Planta 144, 235
Planta
240 (1979)
9 by Springer-Verlag 1979
High-Frequency Oscillations and Circadian Rhythm of the Membrane Potential in Spinach Leaves B. Novak and H. Greppin Laboratoire de Physiologic V6g6tale, 3, place de l'Universit~, CH-1211 Gen+ve 4, Switzerland
Abstract. The microelectrode technique was used to follow oscillations in membrane potential in mesophyll cells of spinach (Spinacia oleracea L.) during exposure do different photoperiodic conditions. Both high-frequency oscillations and circadian variations were observed. The circadian rhythm was imposed on the period of high-frequency oscillation during short days as well as in continuous light: The freerunning period was 25.2 h. The average period of high-frequency oscillation increased from 7.64 min in the dark to 19.95 rain in the light within several minutes after dark to light transition. This period length coincides with the established period length for oscillations in the redox potential in the chloroplast suspensions of spinach. Key words: Circadian rhythm-Floral
inductionHigh-frequency oscillation-Membrane potentialSpinacia.
Introduction
Many forms of rythms and oscillations exist in eukaryotic organisms possessing highly spezialized hierarchic organization and compartmentation (B/inning, 1967; Rensing, 1973). It has been established that these rhythms are generated at a subcellular level and that a common control mechanism is responsible. Three models have been proposed to explain the oscillatory mechanism: 1. The replicon-chronon model of Ehret and Trucco (1967) explained the rhythmicity as a consequence of sequential gene expression (polycistronic replicons of optimal length: chronons) at the transcription level through a periodic functioning. The linear sequential replication, cistron by cistron, of CL = continuous light; SD = membrane potential
Abbreviations."
short day; MP
a very long polycistronic unit provides the cell with a remarkably simple interval-measuring and eventenumerating device; the last step (recycling factor) initiates a new transcription round. 2. Another model proposed by Pavlidis (1969), Sel'kov (1968), and also Chance et al. (1973), considered the self-sustained oscillations of a biochemical network with feedback loops causing circadian rhythmicity. However, an explanation of how the highfrequency biochemical oscillations are transformed into low-frequency circadian rythms is lacking. 3. Njus et al. (1974) suggested that rhythm was related to the membrane-bound ion transport systems. If the ionic concentrations of the cytoplasmic compartments oscillate, the transmembrane ionic flux and local membrane potential should also show a circadian periodicity, as has been shown in Acetabularia rned. (Novak and Sironval, 1976). Thus the interaction of the energy-converting compartments is involved in the generation of rhythms (Wagner, 1976). Since the sites of perception for the photoperiodic signals are localized in leaves (Knott, 1934; Biinning and Moser, 1966), we decided to test their influence on the membrane potential of the mesophyll cells. The membrane potential measured over long-term intervals provides unique information about the processes going on in the mesophyll cells, and with the symplastic organization of cells in plants, allowing the immediate transmission of signals through plasmodesmata (Lfittge and Pallaghi, 1969; Pickard, 1973), it is possible to obtain information on the general physiologic state of the plant. Three types of regulatory responses can be observed in plants: 1. Immediate, controlled by electrochemical processes, 2. Short-term, under the control of hormones or metabolite movement, and 3. Slow, controlled by specific proteins after neosynthesis.
0032-0935/79/0144/0235/$01.20
236
B. Novak and H. Greppin: High-Frequency Oscillations and Circadian Rhythm
Floral induction has been shown to be regulated by short-term processes (Penel and Greppin, 1973, 1974); the synthesis of new specific proteins after floral induction appears to be too late to intervene in the initial processes (Greppin, 1975; Balet and Grepping, 1976, 1977). The photoperiodic flowering process in spinach involves the immediate transmission of an electrochemical signal (Greppin, 1975; Greppin and Horwitz, 1975). Bearing these findings in mind, we assume that regulating processes leading to floral induction are of a biophysical nature (Lenk et al., 1978; Greppin et al., 1978). We describe the immediate changes of the period in the high-frequency oscillations upon photoperiodic stimulation.
Materials and Methods Plants and Cultivation Spinach plants (Spinacia oleracea, var. Nobel), were selected after cultivation for 4N 5 weeks in a phytotron under standard conditions: temperature 20_+0.15~ relative humidity during light period, 50 _+2% and during dark period, 70 + 1.5% (periodic fluctuations of temperature and humidity in the phytotron: t t min in clark and 7 min in light). Illumination by fluorescent tubes (Sylvania, TL-33, Daylight, 40 W) grave 6000 lx at plant level. The following conditions for the photoperiod and the relative humidity were imposed on the plants: 1. Short days (SD) of 8 h light with 50% relative humidity (from 8 a.m. to 4 p.m.) and 16 h of dark with 70% relative humidity (from 4 p.m. to 8 a.m.). In SD, Spinacia oleracea remains in a vegetative state for 6 weeks. The transition to flowering was monitored by following ultra structural criteria (Auderset and Greppin, 1977). 2. Continuous light (CL) with relative humidity of 50%. Plants subjected from germination to 4 weeks of continuous light were used to define the fully induced and floral state verified by the appearance of the floral primordia visible in the light microscope (Auderset and Greppin, 1976). 3. The transfer from SD to CL induces flowering. Onset of the induction in leaves is 3 h after the termination of the SD period (critical photoperiod : 11 h). The flowering signal is transmitted from leaves to the shoot apex. The first changes of the apex resulting from the arrival of this signal have been called evocation (Evans, 1969), which begins 11 h after the onset of the photoperiodic induction of leaves (11 h light), in all after 22 h of light. The floral embryogenesis, properly so called, starts only 26 h after evocation (Auderset and Greppin, 1976, 1977). 4. Four weeks of CL is followed by transfer to SD in order to test the degree of reversibility in photoperiodic treatment. The measurements made under the above-stated conditions were repeated at least ten times.
