Photochemistry and Phorob~ology, 1976, Vol. 23. pp. 125-130.
Pergamon Press
Printed in Great Britain
DEVELOPMENT OF THERMOLUMINESCENCE BANDS DURING GREENING OF WHEAT LEAVES UNDER CONTINUOUS AND INTERMITTENT ILLUMINATION YORMAOINOUE, TETSUOICHIKAWAand KAZUOSHIBATA Laboratory of Plant Physiology, The Institute of Physical and Chemical Research, Wako-shi, Saitama 351, Japan
(Received 7 July 1975; accepted 21 October 1975) Abstract-Mature wheat leaves excited by 1-min illumination at a low temperature of -60°C showed five thermoluminescence bands at -45, -10, +25, +40 and +55"C (denoted as Z,, A , B,, B , and C bands, respectively). The development of these bands during greening of etiolated wheat leaves under continuous and intermittent illumination was investigated, and the following results were obtained. (1) Etiolated leaves showed only the C band. When these leaves were greened under continuous light, the B , and B2 bands appeared at 3 h and the Z, band appeared at 10 h. The B , and 8 , bands were intensified during prolonged greening under continuous illumination to be the strong bands observed for mature leaves. The A band and the group of B , and B , bands were alternative: Similar experiments by excitation of thermoluminescence at - 20°C showed the development of the A band instead of these B , and B , bands. (2)When the etiolated leaves were greened under 5-min dark), the 2, band first appeared after 5 h of illumination intermittent illumination (1-ms light (60 flashes) and was gradually intensified during further illumination with 340 flashes but, interestingly, neither the B , nor the B , band appeared even after 2428 h of illumination with 28Cb340 flashes. (3) On exposure of such leaves greened under intermittent illumination to continuous light, the B , and B , bands were rapidly developed. The appearance of these bands was accompanied by the generation of the Hill activity (DCIP photoreduction with water as electron donor). (4)These results were discussed in relation to the previous study on photoactivation of the latent water-splitting system accumulated in the leaves greened under intermittent illurnination. It was deduced that the structure responsible for the A band or the group of B , and B , bands is involved in the evolution of oxygen in chloroplasts, and probably involves cations of the MnZ+catalyst generated by the action of light.
+
cence variations and delayed light emission (PhungNhu-Hung et al., 1970; Dujardin et al., 1970). Recent The development of photosynthetic apparatus in studies by Remy (1973) and by Sironval and his the plastids of angiosperms depends on light. Illuco-workers (Sironval et al., 1971; Michel and Sironmination of etiolated leaves with strong continuous Val, 1972; Strasser and Sironval, 1972) elucidated that light converts protochlorophyllide to chlorophyll a, the water-splitting system is not functional in these which triggers the biosynthesis of various pigments chloroplasts, while the reaction centers of both photoand proteins necessary for membrane formation. The systems have been developed almost completely. In electron transport from water to a Hill oxidant via fact, exposure of such intermittently illuminated photosystem I1 becomes observable after several h of leaves to continuous light rapidly induced the Hill continuous illumination (Virgin, 1972; Remy e t al., activity of DCIP photoreduction with water as elec1972; Ogawa et al., 1973), and all of the photosynthetron donor (Remy, 1973; Inoue et al., 1974b; Inoue tic activities are developed in 1@15 h (Boardman et et al., 1975) and the activation was accompanied by ul., 1970; Boardman et al., 1971). a marked increase of delayed fluorescence (Ichikawa Under intermittent illumination with flashes at long et a!., 197$a). Kinetic analysis of these photoactivaintervals of several min or longer, however, the development proceeds differently. The chloroplasts in such tion and delayed fluorescence enhancement by means of multiple flashes at short intervals of the order of intermittently illuminated leaves are capable of bringing about photosystem-I reactions and DCIP seconds indicated that the process involves three con(2,6-dichlorophenolindophenol)*photoreduction with secutive photoreactions (Inoue, 1975). The photoactian artificial electron donor, DPC (diphenylcarba- vation of the latent water-splitting system in higher zide)*, but incapable of bringing about some of pho- plant leaves seems to be similar in mechanism to the tosystem-I1 reactions such as oxygen evolution, DCIP photoactivation of MnZ+catalyst in Mn2+-deficient photoreduction with water as electron donor, fluores- algal cells studied by Cheniae and Martin (1969; 1971; 1972; 1973)and by Radmer and Cheniae (1971). The thermoluminescence from plant materials, which was first Observed by Arnold and his co* Abbreviations: DCIP, 2,6-dichlorophenolindophenol; DPC, diphenylcarbazide. workers (Arnold and Shenvood, 1957; 1959; Arnold, INTRODUCTION
