Planta
9 Springer-Verlag 1981
Planta (1981) 153:1-5
Bioelectrie potential changes in the style of Lilium longiflorum Thunb.
after self- and cross-pollination of the stigma
A.W. Spanjers
Botanisch Laboratorium, KatholiekeUniversiteit, Toernooiveld, 6525 ED Nijmegen, The Netherlands
Abstract. Two different types of bioelectric potential
changes have been registered in the style of Liliurn
longiflorurn cv. Arai 5 after different treatments of
the stigma. Self-pollination induces a bioelectric potential change different from the potential change induced by cross-pollination. Removal of the stigma
or apllication of killed compatible pollen induced a
bioelectrical response similar to the potential change
recorded after cross-pollination. No bioelectric potential change was recorded after application of pollen
of Petunia hybrida, pollen of Haernanthus katherinae,
killed self-pollen, or no treatment at all.
The mean generation time of the bioelectric potential change was 338 min after the treatment. The
translocation velocity varied between 1.2 and
5.4 crn h - 1. Both responses are similar to the potential changes registered in Mimosa pudica after different types of stimulation and to the potential changes
generated by the phytochrome in Arena coleoptiles.
The translocation of the signals is discussed in relation
to the models on the translocation in Mimosa. The
relation between the bioelectric potential changes and
the incompatibility reaction in Liliurn longiflorurn is
discussed.
Key words: Electrical potential changes - Incompatibility - Liliurn - Pistils - Pollination.
Introduction
Post-pollination effects such as the induction of enlargement of the ovary and wilting of the corolla
occur long before the actual fertilization. During the
progamic phase an increase of ribosomal RNA was
measured in the ovaries of Petunia hybrida (Linskens
1973). Also, in Petunia ovaries, the first increase in
polyribosome activity was observed 6 to 12 h after
pollination (Deurenberg 1976). This means that the
ovaries receive information about the presence of pollen on the stigma or of pollen tubes in the style.
At the same time, even information about the compatible or self-incompatible nature of the pollen (-tubes)
(Deurenberg 1977), is recognized by the ovary before
the pollen tubes have reached that ovary.
With regard to wilting of the corolla of Digitalis
purpurea, the first visual reaction takes place at about
8 h after pollination (Stead and Moore 1979). The
induction of corolla wilting of Petunia hybrida flowers, after either compatible or self-incompatible pollination is controlled by the style (Gilissen 1976;
1977).
So, somehow, transport of information about the
presence and type of pollen must pass the style from
the stigma to other parts of the pistil and the flower.
The mechanism for this information transport is as
yet not understood. Evidence for hormonal and electrical signals have been described in the literature.
Apparently the influence of hormones on the wilting
of the corolla is cooperative. Hormones do not initiate
this post-pollination effect (Kende and Hanson 1976,
Mayak et al. 1977). Bioelectrical signals, regulating
movements in plants such as Mimosa and Biophyturn
and some carnivorous species, transmit information
about the nature of the stimulus (Sibaoka 1969; Pickard 1973).
In pistils of plants belonging to different genera
diverse types of electrical responses after pollination
have been reported. Sinyukhin and Britikov 1967 reported the generation of action potentials in the pistils
of Incarvillea and Liliurn in conjunction with movement of the stigma and fertilization. Efforts to reproduce these results have not been succesful (Pickard
1973). Linskens and Spanjers (1973) have reported
the influence of self-incompatible and cross-compatible pollination on the electric potential curve immediately after insertion of the electrode in the style of
Petunia hybrida.
0032-0935/81/0153/0001/$01.00
A.W. Spanjers : Bioelectric potential changes in Lilium
2
Previously the generation of a "Variation Potential" in the style of Lilium longiflorum cv. M o u n t
Everest after self-pollination was demonstrated
(Spanjers 1978). This paper deals with a more extended study on this subject. It presents the different
bioelectric potential changes in styles of Lilium longiflorum cv. Arai 5 after cross-compatible pollination,
after self-incompatible pollination, and after other
treatments of the stigma.
I
D.C. Amplifier
area 2
Materials and methods
I
Oscilloscope
area 1
Bulbs of Lilium longiflorum Thunb. cv. Arai 5 were stored in a
cold room at 1~ C. To have flowers available every day of the
year, 10 bulbs were removed from the cold r o o m every week and
were planted in 16-cm filled with normal potting soil. The plants
were grown in a controlled climate chamber at a temperature of
20_+1 ~ C, relative humidity 50 to 70% and a long-day regime
of 16 h of fluorescent light, intensity 15,000 lx on plant level. Day
and night corresponded to the natural day and night cyclus. Since
the properties of the flowers of hilium longiflorum resemble those
of the so-called m o t h blossoms (Faegri and van der Pijl 1979),
natural pollination can be expected to take place at sunset. Therefore, pollen or other treatment were applied to the stigma at the
onset of the dark period.