Membrane Potential Measurements Membrane potential (MP) measurements in the mesophyll cells were made using a specially constructed Plexiglas cuvette (Fig. 1). It consists of a vessel and a cover that are gently pressed together. The cuvette served as a holder for a leaf in a fixed position between the vessel and the cover. The leaves were dipped in the perfusing nutrient solution o f pH 7.0 (for details see Etherton, 1968).
/
/ s///
\L
Fig. 1. Plexiglas cuvette with a plant and the principal circuit for measurement and registration of the membrane potential. CU cuvette; K cover; S silicon rubber layers attached to Plexiglas pieces; L leaf; M E glass microelectrode; RE reference electrode; C compensation of the junction potential; E electrometer (R~ = 1012 ~?) ; PR pen recorder
The cuvette allowed measurements on leaves that were still attached to the plant. After the leaf was fixed in the cuvette, the plant was allowed to equilibrate for at least 12 h in the dark. Details of the measuring equipment are shown in Figure 1. Silver wires that had been coated with a silver chloride layer were used for both the microelectrode and for the reference electrode. The glass microcapillaries were made by the vertical puller of Getra Inst., Munich W-Germany. The 1.4-mm diameter microcapillaries were filled with 0.5 M KCI solution. The tip resistance was 5 ~ 30 M~2. The tips of the glass microcapillaries were impaled into the cells using manually operated micromanipulators supplied by Narishige, Japan. The signals were measured using a solid state electrometer (Keythley Inst) with an imput resistance of 10 ta s'2 and registered by a pen-recorder (Watanabe, Japan). The membrane potential of a mesophyll cell was monitored for periods of 2 N 5 days under the different light/dark and humidity programs.
Results
The electrophysiologic approach appeared to be advantageous compared to biochemical methods, since we were able to monitor the cells of intact leaves for a long interval with only very few mechanical and electrical disturbances. The stability of the MP was good during the measurements, in comparison to that when an artifical disturbance (mechanical, electric, thermic) was applied to the plant. The resting MP o f - 84.8_+ 16.3 mV (number of measurements = 53) inside the mesophyll cell was detected just after the impalement of the glass microelectrode (Fig. 2). An exponential decay in MP was then observed within several minutes, and it finally stabilized around a mean value of - 38.5 + 9.6 mV (n = 53). The lowering of the MP might be caused by the leak of ions around the wound made by the penetration of the tip into the cell. Within several hours the wound healed up and the MP remained fairly stable (Fig. 3). The recorded potentials returned to zero level + 4 mV when the microelectrode was withdrawn from the tissue.
B. Novak and H. Greppin: High-Frequency Oscillations and Circadian Rhythm
237
Table 1. The average period of high-frequency oscillation as a function of the state of the plants and the light/dark conditions
-U (mY)
[ 10(mV)
State
Photoperiod
Illumination
Average period (min)
Vegetative
SD
Dark Light
TsD -- 7.64_+0.35 Tsc -- 19.95 • 0.22
lnduced
CL
Light, 1st phase Light, 2nd phase
Tm = 13.37 + 1.08 TL2 =21.19--+1.63
CL
Light, 1st phase Light, 2nd phase
Tw =12.54_+0.56 Tv2 =19.63_+2.41
Dark 1st light 2nd light
TFD = 7.24_+0.16 TFL1 =33.53_+3.77 TFL2 =24.71 _+1,88
60 l(min)
,
t Cmin)
Fig. 2. Short-term decay of the membrane potential after the im-
Floral
palement of a microelectrode into the cell. I instant of the impalement
SD
S D = s h o r t day; CL=continuous light; 1st phase of CL=time interval between 4 p.m. and 8 a.m.; 2nd phase of CL =time interval between 8 a.m. and 4 p.m.
'UCmV)
22
t
[min)
4C 20
o
o
o
o
o
o
o
o
T
20
T
o o
18 i
lh
i
16 14
m
t (h)
Fig. 3. Reproduction of the original record of the membrane potential. Plant: 4 weeks old, in the vegetative state. Measurement in SD, left arrow." light on, right arrow." light off
12 I0 o o
When recorded continuously, the MP of a mesophyll cell appeared to vary periodically. The recording of the MP consisted of two different oscillations (Fig. 3): 1. The sustained light-sensitive high-frequency oscillations of the MP with an amplitude in the range of 10 mV in the light and of 6 mV in the dark superimposed on the resting membrane potential (Table 1). 2. An alternative circadian change (commutation) between two different high-frequency oscillations (see Fig. 6, left part). The amplitude of the oscillations fluctuated much more than the period and did not appear to be directly related to external conditions (Fig. 3). If one observates the MP oscillations of plants cultivated in SD (Fig. 4; noninduced state), there is in dark a period of 7.64_+0.35 win; after the onset of illumination, the period increased abruptly to 19.95+0.22 min. Such alternations occurring within several minutes were detected along several light/dark cycles. Upon the transfer from SD to CL (Fig. 5; Table 1), an intermediate period of 13.37_+ 1.08 win
12
14
16
18
o
o
20
o
o
o
o
22
24
o
o
o
o
o
o
2
4
6
8
lb
lJ2
1~,
Daytime (h)
Fig. 4. The average period of the high-frequency oscillations plotted as a function of time for a noninduced plant monitored in SD (L/D: 8/16 h). Every point corresponds to the period averaged for one hour per interval. Average vaIues of the periods: in the dark 7.64_+0.35 min (f=2.18 10 -3 Hz), in the light 19.95_+0.22 min (f=8.35 10 -4 Hz)
appeared between 4 p.m. and 8 p.m, of the first long day. Then a longer period of 21.19 + 1.63 min, similar to that in SD, was observed; however, the commutation between both of these periods is not so abrupt as in SD (Zeitgeber). The plants cultivated 4 weeks in CL (floral state) also produced diurnal oscillations of the MP period, as they did after transfer from SD to CL (Fig. 6; Table 1). The period alternated regularly from 12.54+0.56 win to 19.63+2.41 win, every 25.2_+ 0.9 h (circadian rhythm). The increase and the decrease of the period between both steady-state values took about 3.5 h. The oscillator functioned 16 h at the short period and 9 h at the long one (Fig. 6).