125
126
YORINAOINOUE,TETSUO ICHIKAWA and KAZUOSHIBATA
1966) after the discovery of delayed emission by Strehler and Arnold (1951), has been interpreted as resulting from recombination of electrons and positive holes at the reaction center of photosystem I1 in chloroplasts (Arnold, 1965; Arnold and Azzi, 1968; Rubin and Venediktov, 1969; Shuvalov and Litvin, 1969; Lurk and Bertsch, 1974a, 1974b). Previous measurements (Inoue et al., 1974a) by a sensitive photo-electron counting method of mature leaves excited a t - 60°C showed five thermoluminescence bands emitted at -45, - 10, +25, +40 and +55"C, and these bands were denoted as Z,, A , B , , B 2 and C bands, respectively, according to our previous assignments for designation (Ichikawa et a!., 1975b) based on the nomenclature by Arnold (Arnold and Azzi, 1968) with partial modification. In the present study, we have investigated the thermoluminescence of wheat leaves being greened under continuous and intermittent illumination in order to elucidate the process of development of these bands. The courses of development of various bands were compared with the development of the photosystem-I1 activity measured for similar wheat leaves being activated under the same conditions. MATERIALS AND METHODS
Wheat seeds (Triticum aestiuum L.) were germinated and grown on moist filter paper in darkness at 24 & 1°C. Seven-day old etiolated leaves were harvested and cut into segments 3.5 cm long from apexes and spread on moist filter paper to be illuminated for greening with continuous light from white fluorescent lamps or with intermittent light (I ms light + 5 min dark) from electronic Xe strobes. Intensities of continuous and intermittent light beams were 7.5 J/m2 (750 pW/cm2) and 15 J/m2 (1.5 x lo4 ergs/cm2) per flash, respectively. The greening was continued for 24-28 h. The intermittently illuminated leaves obtained after exposure to 29Cb340 flashes were further subjected to photoactivation of their latent water-splitting system by continuous red light (680 & 5 nm, 0.14 Jim') for 1Cb30 min. Light energy from various light sources were measured with a Kipp & Zonen thermopile (model E-2) or with a Quantronics thermocouple (model 500). The green leaves illuminated under various light conditions were kept in darkness at room temperature for more than 20 min before use. Measurement of thermoluminescence. Thermoluminescence from intact leaves was measured by the method reported previously (Inoue et al., 1974) which is briefly described below. Ten segments of wheat leaves harvested at various stages of greening were arranged close together on moist filter paper and sandwiched between a heater and a glass plate to be mounted on an aluminum plate. The sample leaves were, then, cooled to -60°C in a Dewar bottle with an end of the aluminum holder dipped in cold nitrogen gas. The temperature of the leaves was monitored with a Cu-constantan thermocouple inserted between the sample and the glass plate. The leaves at - 60°C were then illuminated with red light longer than 630 nm (J/m2) for 1 rnin and, then, rapidly cooled down to - 196°C by dipping the holder into liquid N,. In some experiments, leaves were illuminated at a higher temperature of -20°C. The sample was transferred with the Dewar bottle to a housing of a photon-counting device (Jasco model KC-200) equipped with a 30 Hz mechanical chopper and a redsensitive photomultiplier (EM1 9659QB). The leaves were
heated slowly at a rate of 0.5"C/s and the photons emitted from the leaves during heating were counted through a red glass filter (VR-63, Toshiba Kasei Co.). The digital photon-count in every 32 Hz was converted to an analogue signal and recorded against temperature on an X Y recorder. Activity measurements and chlorophyll assay. Chloroplasts for the measurement of the Hill activity were prepared from the sample leaves with 0.5 M Tricine buffer (pH 7.5) containing 0.4 M sucrose and 0.01 M MgCI2. The activity of DCIP photoreduction with water or DPC as electron donor and the chlorophyll concentration in the sample suspension was measured by the procedure described previously (Inoue et al., 1974b).