Compatible pollen of Lilium Iongiflorum cv. Ace and pollen
of Petunia hybrida Vilm. were stored in a refrigerator at 4 ~
until use, which did not affect their ability to germinate. Pollen
of Haemanthus katherinae Bak. and pollen of L. longiflorum cv.
Arai were used fresh. W h e n necessary pollen was killed by heating
them in a closed petri dish lot 1.5 h at 150 ~ C.
The bioelectric potential changes were measured only in Lilium
longiflorum cv. Arai 5 styles with glass micro-pipettes, pulled from
glass capillary tubes with an inside diameter of 1 m m by a microelectrode puller (C.F. Palmer Ltd. London U.K.) to a tip diameter
smaller than 2.5 ~tm. The pipettes were filled with 3 M KC1 by
the boiling technique (Geddes 1972). A chlorided silver wire, connected with the micro-pipette via a bridge of 0.1 M KC1, was
used as the electrode (Chloration method according to Barry and
D i a m o n d 1970). The signals were monitored with a DC-microelectrode amplifier (Grass Instruments, Type P-18) and simultaneously
recorded on a pen recorder (Servogor, type RE 511) and a F.M.tape recorder (SE-labs). A n osciiloscope (Philips type PM 3234)
was used for direct control. Except for three tracks concerning
the application of Haemanthus katharinae pollen to the stigma,
the recordings were obtained by so-called differential recording.
A diagram of the set-up is presented in Fig. 1.
Flowers of the age of two days after anthesis were used for
the experiments. The corolla and staminae were removed and the
gynaecium remained on the plant.
The two electrodes were inserted immediately after pollination
or other treatments of the stigma. Electrode 1 was inserted in
the style at a mean distance of 1 cm from the ovary (area 1 in
Fig. 1) and electrode 2 at a mean distance of 3 cm from the ovary
(area 2 in Fig. 1). Both tips of the electrodes were near one of
the three vascular bundles. A grounded reference electrode, a chlorided silver wire, was inserted just below the ovary.
The plant to be measured and the amplifier were placed in
a Faraday cage. The whole set-up was placed on pneumatic suspensions (Micro Controle, suspensions pneumatic, type VX 95), in
order to prevent artefacts caused by vibrations of the climate
chamber.
l
I
Tape Recorder
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/
Fig. 1. Diagram of the set-up for the registration of bioelectric
potential changes in pistils of Liliurn longiflorum cv. Arai 5
Results
The recordings after different treatments of the stigma
of Lilium longiflorum cv. Arai 5 are listed in Table 1.
Two types of bioelectric potential changes were
recorded. Examples of response type 1 and response
tape 2 are presented in Fig. 2. The potential shift
of response type 1 (Fig. 2a) varies between - 2 and
- 6 mV, the mean duration is about 12 s. The return
to the initial level takes between 20 and 50 s. The
complete bioelectric potential change takes about
60 s. Response type 2 is presented in Fig. 2b and
Fig. 2c. The potential shift is similar to the one of
response type 1. However, the return is incomplete
and takes about 30 s. This return is followed by a
second, slower potential shift. The potential returns
to the initial level in about 10 rain or more. The mean
time of recording a bioelectric response was 338 rain
after the treatment of the stigma.