238
B. N o v a k and H. Greppin: High-Frequency Oscillations and Circadian R h y t h m tfmin)
Table 2. Dependence of period enhancement A C and of normalized period enhancement P on the state of plants during different photoperiodic conditions
o o
35
o
o o
3(3
o
AC
o
25
o
State
AC
P = A Csc-
Vegetative
A Csc -
o o
2C
OoO
oo o o
15
o
oo
oo OoOooO
oOO
oo
Ts~ Ts~
=2.61
1
oO~176
ooo
TL1 =1.75 A CL1 - - -
0.67
T~
10
Induced 22::::::':':':2:':':':':':':'::~" ~; 10
t4- 18 2'2
2
6
10 1~,
18
2'2
2
g Daytime (h)
Fig. 5 The m e a n period of the high-frequency oscillations during the transfer of a noninduced plant 4 weeks old from SD to CL condition. / = t i m e corresponding to the beginning of the floral induction (after 11 h of light) ; E = time related to the floral evocation (after 22 h of light). The average value for the intermediate period: 13.37 + 1.08 rain ( f = 1.25 10 . 3 Hz)
A CL2
---- -
TL2 = 2.77
1.06
A CF1
=T~ =1.73 TFD
0.66
Z~ C F 2
=T~ = 2.71 TFD
1.04
TFL1 =4.63
1.77
TFL2=3.41
1.31
Tso
Floral CL
ZJ CFL 1 - - TFD
Floral SD
The interesting case arose when plants grown in CL were transfered to SD. When darkness was imposed on the plant the period decreased dramatically within several minutes to the lowest value of 7.24 + 0.16 rain. This level was retained with remarkable constancy until the next light period. The first period caused an increase of the period to 33.53 _+3.77 rain, while the second one caused an increase to only 24.71 + 1.88 rain (Fig. 6, Table 1). The period of the circadian rhythm was entrained to 24 h. In an attempt to analyze these results quantitatively, we introduced two parameters to characterize the alternation of the period (Table 2). The relative differences in the periods are expressed by the coefficient of the period enhancement A C. This coefficient is defined by the ratio of the period T in light for any state of the plant (see Table 1) either to that in dark (SD, vegetative: see Table 1)
Z] C F L 2 = TFD
For definition of indexes see Table 1
or to that in dark (SD, floral state: see Table 1)
AC-
T
TvD"
The changes of the different coefficients AC expressed in relation to A CSL (vegetative state) defines the normalized period enhancement P:
p_AC.
T AC=-Tsi) '
AsL
The values of P characterize the alternation of the period relative to that in the vegetative state. If P > 1, the alternation of the period in this state is greater than that in the vegetative state, if P ~ 1, the opposite is true (Table 2). The normalized period enhancement
t (mln)
O ooo O DO
3~ 3C
0 0
25 O~ o~
20
oo o o o
15 10
0 O0
o
oOooooooOooo
oo
o
O0
oo
o
o ~176176176 OoooOOOoOOOOOOOo
Oooooooooooooooo
Daytime
(h)
Fig. 6. The mean period of the high-frequency oscillations of plant, 4 weeks old, cultivated in CL. Left part of the picture : measur~ement performed in CL. Right part: Iarge variation of the periods under SD conditions. The plant was exposed to the dark for the first time
B. Novak and H. Greppin: High-FrequencyOscillationsand Circadian Rhythm for induced and floral states in CL does not differ at all (Table 2). However, it increases to 77% when plants in the floral state were placed in SD.
Discussion
The membrane potentials of mesophyll cells in spinach leaves were continuously recorded for several days under precisely defined conditions (constant temperature). Our data can be explained assuming the existence of two distinct periodic processes, since we have observed two types of periodicities: (1) High-frequency oscillations (a) in light, (b) in dark. (2) Circadian rhythm related to the variation of the period in the high-frequency oscillations. The high-frequency oscillation persisted with a notable constancy over all of the measurements. The oscillatory character of the metabolic pathway is regarded as a general feature of a dynamic system (see review, Chance et al., 1973). High-frequency oscillations have been demonstrated in photosynthesis (Chernavskaya and Chernavskii, 1961), phosphorylation (Fukushima and Tonomura, 1972), glycolysis (Sel'kov, 1968), purine nucleotide cycle (Tornheim and Lowenstein, 1973), and peroxydase enzymes (Yamazaki and Yokota, 1967). The chain linking metabolic pathway oscillations and the membrane potential oscillation could be accounted for by the application of the Mitchell chemiosmotic theory (Mitchell, 1957). This theory involves the vectorial transport of protons across membranes and the redox or photoredox system localized in the organelle membrane. Such membrane systems in eukaryotic cells may be sites for the control of the cellular oscillatory processes especially in the MP oscillations and in the energy transduction in relation to different internal and external signals (See Wagner, 1977). High-frequency oscillations have also been demonstrated in isolated mitochondria (Gooch and Packer, 1974). In this system it is possible that multienzyme systems function as the high-frequency oscillators. High-frequency oscillations of catalytic activity have recently been observed in a chloroplast suspension isolated from spinach (Puu, 1976a, b). The most striking coincidence is the similarity of the oscillatory period monitored by Puu for the redox potential of chloroplast suspensions and of that for the resting potential in mesophyll cells. This suggests that the membrane potential oscillations observed here may be linked with the enzymatic system oscillations and/or with the periodic fluctuations of the redox potential of chloroplasts.
