tip and do
not change significantly. The
50
50
peak fluorescence intensity is
0
0
more similar in magnitude to
0
10
20
30
40
50
0
10
20
30
40
50
60
the fluorescence intensity
Imaged Length (µm)
further back in the tube than in
the Calcium Green profiles.
Profiles were generated by
drawing a line 20 pixels wide on top and parallel to the long axes of the images of the tubes. The 20 pixels were averaged to return a single
value to each point on the line. The data for each tube were saved to a spreadsheet and graphed.
uted; growth pulses are also small and irregular in shorter
tubes. The increase in the size and regularity of the Ca2+ pulses
in longer tubes is accompanied by a similar change in the
growth pulses. The mean period of the Ca2+ pulses, 40 seconds
with a range of 29 to 57 seconds, is not significantly different
(P=0.5) from the period of the growth pulses, 42 seconds with
a range of 38 to 55 seconds.
A rough estimate of the Ca2+ concentration change due to
the pulses can be made by assuming that the baseline level of
light emission from a 1 mm long tube is about 2.5
photons/second (2/3 tube and grain signal minus instrumental
background, Fig. 3A,B), with a basal Ca2+ level of about 200
nM (Miller et al., 1992; Pierson et al., 1996; Rathore et al.,
1991). The Ca2+ pulses are localized to the distal 20 µm of the
tube (Miller et al., 1992; Pierson et al., 1996) and the peaks of
the unaveraged pulses are commonly 150 photons/second. This
60-fold increase in light occurs in about 1% of the tube volume;
i.e. in the distal 20 µm that we model as a hemisphere
connected to the 1 mm long cylindrical tube. Thus, the peak
light emission per unit volume from the tip is about 6,000 times
as high as that from the rest of the tube. As the light emission
increases with the 2.2 power of [Ca2+], [Ca2+] at the peak of
the pulses is about 50 times the basal level concentration found
in the tube, or 10 µM. A similar calculation indicates that
[Ca2+] at the tip is about 3 µM between pulses. While these
estimates of absolute [Ca2+] levels are crude, it should be noted
that the values obtained are fairly insensitive to changes in the
starting assumptions due to the 2.2 power dependence.
The other method that we have used to detect Ca2+ changes,
scanning confocal imaging of Calcium Green fluorescence,
allowed us simultaneously to measure Ca2+ and growth rates.
While the temporal resolution of this method was not as good
as the aequorin measurements, it was sufficient to establish the
phase relationship between growth and Ca2+. Parallel experiments done with fluorescein make it clear that the changes
revealed by Calcium Green are due to changes in Ca2+, and
not to the amount of dye in the optical path. We regard
the aequorin-detected pulses as a better representation of
the temporal aspects of Ca2+ changes than the Calcium Green
measurements. The aequorin measurements had a temporal
resolution of 1 second, while the fluorescence measurements
had a temporal resolution of 5 seconds. For this reason the Ca2+
dynamics are better described as pulses rather than oscillations.
The spatial averaging and the reduced temporal resolution of
the Calcium Green images made the Ca2+ appear as oscillations rather than pulses.
Our results are connected in an interesting way with earlier
measurements of ionic current in lily pollen. Weisenseel et al.
(1975) found a steady ionic current that entered the pollen grain,
predicting the site of germination. Current then entered the
growing tip of the elongating tube. In some older tubes this
steady current continued to enter the growing tip of the tube, but
a new phenomenon appeared, which they describe as ‘an endless
train of spontaneous monophasic pulses [riding] on top of the
steady current’. The current pulses entered only the growing tip
and continued until growth stopped. The pulses were sym-
1278 M. Messerli and K. R. Robinson
metrical and had periods of about 46 seconds. We are confident
that these current pulses are the electrical correlate of the calcium
and growth pulses that we report. The only substantial difference
is that they did not see current pulses in every case while we
detected Ca2+ pulses in every pollen tube studied. Interestingly,
all three quantities, growth, tip Ca2+ concentration and ionic
current, have steady components on which the pulsatile components are superimposed. The Ca2+ concentration is always higher
in the tip than in more proximal regions, even between the Ca2+
pulses; growth continues between growth pulses, and there is
substantial current entry between current pulses.