In 21 out 64 tracks, after self- or cross-pollination
or application of killed cross-compatible pollen or
removal of the stigma, a response was recorded. Six
times the response was measured only in area 2 and
eleven times a response occurred in area 1. Fourtimes
a response occurred in both area 1 and area 2. One
A.W. Spanjers: Bioelectric potential changes in Lilium
3
Table 1. Survey of the bioelectric responses in the pistil of Liliurn longiflorum cv. Arai 5 after different treatments of the stigma ( ( - ) = n o
response)
Treatment of the
stigma
Self-pollination
Number of
experiments
with positive
response
Type of
response
3 out of 11
Cross-pollination with pollen
of Lilium longiJlorum cv. Ace
14 out of 40
No treatment
none out of 6
Application of pollen
of Petunia hybrida
none out of 8
Application of pollen
of Haemanthus katherinae
none out of 15
Removal of the stigma
2 out of 5
Application of killed
pollen of L, longiflorum cv. Ace
2 out of 8
cv. Arai
none out of 7
Velocity
cm/h-*
Area 1
Area 2
Response
time after
treatment
(min)
Depolarisation
(mV)
Response
time after
treatment
(min)
1
353
308
332
2.2
6.0
2.5
(-)
(-)
(-)
2
2
2
2
2
2
288
381
296
392
384
302
2.2
3.6
2,8
6.8
4.7
2.6
(-)
(--)
(--)
(--)
(-)
(-)
2
2
(-)
(-)
224
340
2.1
4.4
2
2
2
2
2
2
(-)
(-)
374
365
354
343
2.4
2.0
3.7
2.9
290
345
406
318
304
359
6.6
8.0
1.5
2.0
2.9
3.6
2
2
(-)
365
402
(-)
2.9
4.9
2
2
302
(-)
o f these responses is p r e s e n t e d in Fig. 2c. T w o times
the d i r e c t i o n o f the t r a n s l o c a t i o n o f the p o t e n t i a l
c h a n g e was t o w a r d the o v a r y a n d two times in the
o p p o s i t e direction. T h e speed was respectively 1.9 a n d
1.2 c m h - 1 a n d 5.4 a n d 4.1 c m h - ~ .
S t i g m a treatment and response type. Eleven tracks
were r e c o r d e d after s e l f - p o l l i n a t i o n o f the Lily stigma.
In three cases a bioelectric p o t e n t i a l c h a n g e t y p e 1
(Fig. 2 a) was f o u n d a n d eight t r a c k s s h o w e d n o response.
F o u r t e e n t r a c k s o u t o f f o u r t y r e c o r d e d after poll i n a t i o n with c o m p a t i b l e pollen o f Lilium longiflorum
cv. A c e s h o w e d r e s p o n s e t y p e 2 (Fig. 2 b a n d c). In
t w e n t y - e i g h t t r a c k s n o b i o e l e c t r i c a l r e s p o n s e was recorded.
5.1
1.9
1.2
4.1
6.4
(-)
328
Depolarisation
(mv)
4.0
N e i t h e r o f these r e s p o n s e types were r e c o r d e d in
the six c o n t r o l s w i t h o u t p o l l i n a t i o n . Also, no bioelectric p o t e n t i a l c h a n g e was registered in the eight tracks
after a p p l i c a t i o n o f Petunia p o l l e n a n d in the fifteen
t r a c k s after a p l l i c a t i o n o f H a e m a n t h u s pollen on the
Lily stigma.
R e m o v a l o f the s t i g m a generates a bioelectric p o tential c h a n g e type 2, which was r e c o r d e d two times
out o f five tracks.
The bioelectrical r e s p o n s e in the style after applic a t i o n o f killed p o l l e n on the s t i g m a is d e p e n d e n t
on the c o m p a t i b l e o r s e l f - i n c o m p a t i b l e n a t u r e o f the
pollen. A f t e r a p p l i c a t i o n o f killed c o m p a t i b l e pollen
o f Lilium longiflorum cv. A c e two o u t o f eight t r a c k s
s h o w e d a bioelectric p o t e n t i a l c h a n g e type 2. N o n e
o f the seven tracks after a p p l i c a t i o n o f killed self-
4
A.W. Spanjers: Bioelectric potential changes in Lilium
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Fig.2A-C. Three examplesof the bioelectricalresponsesregistered
in the pistils of Lilium longiflorum cv. Arai 5. A An example of
the bioelectrical response after self-pollination.B An example of
the bioelectrical response after cross- pollination with pollen of
Lilium longiflorum cv. Ace. C An example of the bioelectrical response after cross-pollinationrecorded at both electrodes, showing
the transport of the response
incompatible pollen recorded a bioelectric potential
change.
Discussion
The bioelectric potential change induced by self-pollination, presented in Fig. 2a, is similar to those described previously (Spanjers 1978). The mean response time after self-pollination in the present investigation was 338 rain, whereas the previously reported
bioelectrical response in the style of Lilium IongifIorum cv. Mount Everest was measured about
120 min after self-pollination. This difference in response time may be attributed to the difference in
the Lilium variety used and/or to the fact that the
responses, presented in 1978, were recorded during
the day under the variable conditions of a green hous
in which the plants were grown.
Both electric potential changes presented in this
paper are also similar in strength, shape, polarity,
and duration to the reported bioelectric potential
changes after activation or after activation followed
by inactivation of the phytochrome system in Arena
coleoptiles (Newmann and Briggs 1972).