239
Complementary interrelations between the cellular compartments have already been observed by several authors, e.g., reciprocal changes of ultrastructure of chloroplasts and mitochondria (K6nitz, 1965), swelling of mitochondria and contraction of chloroplasts in light and vice versa in darkness (Murakami and Packer, 1970). The two different periods of MP oscillation that appeared in our results can be explained by this complementarity between mitochondria and chloroplasts. The synchronization of the periodic phenomena between the cells is then achieved by the symplastic organization through plasmodesmatal bridges including the regulatory feedbacks (Gunning and Robards, 1976). The surface potential oscillations in Vicia faba roots (Gunther and Scott, 1966) have a period similar to that of spinach leaves in the dark. The high-frequency oscillations of the surface potential measured near to the pulvinus were found to have two periods of 12 and 5 min, regardless of light and dark conditions (Aimi and Shibasaki, 1972, 1975). In addition, a slow rhythmicity of the surface potential corresponded to the circadian rhythm of the leaf movements in the secondary pulvinus cells. During the light period, the surface potential rose and then decreased in the dark reaching its original level by the end of night. The circadian rhythm of the period expressed by the commutation of two high-frequency oscillators is better related to the hypothesis of compensatory control between glycolysis, oxidative phosphorylation, and phot0phosphorylation (Wagner and Cumming, 1970; Wagner, 1976, 1977) than to the hypothesis of the superposition of a population of oscillators (Pavlidis, 1969). During the transfer from SD to CL, the induction takes place concomitantly with the intermediate period. After induction some irreversible modification of the feedback system running the MP oscillations seems to have occurred, as we can infer from the transfer of CL plants to SD (Fig. 6; Tables 1 and 2). This modification was quantitatively expressed by the coefficient P (Table 2: Floral SD).
References
Aimi, R., Shibasaki, S.: Circadian rhythm and light control of the diurnalleafmovementof Phaseolusin relationto bioelectric responses. Proc. of the VI Intern. Congr. on Photobiology. The Abstract193 (1972) Aimi, R., Shibasaki, S.: Diurnal change in bioelectric potential of Phaseolus plant in relation to the leaf movement and light condition. Plant Cell Physiol.16, 1157 62 (1975) Auderset, G., Greppin, H. : Etude de l'apex caulinairede l'6pinard avant et apr~s l'induction florale.-Saussurea7, 73 103 (1976)
240
B. Novak and H. Greppin: High-Frequency Oscillations and Circadian Rhythm
Auderset, G., Greppin, H. : Effet de l'induction florale sur l'6volution ultrastructurale de l'apex caulinaire de Spinacia oleracea, Nobel. Protoplasma 91,281-301 (1977) Balet, A., Greppin, H.: Etude immunochimique de l'induction photop6riodique chez Spinacia oleracea. Saussurea 7, 105-120 (1976) Balet, A., Greppin H. : Etude immunochimique de l'induction florale chez Spinacia oleracea. Saussurea 8, 57-64 (1977) B/inning, E., Moser, I. : Unterschiedliche photoperiodische Empfindlichkeit der beiden Blattseiten von Kalanchoe blossfeldiana. Planta 69, 296-98 (1966) B/inning, E.: The physiological clock. Berlin, Heidelberg, New York: Springer 1967 Chance, B., Pye, E.K, Ghosh, A.K., Hess, B. : Biological and Biochemical Oscillators. New York: Academic Press 1973 Chernavskaya, N.M., Chernavskii, D.S.: Periodic phenomena in photosynthesis. Soviet. Phys. Uspekhi 4, 850-65 (1961) Ehret, C.F., Trucco, E. : Molecular models for the circadian clock. I. The chronon concept. J. Theor. Biol. 15, 240-258 (1967) Etherton, B. : Vacuolar and cytoplasmic potassium concentrations in Pea roots in relation to cell-to-medium electrical potentials. Plant Physiol. 43, 838 40 (1968) Evans, L.T. : The Nature of Flower Induction. In: The induction of flowering. Evans, L.L., ed., pp. 457~481. Australia: MacMillan 1969 Fukushima, Y., Tonomura, Y. : Oscillation of an amount of phosphorylated protein and the ATPase activity of microsomes. Effects of calcium ions and cyclic AMP. Biochem. 77, 623-34 (1972) Gooch, D., Packer, L. : Oscillatory states in mitochondria. Studies on the oscillatory mechanism of liver and heart mitochondria. Arch. Biochem. Biophys. 163, 759-68 (1974) Greppin, H. : La floraison : 6bauche d'une nouvelle strat6gie. Saussurea 6, 245 52 (1975) Greppin, H., Horwitz, B.A.: Floral induction and the effect of red and far-red preillumination on the light stimulated bioelectric response of spinach leaves. Z. Pflanzenphysiol. 75, 243-9 (1975) Greppin, H., Auderset, G., Bonzon, M., Penel, C.: Changement d'6tat membranaire et m~canisme de la floraison. Saussurea, 9, 83-101 (1978) Gunning, B.E.S., Robards, A.W., eds.: Intracellular communication in plants: studies on plasmodesmata. Berlin, Heidelberg, New York: Springer 1976 Gunther, R.L., Scott, B.I.H. : Effect of temperature on bioelectric oscillations of bean roots. Nature 211, No. 5052, 967 8 (1966) Knott, J.E.: Effect of a localized photoperiod on spinach. Proc. Am. Soc. Hort. Sci. 31, 152 4 (1934) K6nitz, W. : Etektronenmikroskopische Untersuchungen an Euglena gracilis im tagesperiodischen Licht-Dunkel-Wechsel. Planta 66, 345-73 (1965) Lenk, R., Bonzon, M., Descouts, P~; Greppin; H.: Investigation of the floral induction in spinach leaves by the Nuclear MagneticResonance. 1st Meeting of Fed. Eur. Soc. Plant Physiol. Abstract, 324=325 (1978) L/ittge, U., Pallaghi, C.K.: Light triggered transient changes