Another correlate of our Ca2+ pulses may have been detected
by Jaffe et al. (1975). They exposed growing lily pollen tubes
to 45Ca, then did low-temperature autoradiography on washed,
frozen tubes. They always detected higher levels of 45Ca at the
tip, but in some cases the tips were extremely hot, resulting in
autoradiographs with high grain density at the pollen tube tips.
It may be that the exposure to 45Ca was terminated during, or
just following, a Ca2+ pulse in these cases.
There are two other examples of oscillating changes in Ca2+
associated with definite events in higher plants. The self-incompatibility response of Papaver rhoeas pollen tubes appears to be
mediated by cytosolic free Ca2+ (Franklin-Tong et al., 1993) and
a slow-moving wave can be triggered in these pollen tubes by
localized elevation of inositol(1,4,5)trisphosphate (FranklinTong et al., 1996). Upon reaching the growing tip, the wave of
elevated calcium inhibits growth, perhaps by disrupting the Ca2+
gradient there. Calcium spiking is also induced in legume root
hairs in response to nodulation factors released by Rhizobium
bacteria, and the calcium response seems to be necessary for
inducing the nodulation response (Ehrhardt et al., 1996).
The situation in growing lily pollen is quite different. No
external signal is required for growth or for the Ca2+ pulses.
Furthermore, the pulses do not spread in a wave down the
pollen tube, but remain localized at the tip. Entry of external
calcium is necessary for both growth and Ca2+ pulses. At this
point, we do not know if the Ca2+ pulses also involve the
release of Ca2+ from internal stores. The La3+ data indicate that
at least some component of the Ca2+ signal involves Ca2+
influx. In some ways, the more analogous situation is that of
neurotransmitter release. In both pollen and neurons, there is a
massive, calcium-regulated exocytosis of vesicles that must be
restricted spatially. In the case of neurons, action potentials
open voltage-gated calcium channels allowing Ca2+ influx into
the presynaptic cell. The interaction of Ca2+ with neuronal
proteins leads to the secretion of neurotransmitter-containing
vesicles. The proteins involved in this process are beginning to
be understood, and it is clear that yeast and non-neuronal
secretory cells have homologous proteins (Calakos and
Scheller, 1996). It may be that similar proteins are involved in
transducing Ca2+ regulated growth in pollen.
We thank Theresa Kirk of Purdue University, Horticulture Department, for supplying the pollen for this work. We also thank J. Paul
Robinson and Huw S. Kruger Gray of the Purdue Cytometry Laboratory for their help with the scanning confocal imaging. This research
was funded by the US Department of Agriculture (95-37304-2222).
REFERENCES
Blinks, J. R., Wier, W. G., Hess, P. and Prendergast, F. G. (1982).
Measurement of Ca2+ concentrations in living cells. Prog. Biophys. Mol.
Biol. 40, 1-114.
Brewbaker, J. L. and Kwack, B. H. (1963). The essential role of calcium ion
in pollen germination and pollen tube growth. Am. J. Bot. 50, 859-865.
Calakos, N. and Scheller, R. H. (1996). Synaptic vesicle biogenesis, docking,
and fusion: a molecular description. Physiol. Rev. 76, 1-29.
Cork, R. J., Cicirelli, M. F. and Robinson, K. R. (1987). A rise in cytosolic
calcium is not necessary for maturation of Xenopus laevis oocytes. Dev. Biol.
121, 41-47.
Ehrhardt, D. W., Wais, R. and Long, S. R. (1996). Calcium spiking in plant
root hairs responding to Rhizobium nodulation signals. Cell 85, 673-681.