RESULTS AND DISCUSSION
Activity generation and chlorophyll formation by continuous and intermittent illumination
The curves in Fig. l a show the development of photosystem-I1 activities and the formation of chlorophylls during greening of etiolated wheat leaves under continuous and intermittent illumination. Under continuous illumination with strong light, chlorophylls were rapidly synthesized after a few hours of lag period as shown by curve A. Both activities of DCIP photoreduction with water as electron donor (the Hill activity) and DCIP photoreduction with DPC as the donor became observable after 6 h of illumination and rapidly increased between 6 and 12 h to reach saturation after 18 h (curves B and C). Under intermittent illumination with strong Xe flashes, however, chlorophyll was synthesized at a lower rate (about 30% of the rate under continuous illumination) to yield pale green leaves after 28 h of intermittent illumination (curve D). The activity with DPC as electron donor was generated and increased at a slower rate (curve F) but the Hill activity with water as the donor was not generated a t all (curve E) even after 28 h of the intermittent illumination. When such intermittently illuminated leaves were exposed to continuous light, the Hill activity was rapidly generated, as shown by curve H in Fig. lb. This process of photoactivation proceeded first rapidly and then more slowly to reach saturation after about 60 min of continuous illumination with weak red light (680 & 5 nm, 0.14 J/m2). The activity of DCIP photoreduction with DPC as electron donor was also enhanced about 3 times (curve I), while the chlorophyll content did not increase much during this continuous illumination (curve G). These results are in good agreement with the previous observations (Dujardin et al., 1970; Sironval et al., 1971; Michel and Sironval, 1972; Remy, 1973) that the leaves greened under intermittent illumination are devoid of the water-splitting activity whereas the reaction centers for photosystems I and I1 have been developed almost completely, and that the latent watersplitting system accumulated in the intermittently illuminated leaves is activated by exposure to continuous light.
Thermoluminescence during chloroplast development
-
c
,
I
,
Development of' thermoluminescence batids hy tinuous illumination of etiolated leaves
I
,
a) Greening
b) Photoactivation
Time in h
Time in min
Figure l.(a) Chlorophyll formation (curves A and D) and the development of the activities of DCIP photoreduction with water (curves B and E) and DPC (curves C and F) as electron donor during greening under continuous (solid curves) and intermittent (dotted curves) illumination. Excised leaves of 7-day old etiolated wheat seedlings were exposed to continuous white light (7.5 J/m2; 750 pW/cm2) from fluorescent lamps or to intermittent light (1 ms light + 5 min dark) from electronic Xe flashes (15 J/m2; I .5 x lo4 ergs/cm*/flash). Photosystem-I1 activities measured with chloroplasts isolated from the leaves at various stages of greening are expressed on chlorophyll basis. (b) Generation of the activity of DCIP photoreduction with water (curve H) or DPC (curve I) as electron donor during photoactivation. Intermittently illuminated leaves obtained after exposure to 340 flashes (28 h) were further exposed to continuous red light (680 5 nm, 0.14 Jimz; 14 pW/cm2) in order to activate the latent watersplitting system. The measurements of photosystem-I1 activities were carried out in the same manner as in Fig. la.