of membrane potentials in green cells in relation to photosynthetic electron transport. Z. Pflanzenphysioi. 61, 58-67 (t969) Mitchell, P. : A general theory of membrane transport from studies of bacteria. Nature (Loud.) 180, 134-6 (1957) Murakami, S., Packer, L. : Light-induced changes in the conformation and configuration of the thylakoid membrane of Ulva and Porphyra chloroplasts in vivo. Plant Physiol. 45, 2892-99 (1970) Njus, D., Sulzman, F.M., Hastings, J.W.: Membrane model for the circadian clock. Nature 248, No. 5444, 1 i6 120 (1974) Novak, B., Sironval, C. : Circadian rhythm of the transcellular current in regenerating enucleated posterior stalk segments of Acetabularia rnediterranea. Plant Sci. Lett. 6, 273-83 (1976) Pavlidis, T. : Populations of interacting oscillators and circadian rhythms. J. Theor. Biol. 22, 418 36 (1969) Penel, C., Greppin, H.: Action des lumi6res rouge et infrarouge sur l'activit6 peroxydasique des feuilles d'6pinards avant et apr6s l'induction florale. Ber. Schweiz. Bot. Ges. 83, 253-61 (1973) Penel, C., Greppin, H. : Variation de la photostimulation de l'activit6 des peroxydases basiques chez l'6pinard. Plant. Sci. Lett..3, 75-80 (1974) Pickard, B.G.: Action potentials in higher plants. Bot. Rev. 39, 172 201 (1973) Puu, G. : Oscillatory catalytic activity in chloroplasts from spinach cotyledons. IUB. Tenth Inter. Congress of Biochemistry, Abstact 14-6-025, Hamburg 1976a Puu, G. : Oscillatory phenomen in chloroplasts from spinach cotyledons. Ph.D. Thesis, Univ. of Umea, Sweden (1976b) Reusing, L.: Biologische Rhythmen und Regulation. Stuttgart: Fischer 1973 Sel'kov, E.E. : Self-oscillation in glycolysis I. A simple kinetic model. Eur. J. Biochem. 4, 79-86 (1968) Tornheim, K., Lowenstein, J.M.: The purine nucleotide cycle III. Oscillation in metabolite concentration during the operation of the cycle in muscle extracts. J. Biol. Chem. 248, 2670-7 (1973) Wagner, E., Cumming, B.C.: Betacyanin accumulation, chlorophyll content and Flower initiation in Chenopodium rubrurn as related to endogeneous rhythmicity and phytochrome action. Can. J. Bot. 48, 1 18 (1970) Wagner, E. : Endogeneous rhythmicity in energy metabolism: basis for timer photoreceptor interaction in photoperiodic control. In: The Molecular Basis of circadian Rhythms, Report of the Dahlem Workshop on the Molecular Basis of Circadian Rhythms, Berlin, 1975. Hastings, J.W., Schweiger, H.G., eds. pp. 215 38. Berlin: Abakon 1976 Wagner, E.: Molecular Basis of physiological Rhythms. In: Integration of Activity in the Higher Plant, Symposium XXXI of the Society for experimental Biology, Cambridge, 1976. Jennings, D.H., eds. pp. 33-72. Cambridge: Univ. Press 1977 Yamazaki, I., Yokota, K. : Analysis of the conditions causing the oscillatory oxidation of reduced nicotinamide-adenine dinucleotide by horse radish peroxidase. Biochem. Biophys. Acta 132, 310-20 (1967) Received 5 May; accepted 23 October 1978
Planta
240 (1979)
9 by Springer-Verlag 1979
High-Frequency Oscillations and Circadian Rhythm of the Membrane Potential in Spinach Leaves B. Novak and H. Greppin Laboratoire de Physiologic V6g6tale, 3, place de l'Universit~, CH-1211 Gen+ve 4, Switzerland
Abstract. The microelectrode technique was used to follow oscillations in membrane potential in mesophyll cells of spinach (Spinacia oleracea L.) during exposure do different photoperiodic conditions. Both high-frequency oscillations and circadian variations were observed. The circadian rhythm was imposed on the period of high-frequency oscillation during short days as well as in continuous light: The freerunning period was 25.2 h. The average period of high-frequency oscillation increased from 7.64 min in the dark to 19.95 rain in the light within several minutes after dark to light transition. This period length coincides with the established period length for oscillations in the redox potential in the chloroplast suspensions of spinach. Key words: Circadian rhythm-Floral
inductionHigh-frequency oscillation-Membrane potentialSpinacia.
Introduction
Many forms of rythms and oscillations exist in eukaryotic organisms possessing highly spezialized hierarchic organization and compartmentation (B/inning, 1967; Rensing, 1973). It has been established that these rhythms are generated at a subcellular level and that a common control mechanism is responsible. Three models have been proposed to explain the oscillatory mechanism: 1. The replicon-chronon model of Ehret and Trucco (1967) explained the rhythmicity as a consequence of sequential gene expression (polycistronic replicons of optimal length: chronons) at the transcription level through a periodic functioning. The linear sequential replication, cistron by cistron, of CL = continuous light; SD = membrane potential
Abbreviations."