The electric potential changes are also similar in
shape, polarity, and duration to the registered bioelec-
tric potential changes in Mimosa pudica (Houwink
1935; Sibaoka 1953; Umrath 1959). Response type
1 (Fig. 2a) in the Lily pistil is similar to the action
potential in Mimosa, induced by a drop of very cold
water or by vibration. The response type 2 (Fig. 2b
and c) is similar to the "variation potential", preceded by an action potential in Mimosa. This type
of response is induced in Mimosa by cutting the stem
or by holding a flame close to the tissue.
It is noteworthy that specific stimulation of different organs of different plants evoke similar bioelectrie
potential changes. The low frequency in recording
a bioelectrical response may be due to (a) time limits
of the registrations, (b) the position of the electrode
tips near to one of the three vascular bundles in the
style. Possibly only "special cells" in or near the
bundles are able to generate potential changes. Studies which provide evidence for the existence of such
special cells are in progress.
The conduction rate of the bioelectric potential
change type 2 is 1-5 cm h-1. This speed is very low
even in comparison to the conduction rate of the
variation potentials in Mimosa, which are 0.20.6 cm s -1. (Houwink 1935; Sibaoka 1953; Umrath
1959), or to the translocation of action potentials
in Lupinus angustifolius (Paszewski and Zawadzki
1973). Two different models for the electrical conduction in Mimosa pudica have been proposed (Houwink
1935; Sibaoka 1953, 1969; Umrath 1959). The experiments presented show that in all four cases of registered transport of response type 2 the action potential
part of the potential change is at both electrodes immediately followed by the variation potential part
of the response. They also show that the conduction
can take place in two directions, i.e., the direction
of the stimulus as well as in the opposite direction;
and the conduction rate in the direction of the stimulus is about 4 times the conduction rate in the opposite
direction. These facts support the model proposed
by Umrath 1959; that is that the excitation takes
place by the effect of the wound hormone present
in the xylem on the neighboring living cells and that
the excited cells themselves form new wound hormone
by which the conduction of bioelectric potential
changes is supported.
The bioelectric potential changes in relation to incompatibility. The generation of the bioelectrical response
was at a mean time of 338 rain after pollination. This
means that the pollen are recognized early in the
progamic phase, which has been suggested previously
for Lilium longiflorum (Hiratsuka and Tezuka 1979)
and for Petunia hybrida (Linskens 1973; Deurenberg
1976).
The experiments without pollination or applica-
A.W. Spanjers: Bioelectric potential changes in Lilium
tion of pollen from plants of different genera, which
did not generate bioelectric potential changes, and
the different types of responses after cross- and selfpollination suggest that a recognition of both selfincompatible pollen and compatible pollen occurs.
It also supports the suggestion of the existance of
two different control mechanisms on the growth of
the pollen tube in the style (Ascher 1975; Hopper
et al. 1967; Ascher and Peloquin 1970).
The results of the experiments with killed pollen
are of interest with regard to the recognition-reaction
as well as the rejection reaction of the pollen tube.
Killing compatible pollen by heat treatment up to
150~ C does not affect the bioelectrical response, suggesting that the recognition of the killed pollen is
still possible. The same treatment of the self-incompatible pollen prevents the generation of any bioelectrical response, suggesting that the pollen is not recognized at all or is no longer able to excitate a response
in the style. Together these results are in accordance
with the expectations based on the knowledge about
the use of killed compatible pollen as a mentor pollen
for the incompatible pollen tubes to make them
pseudo-compatible (de Nettancourt 1977).
The transport of the bioelectric potential changes
was only occasionally recorded and such transport
occurred in both directions. In particular, the fact
that the signals are not unidirectional suggest that
the recorded bioelectric potential changes probably
do not represent the specific signal for transmission of
information concerning the presence and type of pollen on the stigma. The bioelectrical responses rather
contribute to the spreading or strengthening of a
change in the style. This change of the gene expression
of the style, which in turn affects pollentube growth,
can be explained by the assumption of the extra release of a stimulating agent, in analogy to the hypothesis put forward by Umrath (1959). This assumption
on action potentials and its related extra release of
a stimulating agent may also be in analogy to the
model presented by Kende and Hanson (1976) and
Mayak et al. (1977) with regard to the wilting of the
flowers.
The author is much endebted to Prof. Dr. H.F. Linskens for his
stimulating interest and to Dr. G. Barendse for correcting the
manuscript. Thanks are also due to Prof. Dr. P.L. Pfahler, University of Florida, USA, and to Mr. S.E. Bottema, Berkhout, NL,
for sending bulbs of Lilium longiflorum cv. Ace. The author also
wishes to thank Mr. B.M. van Meurs and Mr. A.H. Glaap for
growing excellent plant material.