+
I27 COIZ-
Figure 2a shows the change of thermoluminescence profile of etiolated wheat leaves during greening under continuous illumination. The temperature for actinic illumination of leaves for the measurement was -60°C. The profile of etiolated leaves (curve A) showed a single band around +55"C. Unlike othcr bands, the C band was observable without actinic illumination for all of tthe sample leaves as shown by broken curves in Fig. 2. The height of the C band fluctuated considerably depending on freezing conditions, but the height without actinic illurnination was generally higher than that after the illumination. Considering these, it seems likely that this band is not concerned with photosynthetic activities. The profile of fully greened leaves (curve G) showed three distinct luminescence peaks. The peaks around -45 and +55"C were denoted previously as Z, and C band, respectively, and the middle peak around + 30°C with a shoulder around 40°C are a composite of B1 and B, bands. The assignment for designation of these thermoluminescence bands used in the present study was mainly based on the nomenclature by Arnold (Arnold and Azzi, 1968) and the correspondence between these bands is summarized in Table 1. together with the data obtained by previous investigators. The emission temperature of the Z,. band was found to be variable depending on the illumination temperature, whereas those of the other bands were constant, being independent of the illumination temperature. The Z , band is a newly found one and some of its characteristics including its response to the
+
Table 1. Correspondence of thermoluminescence bands in the present study to those reported previously by various investigators Author
Thermoluminescence bands
z
Arnold L A z z i (-
(1968)
155 o c )
B (t3OoC)
A (-6°C)
C (f550C) n
Shuvalov
&
Litvin
(1969)
Component I1 (-160OC) 1, (Component I)
Component I11 (-15'C)
Sane et al.
peak
(19n)-
(118'K)
peak
Component V
(t2OOC)
peak 1 (-10°C)
Lurie L Bertsch (1974a)
Component Iv
*
(254OK)
peak 2 (+3OoC)
peak
*
(257'K)
peak 3 (+40°C)
__
peak
peak
(290°K:
(320'Kj
pi
Inoue g &. (present paper) L
1
Ichikawa et al. (1975b)- -
Zt
(-160'C)
+
t
zv (variable)
r a l t e r n a t i v e-
L
A (-1OOC)
B1 (t25'C)
B2 (t4OoC)
C (+550C)
* These temperatures were read on the glow curve in Sane's paper. t The relationship between the Z and the Z , bands has been discussed previously (Ichikawa et al., 1975b). 5 Either the A band or the group of B, and B2 bands was observed in wheat leaves depending on the excitation temperature. I/ This component is a delayed emission with life time of 5 ms observed below 0°C. 71 Another component of delayed emission with life time of 1@15 s observed above 0°C.
YORINAO INOUE, TETSUOICHIKAWAand KAZUOSHIBATA
128
the A band and the group of B , and B, bands will be discussed later. Curves B, C, D, E and F show light the process of development of these luminescence bands during continuous illumination. The B , and B2 bands were progressively enhanced until 28 h to be the strong bands shown on curve G, whereas the Z , band was not much intensified. The time courses of the appearance of these luminescence bands are summarized in Fig. 3a. Curves B and C show the development of the €3, and B, bands, respectively, which proceeded approxi(A ) mately in parallel with the development of the Hill 4 -50 0 +50 activity shown by curve B in Fig. la. On the other Temperature in "C hand, curve A showing the development of the Z,, band increased to a much slighter extent. Figure 2. Development of thermoluminescence bands durThe above profiles were measured for the leaves ing greening under continuous (Fig. 2a) and intermittent (Fig. 2b) illumination. Sample leaves obtained at various excited by the illumination at -60°C. The profiles stages of greening in the experiment of Fig. l a were illu- obtained for similar samples excited at higher temminated at - 60°C for excitation with red light (above 630 peratures are considerably different. Curves A and B nm, 6 J/mz; 600 pW/cm2) for 1 min before the measure- in Fig. 4,which are typical examples for comparison, ment of thermoluminescence. Curves A, B, C, D, E, F and G were the profiles obtained for leaves after continuous are the profiles of mature leaves excited at -60°C illumination for 3, 5, 10, 15 and 28 h, respectively, and and -2o"C, respectively. When the leaves were curves H, I and J are those obtained after intermittent excited at -20"C, a strong A band with weaker B , illumination for 5, 12 and 28 h, respectively. The broken and B, bands were