short day; MP
a very long polycistronic unit provides the cell with a remarkably simple interval-measuring and eventenumerating device; the last step (recycling factor) initiates a new transcription round. 2. Another model proposed by Pavlidis (1969), Sel'kov (1968), and also Chance et al. (1973), considered the self-sustained oscillations of a biochemical network with feedback loops causing circadian rhythmicity. However, an explanation of how the highfrequency biochemical oscillations are transformed into low-frequency circadian rythms is lacking. 3. Njus et al. (1974) suggested that rhythm was related to the membrane-bound ion transport systems. If the ionic concentrations of the cytoplasmic compartments oscillate, the transmembrane ionic flux and local membrane potential should also show a circadian periodicity, as has been shown in Acetabularia rned. (Novak and Sironval, 1976). Thus the interaction of the energy-converting compartments is involved in the generation of rhythms (Wagner, 1976). Since the sites of perception for the photoperiodic signals are localized in leaves (Knott, 1934; Biinning and Moser, 1966), we decided to test their influence on the membrane potential of the mesophyll cells. The membrane potential measured over long-term intervals provides unique information about the processes going on in the mesophyll cells, and with the symplastic organization of cells in plants, allowing the immediate transmission of signals through plasmodesmata (Lfittge and Pallaghi, 1969; Pickard, 1973), it is possible to obtain information on the general physiologic state of the plant. Three types of regulatory responses can be observed in plants: 1. Immediate, controlled by electrochemical processes, 2. Short-term, under the control of hormones or metabolite movement, and 3. Slow, controlled by specific proteins after neosynthesis.
0032-0935/79/0144/0235/$01.20
236
B. Novak and H. Greppin: High-Frequency Oscillations and Circadian Rhythm
Floral induction has been shown to be regulated by short-term processes (Penel and Greppin, 1973, 1974); the synthesis of new specific proteins after floral induction appears to be too late to intervene in the initial processes (Greppin, 1975; Balet and Grepping, 1976, 1977). The photoperiodic flowering process in spinach involves the immediate transmission of an electrochemical signal (Greppin, 1975; Greppin and Horwitz, 1975). Bearing these findings in mind, we assume that regulating processes leading to floral induction are of a biophysical nature (Lenk et al., 1978; Greppin et al., 1978). We describe the immediate changes of the period in the high-frequency oscillations upon photoperiodic stimulation.
Materials and Methods Plants and Cultivation Spinach plants (Spinacia oleracea, var. Nobel), were selected after cultivation for 4N 5 weeks in a phytotron under standard conditions: temperature 20_+0.15~ relative humidity during light period, 50 _+2% and during dark period, 70 + 1.5% (periodic fluctuations of temperature and humidity in the phytotron: t t min in clark and 7 min in light). Illumination by fluorescent tubes (Sylvania, TL-33, Daylight, 40 W) grave 6000 lx at plant level. The following conditions for the photoperiod and the relative humidity were imposed on the plants: 1. Short days (SD) of 8 h light with 50% relative humidity (from 8 a.m. to 4 p.m.) and 16 h of dark with 70% relative humidity (from 4 p.m. to 8 a.m.). In SD, Spinacia oleracea remains in a vegetative state for 6 weeks. The transition to flowering was monitored by following ultra structural criteria (Auderset and Greppin, 1977). 2. Continuous light (CL) with relative humidity of 50%. Plants subjected from germination to 4 weeks of continuous light were used to define the fully induced and floral state verified by the appearance of the floral primordia visible in the light microscope (Auderset and Greppin, 1976). 3. The transfer from SD to CL induces flowering. Onset of the induction in leaves is 3 h after the termination of the SD period (critical photoperiod : 11 h). The flowering signal is transmitted from leaves to the shoot apex. The first changes of the apex resulting from the arrival of this signal have been called evocation (Evans, 1969), which begins 11 h after the onset of the photoperiodic induction of leaves (11 h light), in all after 22 h of light. The floral embryogenesis, properly so called, starts only 26 h after evocation (Auderset and Greppin, 1976, 1977). 4. Four weeks of CL is followed by transfer to SD in order to test the degree of reversibility in photoperiodic treatment. The measurements made under the above-stated conditions were repeated at least ten times.
Membrane Potential Measurements Membrane potential (MP) measurements in the mesophyll cells were made using a specially constructed Plexiglas cuvette (Fig. 1). It consists of a vessel and a cover that are gently pressed together. The cuvette served as a holder for a leaf in a fixed position between the vessel and the cover. The leaves were dipped in the perfusing nutrient solution o f pH 7.0 (for details see Etherton, 1968).
/
/ s///
\L
Fig. 1. Plexiglas cuvette with a plant and the principal circuit for measurement and registration of the membrane potential. CU cuvette; K cover; S silicon rubber layers attached to Plexiglas pieces; L leaf; M E glass microelectrode; RE reference electrode; C compensation of the junction potential; E electrometer (R~ = 1012 ~?) ; PR pen recorder
The cuvette allowed measurements on leaves that were still attached to the plant. After the leaf was fixed in the cuvette, the plant was allowed to equilibrate for at least 12 h in the dark. Details of the measuring equipment are shown in Figure 1. Silver wires that had been coated with a silver chloride layer were used for both the microelectrode and for the reference electrode. The glass microcapillaries were made by the vertical puller of Getra Inst., Munich W-Germany. The 1.4-mm diameter microcapillaries were filled with 0.5 M KCI solution. The tip resistance was 5 ~ 30 M~2. The tips of the glass microcapillaries were impaled into the cells using manually operated micromanipulators supplied by Narishige, Japan. The signals were measured using a solid state electrometer (Keythley Inst) with an imput resistance of 10 ta s'2 and registered by a pen-recorder (Watanabe, Japan). The membrane potential of a mesophyll cell was monitored for periods of 2 N 5 days under the different light/dark and humidity programs.