Franklin-Tong, V. E., Ride, J. P., Read, N. D., Trewavas, A. J. and Franklin,
F. C. (1993). The self-incompatability response in Papaver rhoeas is
mediated by cytosolic free calcium. Plant J. 4, 163-177.
Franklin-Tong, V. E., Drøbak, B. K., Allan, A. C., Watkins, P. A. and
Trewavas, A. J. (1996). Growth of pollen tubes of Papaver rhoeas is
regulated by a slow-moving calcium wave propagated by inositol 1,4,5trisphosphate. Plant Cell 8, 1305-1321.
Jaffe, L. A., Weisenseel, M. H. and Jaffe, L. F. (1975). Calcium
accumulations within the growing tips of pollen tubes. J. Cell Biol. 67, 488492.
Keating, T. J., Cork, R. J. and Robinson, K. R. (1994). Intracellular free
calcium oscillations in normal and cleavage-blocked embryos and artificially
activated eggs of Xenopus laevis. J. Cell Sci. 107, 2229-2237.
Kiehart, D. P. (1982). Microinjection of echinoderm eggs; apparatus and
procedures. Meth. Cell Biol. 25, 13-33.
Lancelle, S. A. and Hepler, P. K. (1992). Ultrastructure of freeze-substituted
pollen tubes of Lilium longiflorum. Protoplasma 167, 215-230.
Larson, D. A. (1965). Fine-structural changes in the cytoplasm of germinating
pollen. Am. J. Bot. 52, 139-154.
López-Franco, R., Bartnicki-Garcia, S. and Bracker, C. E. (1994). Pulsed
growth of fungal hyphal tips. Proc. Nat. Acad. Sci. USA 91, 12228-12232.
Malhó, R., Read, N. D., Pais, M. S. and Trewavas, A. J. (1994). Role of
cytosolic free calcium in the reorientation of pollen tube growth. Plant J. 5,
331-341.
Miller, D. D., Callaham, D. A., Gross, D. J. and Hepler, P. K. (1992). Free
Ca2+ gradient in growing pollen tubes of Lilium. J. Cell Sci. 101, 7-12.
Miller, A. L., Karplus, E. and Jaffe, L. F. (1994). Imaging [Ca2+]i with
aequorin using a photon imaging detector. Meth. Cell Biol. 40, 305-338.
Picton, J. M. and Steer, M. W. (1985). The effects of ruthenium red,
lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle
transport, vesicle fusion, and tip extension in pollen tubes. Planta 163, 20-26.
Pierson, E. S., Miller, D. D., Callaham, D. A., Shipley, N. M., Rivers, B. A.
and Hepler, P. K. (1994). Pollen tube growth is coupled to the extracellular
calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type
buffers and hypertonic medium. Plant Cell 6, 1815-1828.
Pierson, E. S., Miller, D. D., Callaham, D. A., van Aken, J., Hackett, G. and
Hepler, P. K. (1996). Tip-localized calcium entry fluctuates during pollen
tube growth. Dev. Biol. 174, 160-173.
Rathore, K. S., Cork, R. J. and Robinson, K. R. (1991). A cytoplasmic
gradient of Ca2+ is correlated with the growth of lily pollen tubes. Dev. Biol.
148, 612-619.
Rosen, W. G., Gawlik, S. R., Dashek, W. V. and Siegesmund, K. A. (1964).
Fine structure and cytochemistry of Lilium pollen tubes. Am. J. Bot. 51, 6171.
Shimomura, O., Musicki, B., Kishi, Y. and Inouye, S. (1993). Light-emitting
properties of recombinant semi-synthetic aequorins and recombinant
flourescein-conjugated aequorin for measuring cellular calcium. Cell
Calcium 14, 373-378.
Weisenseel, M. H. and Jaffe, L. F. (1976). The major growth current through
lily pollen tubes enters as K+ and leaves as H+. Planta 133, 1-7.
(Received 24 January 1997 – Accepted 11 March 1997)