observed, but the Z , band could curve on each profile shows the luminescence observed without actinic illumination. The heights of the C bands not be recognized. The A band was developed during in the profiles of the leaves at early stages of greening greening of etiolated leaves by continuous illuminaunder continuous illumination (curves A to E) or under tion. Either the A band or the group of B , and B, intermittent illumination (curves H to J) were the averages bands was observed as a major band@), depending taken from the data of repeated measurements. on the excitation temperature. It is likely that the energy given by light for excitation is stored in differtreatments with photosynthetic inhibitors have been ent sites in a structure developed by the continuous discussed in the previous paper (Ichikawa et al., illumination. 1975b). The alternative characteristics found between Development of thermoluminescence bands by intermittent illumination of etiolated leaves a) Greening ' b) PAotoac+;vation' ' b)' ' ' Intermittent light
.-
=
(u
I
~
I
c
/
3
Time in h
p9
Figure 2b shows the change of thermoluminescence profile during intermittent illumination of etiolated leaves at intervals of 5 min. In the measurements,
Time in min
Figure 3. Time courses of the development of various thermoluminescence bands during greening under continuous and intermittent illumination (Fig. 3a) and during photoactivation of the intermittently illuminated leaves by continuous red light (Fig. 3b). Solid curves A, B and C indicate the development of the Z,, B , and B2 bands, respectively, during continuous illumination of etiolated leaves with white light, and dotted curves D and E show the development of the 2, band and the group of B1 and B z bands, respectively, during intermittent illumination of etiolated leaves. Curves F, G and H in Fig. 3b show the change in height of the Z,, B I and BZ bands, respectively, during the illumination of intermittently illuminated leaves with continuous red light (680 5 nm, 0.14 J/m2; 14 pW/cm2).
-50
0 +50
-50 0 +50 Temperature in "C
Figure 4. Thermoluminescence profiles of mature (curves A and B) and intermittently illuminated wheat leaves (curves C and D) illuminated for excitation of thermoluminescence at -60°C (curves A and C) and at -20°C (curves B and D), respectively. Broken curve on each profile shows the luminescence observed without actinic illumination, and the heights of the C bands were the averages taken from the data of repeated measurements.
129
Thermoluminescence during chloroplast development
the sample leaves were excited by 1-min illumination at -60°C. As seen from curves H, I and J, the Z , band instead of the B , and B2 bands was developed by intermittent illumination. The Z , band appeared at 5 h and was enhanced during prolonged intermittent illumination up to 28 h. The height of the Z, band on curve J at the end of this intermittent illumination was slightly higher than that in the profile of leaves greened under continuous illumination (curve GI. It is remarkable that the B , and B, bands were not developed at all even after 28 h of the exposure to about 340 flashes, so that the profiles of intermittently illuminated leaves are composed of only two bands (2, and C bands). Curve E in Fig. la shows that the Hill activity was not generated during this intermittent illumination, and curve E in Fig. 3a shows that the B , and B , bands were not developed at all during the illumination. Curve D in the same figure shows the gradual intensification of the Z,, band. The absence of these B , and B, bands in the profile of intermittently illuminated leaves is consistent with the previous observation that the B, and B 2 bands were absent in the profile of the photosystem-I1 particles prepared with Triton X-100 which are devoid of the oxygen-evolving activity (Ichikawa et al., 1975b). The profile of intermittently illuminated leaves measured by excitation at -20°C shows only the C band (curve D in Fig. 4), being different from the profile (curve C) of the same sample measured by excitation at -60°C. This would be expected from the absence of the Z,, band in the profile (curve B) of mature leaves measured by excitation at -20°C. Deuelopmmt of thermaluminescence hands by continuous illurnination of the leaves greened under intermittent illumination