Results
The electrophysiologic approach appeared to be advantageous compared to biochemical methods, since we were able to monitor the cells of intact leaves for a long interval with only very few mechanical and electrical disturbances. The stability of the MP was good during the measurements, in comparison to that when an artifical disturbance (mechanical, electric, thermic) was applied to the plant. The resting MP o f - 84.8_+ 16.3 mV (number of measurements = 53) inside the mesophyll cell was detected just after the impalement of the glass microelectrode (Fig. 2). An exponential decay in MP was then observed within several minutes, and it finally stabilized around a mean value of - 38.5 + 9.6 mV (n = 53). The lowering of the MP might be caused by the leak of ions around the wound made by the penetration of the tip into the cell. Within several hours the wound healed up and the MP remained fairly stable (Fig. 3). The recorded potentials returned to zero level + 4 mV when the microelectrode was withdrawn from the tissue.
B. Novak and H. Greppin: High-Frequency Oscillations and Circadian Rhythm
237
Table 1. The average period of high-frequency oscillation as a function of the state of the plants and the light/dark conditions
-U (mY)
[ 10(mV)
State
Photoperiod
Illumination
Average period (min)
Vegetative
SD
Dark Light
TsD -- 7.64_+0.35 Tsc -- 19.95 • 0.22
lnduced
CL
Light, 1st phase Light, 2nd phase
Tm = 13.37 + 1.08 TL2 =21.19--+1.63
CL
Light, 1st phase Light, 2nd phase
Tw =12.54_+0.56 Tv2 =19.63_+2.41
Dark 1st light 2nd light
TFD = 7.24_+0.16 TFL1 =33.53_+3.77 TFL2 =24.71 _+1,88
60 l(min)
,
t Cmin)
Fig. 2. Short-term decay of the membrane potential after the im-
Floral
palement of a microelectrode into the cell. I instant of the impalement
SD
S D = s h o r t day; CL=continuous light; 1st phase of CL=time interval between 4 p.m. and 8 a.m.; 2nd phase of CL =time interval between 8 a.m. and 4 p.m.
'UCmV)
22
t
[min)
4C 20
o
o
o
o
o
o
o
o
T
20
T
o o
18 i
lh
i
16 14
m
t (h)
Fig. 3. Reproduction of the original record of the membrane potential. Plant: 4 weeks old, in the vegetative state. Measurement in SD, left arrow." light on, right arrow." light off
12 I0 o o
When recorded continuously, the MP of a mesophyll cell appeared to vary periodically. The recording of the MP consisted of two different oscillations (Fig. 3): 1. The sustained light-sensitive high-frequency oscillations of the MP with an amplitude in the range of 10 mV in the light and of 6 mV in the dark superimposed on the resting membrane potential (Table 1). 2. An alternative circadian change (commutation) between two different high-frequency oscillations (see Fig. 6, left part). The amplitude of the oscillations fluctuated much more than the period and did not appear to be directly related to external conditions (Fig. 3). If one observates the MP oscillations of plants cultivated in SD (Fig. 4; noninduced state), there is in dark a period of 7.64_+0.35 win; after the onset of illumination, the period increased abruptly to 19.95+0.22 min. Such alternations occurring within several minutes were detected along several light/dark cycles. Upon the transfer from SD to CL (Fig. 5; Table 1), an intermediate period of 13.37_+ 1.08 win
12
14
16
18
o
o
20
o
o
o
o
22
24
o
o
o
o
o
o
2
4
6
8
lb
lJ2
1~,
Daytime (h)
Fig. 4. The average period of the high-frequency oscillations plotted as a function of time for a noninduced plant monitored in SD (L/D: 8/16 h). Every point corresponds to the period averaged for one hour per interval. Average vaIues of the periods: in the dark 7.64_+0.35 min (f=2.18 10 -3 Hz), in the light 19.95_+0.22 min (f=8.35 10 -4 Hz)
appeared between 4 p.m. and 8 p.m, of the first long day. Then a longer period of 21.19 + 1.63 min, similar to that in SD, was observed; however, the commutation between both of these periods is not so abrupt as in SD (Zeitgeber). The plants cultivated 4 weeks in CL (floral state) also produced diurnal oscillations of the MP period, as they did after transfer from SD to CL (Fig. 6; Table 1). The period alternated regularly from 12.54+0.56 win to 19.63+2.41 win, every 25.2_+ 0.9 h (circadian rhythm). The increase and the decrease of the period between both steady-state values took about 3.5 h. The oscillator functioned 16 h at the short period and 9 h at the long one (Fig. 6).
238
B. N o v a k and H. Greppin: High-Frequency Oscillations and Circadian R h y t h m tfmin)
Table 2. Dependence of period enhancement A C and of normalized period enhancement P on the state of plants during different photoperiodic conditions
o o
35
o
o o
3(3
o
AC
o
25
o
State
AC
P = A Csc-
Vegetative
A Csc -
o o
2C
OoO
oo o o
15
o
oo
oo OoOooO
oOO
oo
Ts~ Ts~
=2.61
1
oO~176
ooo
TL1 =1.75 A CL1 - - -
0.67
T~
10
Induced 22::::::':':':2:':':':':':':'::~" ~; 10
t4- 18 2'2
2
6
10 1~,
18
2'2
2
g Daytime (h)
Fig. 5 The m e a n period of the high-frequency oscillations during the transfer of a noninduced plant 4 weeks old from SD to CL condition. / = t i m e corresponding to the beginning of the floral induction (after 11 h of light) ; E = time related to the floral evocation (after 22 h of light). The average value for the intermediate period: 13.37 + 1.08 rain ( f = 1.25 10 . 3 Hz)
A CL2
---- -
TL2 = 2.77
1.06
A CF1
=T~ =1.73 TFD
0.66
Z~ C F 2
=T~ = 2.71 TFD
1.04
TFL1 =4.63
1.77
TFL2=3.41
1.31
Tso
Floral CL
ZJ CFL 1 - - TFD
Floral SD
The interesting case arose when plants grown in CL were transfered to SD. When darkness was imposed on the plant the period decreased dramatically within several minutes to the lowest value of 7.24 + 0.16 rain. This level was retained with remarkable constancy until the next light period. The first period caused an increase of the period to 33.53 _+3.77 rain, while the second one caused an increase to only 24.71 + 1.88 rain (Fig. 6, Table 1). The period of the circadian rhythm was entrained to 24 h. In an attempt to analyze these results quantitatively, we introduced two parameters to characterize the alternation of the period (Table 2). The relative differences in the periods are expressed by the coefficient of the period enhancement A C. This coefficient is defined by the ratio of the period T in light for any state of the plant (see Table 1) either to that in dark (SD, vegetative: see Table 1)
Z] C F L 2 = TFD
For definition of indexes see Table 1
or to that in dark (SD, floral state: see Table 1)
AC-
T
TvD"
The changes of the different coefficients AC expressed in relation to A CSL (vegetative state) defines the normalized period enhancement P:
p_AC.