References
Ascher, P.D., Peloquin, S.J. (1970) Temperature and the self-incompatibility reaction in Lilium longiflorum Thunb. J. Am. Soc.
Hort. Sci. 95, 586-588
5
Ascher, P.D. (1975) Special stylar property required for compatible
pollen tube growth in Lilium longiJlorum Thunb. Bot. Gaz.
136, 317-321
Barry, P.H., Diamond, J.M. (1970) Junction potentials, electrode
standard potentials and other problems in interpreting electrical
properties of membranes. Junction Membr. Biol. 3, 93-122
Deurenberg, J.J.M. (1976) In vitro protein synthesis with polysomes from unpollinated, cross- and self-pollinated Petunia
ovaries. Planta 128, 29-33
Deurenberg, J.J.M. (1977) Differentiated protein synthesis with
polysomes from Petunia ovaries before fertilization. Planta 133,
201-206
Faegry, K., van der Pijl, L. (1979) The principles of pollination
ecology, edn. 3, pp. 116. Pergamon Press, Oxford New York
Toronto Sydney Paris Frankfurt
Geddes, L.A. (1972) Electrodes and the measurement of bioelectric
events, pp. 162-163, Wiley Interscience, New York London
Syndney Toronto
Gilissen, L.J.W. (1976) The role of the style as a sense-organ in
relation to wilting of the flower. Planta 131, 201-202
Gilissen, L.J.W. (1977) Style-controlled wilting of the flower. Planta 133, 275-280
Hiratsuka, S., Tezuka, T (1979) Pollen reaction in self- or crossmating systems of Lilium longiJlorum. Incomp. Newsletter 11,
55-59
Hopper, J.E., Ascher, P.D., Peloquin, S.J. (1967) Inactivation of
the self-incompatibility following temperature pretreatments of
the styles in Lilium longijlorum. Euphitica 16, 215-220
Houwink, A.L. (1935) The conduction of excitation in Mimosa
pudica. Rec. Tray. Bot. NeerI. 32, 51 91
Kende, H., Hanson, A.D. (1976) Relationship between ethylene
evolution and senescence in Morning-Glory flower tissue. Plant
Physiol. 57, 523-527
Linskens, H.F. (1973) Activation of the ovary. Caryologia. Vol. 25,
Suppl., pp. 2741
Linskens, H.F., Spanjers, A.W. (1973) Changes of the electrical
potential in the transmitting tissue of Petunia-styles after crossand seIf-pollination. Incomp. Newsletter 3, 81 85
Mayak, S., Vaadia, Y., Dilley, D.R. (1977) Regulation of senescence in carnation (Dianthus caryophyllus) by ethylene. Plant
PhysioI. 59, 591-593
de Nettancourt, D. (1977) Incompatibility in Angiosperms. pp. 7071. Springer, Berlin Heidelberg New York
Newmann, I.A., Briggs, W.R. (1972) Phytochrome-mediated electric potential changes in oat-seedlings. Plant Physiol. g0, 687
693
Paszewski, A., Zawadzki, T. (1973) Action potentials in Lupinus
angustifblius L. shoots. J. Exp. Bot. 24, no 82, pp. 804-809
Pickard, B.G. (1973) Action potentials in higher plants. Bot. Rev.
39, 172 201
Sibaoka, T. (1953) Some aspects on the slow conduction of stimuli
in leaf of Mimosa pudica. The science reports of the T6hoku
University, 4-series, Vol. XX, no 1, Sendai, Japan
Sibaoka, T. (1969) Physiology of rapid movements in higher plants.
Annu. Rev. Plant Physiol. 20, 165-185
Sinyukhin, A.M., Britikov, E.A. (1967) Generation of potentials
in the pistil of Inearvillea and Lily in conjuetion with movement
of the stigma and fertilization. Soviet Plant Physiol. 14, 393403
Spanjers, A.W. (1978) Voltage variation in Lilium longiflorum
pistils induced by pollination. Experientia 34, 36-37
Stead, A.D., Moore, K.G. (1979) Studies on flower longevity in
Digitalis. Planta 146, 409-414
Umrath, K. (1959)Der Erregungsvorgang. In: Encyclopedia of
plant physiology, pp. 24 107, W. Ruhland ed. Springer, Berlin
G6ttingen Heidelberg
Received 30 July 1980; accepted 13 May 1981