The changes in the profile during continuous illumination of the leaves greened under intermittent illumination are shown in Fig. 5. The B , and B, bands were developed by a brief illumination with weak continuous red light (3 min, 680 t 5 nm, 0.14 J/m2, curve B) and the profile obtained after 10 min of continuous illumination with such light (curve D) showed the B , and B, bands more strongly and distinctly at +25 and +4o”C, respectively. These bands were enhanced during the prolonged illumination with red light up to 20 min to form the strong bands shown on curve E. Further illumination with strong white light (7.5 J/m2) for 120 min did not change the profile appreciably as seen from curve F. The Z, and C bands in the profile of such fully photoactivated leaves were about @80% in height of the same bands found in the profile of mature leaves. This difference in height may arise from the lower chlorophyll content in the intermittently illuminated leaves. The Z,, band, on the contrary, decreased slightly as the B , and B, bands were intensified. The C band in the profiles in the early stage of photoactivation by continuous illumination was considerably higher than
Temperature in
“C
Figure 5. Development of thermoluminescence bands during photoactivation of the latent water-splitting system in intermittently illuminated wheat leaves by continuous light. Curves A, B, C, D and E show the profiles obtained for the leaves after illumination with continuous red light (680 f 5 nm, 0.14 J/m2; 14 pW/cm2) for 0, 3, 5, 10 and 20 min, respectively. Curve F shows the profile obtained after exposure of the similar sample leaves used for the measurement of curve E to strong white light (7.5 J/m2; 750 pW/cm2) for further 120 min. Broken curve on each profile shows the luminescence observed without actinic illumination, and the heights of the C bands were the averages taken from the data of repeated measurements.
that in the profiles of mature leaves, and was lowered in the later stage of the continuous illumination. It may be deduced from this result that the C band is emitted from some defects in pigment proteins or membrane such as “primary thylakoid” developed by intermittent illumination (Sironval et al., 1971). The development of the B1 and B2 bands during continuous illumination proceeded as shown by curves G and H in Fig. 3b, which are similar to the development of the Hill activity shown by curve H in Fig. lb. The fully photoactivated leaves as well as the leaves during photoactivation when excited at - 20°C showed a strong A band with lower B1 and B2 bands as found on the continuous illumination of etiolated leaves (Fig. 4). The Z , band was also lacking in the profiles. It was demonstrated in the present study that the development of the B, and B, bands proceeds in parallel with the development of the Hill activity during the continuous illumination of etiolated leaves or intermittently illuminated leaves. The close correlation between these bands and the Hill activity is also seen from the fact that these bands and the Hill activity were not developed in the leaves during greening under intermittent illumination (curve E in Fig. l a and curve E in Fig. 3a). It may be concluded from these data that there are at least two different mechanisms for the energy storage in photosystem I1 to emit thermoluminescence; one is the mechanism for the emission of the B , and B , bands or the A band, and the other is the mechanism for the Z , band. The structure required for the former mechanism is developed under continuous illumination but not under inter-
130
YORINAOINOUE,TETSUOICHIKAWAand KAZUOSHIBATA
mittent illumination at intervals of minutes. Considering that the activity of DCIP photoreduction with DPC as the electron donor is developed under such intermittent illumination, this structure responsible for the B , and B, bands or the A band may be closely associated with the oxygen-evolving system. The Z,, band which is developed under intermittent illumination therefore, may not be associated with the system for oxygen evolution. Considering that the photoactivation of the latent oxygen-evolution system in algal cells is the activation of the Mn2+ catalyst (Cheniae
and Martin, 1969; 1971; 1972; 1973), the oxidation of the activated Mn2+ catalyst in the oxygen-evolution system to yield its cation for energy storage may be one of the possible mechanisms involved in the luminescence of the B1 and B2 bands or the A band. Acknowledgements-The present study was supported by a research grant on “Photosynthetic reaction centers” from the Ministry of Education and by a grant for the study of “Life sciences” at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho). The technical assistance of Mr. S. Furuta is gratefully acknowledged.