T AC=-Tsi) '
AsL
The values of P characterize the alternation of the period relative to that in the vegetative state. If P > 1, the alternation of the period in this state is greater than that in the vegetative state, if P ~ 1, the opposite is true (Table 2). The normalized period enhancement
t (mln)
O ooo O DO
3~ 3C
0 0
25 O~ o~
20
oo o o o
15 10
0 O0
o
oOooooooOooo
oo
o
O0
oo
o
o ~176176176 OoooOOOoOOOOOOOo
Oooooooooooooooo
Daytime
(h)
Fig. 6. The mean period of the high-frequency oscillations of plant, 4 weeks old, cultivated in CL. Left part of the picture : measur~ement performed in CL. Right part: Iarge variation of the periods under SD conditions. The plant was exposed to the dark for the first time
B. Novak and H. Greppin: High-FrequencyOscillationsand Circadian Rhythm for induced and floral states in CL does not differ at all (Table 2). However, it increases to 77% when plants in the floral state were placed in SD.
Discussion
The membrane potentials of mesophyll cells in spinach leaves were continuously recorded for several days under precisely defined conditions (constant temperature). Our data can be explained assuming the existence of two distinct periodic processes, since we have observed two types of periodicities: (1) High-frequency oscillations (a) in light, (b) in dark. (2) Circadian rhythm related to the variation of the period in the high-frequency oscillations. The high-frequency oscillation persisted with a notable constancy over all of the measurements. The oscillatory character of the metabolic pathway is regarded as a general feature of a dynamic system (see review, Chance et al., 1973). High-frequency oscillations have been demonstrated in photosynthesis (Chernavskaya and Chernavskii, 1961), phosphorylation (Fukushima and Tonomura, 1972), glycolysis (Sel'kov, 1968), purine nucleotide cycle (Tornheim and Lowenstein, 1973), and peroxydase enzymes (Yamazaki and Yokota, 1967). The chain linking metabolic pathway oscillations and the membrane potential oscillation could be accounted for by the application of the Mitchell chemiosmotic theory (Mitchell, 1957). This theory involves the vectorial transport of protons across membranes and the redox or photoredox system localized in the organelle membrane. Such membrane systems in eukaryotic cells may be sites for the control of the cellular oscillatory processes especially in the MP oscillations and in the energy transduction in relation to different internal and external signals (See Wagner, 1977). High-frequency oscillations have also been demonstrated in isolated mitochondria (Gooch and Packer, 1974). In this system it is possible that multienzyme systems function as the high-frequency oscillators. High-frequency oscillations of catalytic activity have recently been observed in a chloroplast suspension isolated from spinach (Puu, 1976a, b). The most striking coincidence is the similarity of the oscillatory period monitored by Puu for the redox potential of chloroplast suspensions and of that for the resting potential in mesophyll cells. This suggests that the membrane potential oscillations observed here may be linked with the enzymatic system oscillations and/or with the periodic fluctuations of the redox potential of chloroplasts.
239
Complementary interrelations between the cellular compartments have already been observed by several authors, e.g., reciprocal changes of ultrastructure of chloroplasts and mitochondria (K6nitz, 1965), swelling of mitochondria and contraction of chloroplasts in light and vice versa in darkness (Murakami and Packer, 1970). The two different periods of MP oscillation that appeared in our results can be explained by this complementarity between mitochondria and chloroplasts. The synchronization of the periodic phenomena between the cells is then achieved by the symplastic organization through plasmodesmatal bridges including the regulatory feedbacks (Gunning and Robards, 1976). The surface potential oscillations in Vicia faba roots (Gunther and Scott, 1966) have a period similar to that of spinach leaves in the dark. The high-frequency oscillations of the surface potential measured near to the pulvinus were found to have two periods of 12 and 5 min, regardless of light and dark conditions (Aimi and Shibasaki, 1972, 1975). In addition, a slow rhythmicity of the surface potential corresponded to the circadian rhythm of the leaf movements in the secondary pulvinus cells. During the light period, the surface potential rose and then decreased in the dark reaching its original level by the end of night. The circadian rhythm of the period expressed by the commutation of two high-frequency oscillators is better related to the hypothesis of compensatory control between glycolysis, oxidative phosphorylation, and phot0phosphorylation (Wagner and Cumming, 1970; Wagner, 1976, 1977) than to the hypothesis of the superposition of a population of oscillators (Pavlidis, 1969). During the transfer from SD to CL, the induction takes place concomitantly with the intermediate period. After induction some irreversible modification of the feedback system running the MP oscillations seems to have occurred, as we can infer from the transfer of CL plants to SD (Fig. 6; Tables 1 and 2). This modification was quantitatively expressed by the coefficient P (Table 2: Floral SD).
References
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240
B. Novak and H. Greppin: High-Frequency Oscillations and Circadian Rhythm
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