REFERENCES
Arnold, W. (1965) J. Phys. Chem. 69, 788-791. Arnold, W. (1966) Science 154, 1046-1049. Arnold, W., and J . R. Azzi (1968) Proc. Nati. Acnd. Sci. U S . 61, 29-35 Arnold, W., and H. Sherwood (1957) Proc. Natl. Acad. Sci. U.S. 43, 105-114. Arnold, W., and H. Sherwood (1959) J . Phys. Chem. 63, 2 4 . Boardman, N . K., J. M. Anderson, A. Kahn, S. W. Thorne and T. E. Trcfry (1970) In Autonomy arid Biogenesis of’ Mitochondria and Chloroplasts, (Edited by N. K. Boardman, A. W. Linnane and R. M. Smillie) pp. 7&84, North-Holland, Amsterdam. Boardman, N. K., J. M. Anderson, R. G. Hiller, A. Kahn, P. G. Roughan, T. E. Treffry and S. W. Thorne (1971) In Proc. 2nd Intern. Congr. on Photosyn., (Edited by G. Forti, M. Avron and A. Melandri), pp. 2265-2286, W. Junk N.V. The Hague. Cheniae, G. M., and I. F. Martin (1969) Plant Physiol. 44, 351-360. Cheniae, G . M., and I. F. Martin (1971) Biochim. Biophys. Acta 253. 167-181. Cheniae, G . M., and I. F. Martin (1972) Plant Physiol. 50. 87-94. Cheniae, G. M., and I. F. Martin (1973) Photocltem. Photobiol. 11. 441-459. Dujardin, E. Y., de Kouchkovsky and C. Sironval (1970) Photosyrttheticn 4. 223-227. Ichikawa, T., Y. Inoue and K. Shibata (1975a) Plant Sci. Letters 4, 369-376. Ichikawa. T . , Y. Inouc and K. Shibata (397Sb) Biochim. Bioph.ys. Acta. lnoue, Y . (1975) Biochim. Biophys. Actu 396, 402-413. Inoue. Y., T. Ichikawa, Y. Kobayashi and K. Shibata (1974a) In Proc. 3rd Intern. Congr. on Photosyn., (Edited hv M. Avron), pp. 1833-1840, Elsevier, Amsterdam. Inoue, Y., Y. Kobayashi, E. Sakamoto and K. Shibata (1974b) Physiol. Plantarum. 32, 228 232. Inoue, Y., Y. Kobayashi, E. Sakamoto and K. Shibata (1975) Plant Cell Physiol. 16, 687-695. Lurie, S., and W . Bertsch (1974a) Biochim. Biophys. Acta 357, 420-428. Lurie, S., and W. Bertsch (1974b) Biochim. Biophys. Actu 357, 429-438. Michel, J. M., and C. Sironval (1972) FEBS Letters 27, 231-234. Ogawa, T., Y. Inoue, M. Kitajima and K. Shibata (1973) Photochem. Photobiol. 18, 229-235. Phun-Nhu-Hung, S., B. Houlier and A. Moyse (1970) 2. Pjanzenphysiol. 62, 245-248. Radmer, R., and G. M. Cheniae (1971) Biochim. Biophys. Arta 253, 182-186. Remy, R. (1973) Photochem. Photobiol. 18, 409-416. Remy, R., S. Phung-Nhu-Hung and A. Moyse (1972) Physiol. Veg. 10, 269-290. Rubin, A. B., and P. S. Vendiktov (1969) Biofiz. 14, 105-109. Sane, P. V., V. G . Tatake and T. S. Desai (1974) FEBS Letters 45, 29&294. Shuvalov, V. A., and F. F. Litvin (1969) Mol. Biol. USSR 3, 59-73. Sironval, C., J. M. Michel, R. Bronchart and E. Dujardin (1971) In Progress in Photosynrhetic Research, (Edited by H. Metzner), pp. 47-54, H. Laupp, Jr., Tiibingen. Strasser, R., and C. Sironval (1972) FEBS Letters 28, 56-59.
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DEVELOPMENT OF THERMOLUMINESCENCE BANDS DURING GREENING OF WHEAT LEAVES UNDER CONTINUOUS AND INTERMITTENT ILLUMINATION YORMAOINOUE, TETSUOICHIKAWAand KAZUOSHIBATA Laboratory of Plant Physiology, The Institute of Physical and Chemical Research, Wako-shi, Saitama 351, Japan
(Received 7 July 1975; accepted 21 October 1975) Abstract-Mature wheat leaves excited by 1-min illumination at a low temperature of -60°C showed five thermoluminescence bands at -45, -10, +25, +40 and +55"C (denoted as Z,, A , B,, B , and C bands, respectively). The development of these bands during greening of etiolated wheat leaves under continuous and intermittent illumination was investigated, and the following results were obtained. (1) Etiolated leaves showed only the C band. When these leaves were greened under continuous light, the B , and B2 bands appeared at 3 h and the Z, band appeared at 10 h. The B , and 8 , bands were intensified during prolonged greening under continuous illumination to be the strong bands observed for mature leaves. The A band and the group of B , and B , bands were alternative: Similar experiments by excitation of thermoluminescence at - 20°C showed the development of the A band instead of these B , and B , bands. (2)When the etiolated leaves were greened under 5-min dark), the 2, band first appeared after 5 h of illumination intermittent illumination (1-ms light (60 flashes) and was gradually intensified during further illumination with 340 flashes but, interestingly, neither the B , nor the B , band appeared even after 2428 h of illumination with 28Cb340 flashes. (3) On exposure of such leaves greened under intermittent illumination to continuous light, the B , and B , bands were rapidly developed. The appearance of these bands was accompanied by the generation of the Hill activity (DCIP photoreduction with water as electron donor). (4)These results were discussed in relation to the previous study on photoactivation of the latent water-splitting system accumulated in the leaves greened under intermittent illurnination. It was deduced that the structure responsible for the A band or the group of B , and B , bands is involved in the evolution of oxygen in chloroplasts, and probably involves cations of the MnZ+catalyst generated by the action of light.
+
cence variations and delayed light emission (PhungNhu-Hung et al., 1970; Dujardin et al., 1970). Recent The development of photosynthetic apparatus in studies by Remy (1973) and by Sironval and his the plastids of angiosperms depends on light. Illuco-workers (Sironval et al., 1971; Michel and Sironmination of etiolated leaves with strong continuous Val, 1972; Strasser and Sironval, 1972) elucidated that light converts protochlorophyllide to chlorophyll a, the water-splitting system is not functional in these which triggers the biosynthesis of various pigments chloroplasts, while the reaction centers of both photoand proteins necessary for membrane formation. The systems have been developed almost completely. In electron transport from water to a Hill oxidant via fact, exposure of such intermittently illuminated photosystem I1 becomes observable after several h of leaves to continuous light rapidly induced the Hill continuous illumination (Virgin, 1972; Remy e t al., activity of DCIP photoreduction with water as elec1972; Ogawa et al., 1973), and all of the photosynthetron donor (Remy, 1973; Inoue et al., 1974b; Inoue tic activities are developed in 1@15 h (Boardman et et al., 1975) and the activation was accompanied by ul., 1970; Boardman et al., 1971). a marked increase of delayed fluorescence (Ichikawa Under intermittent illumination with flashes at long et a!., 197$a). Kinetic analysis of these photoactivaintervals of several min or longer, however, the development proceeds differently. The chloroplasts in such tion and delayed fluorescence enhancement by means of multiple flashes at short intervals of the order of intermittently illuminated leaves are capable of bringing about photosystem-I reactions and DCIP seconds indicated that the process involves three con(2,6-dichlorophenolindophenol)*photoreduction with secutive photoreactions (Inoue, 1975). The photoactian artificial electron donor, DPC (diphenylcarba- vation of the latent water-splitting system in higher zide)*, but incapable of bringing about some of pho- plant leaves seems to be similar in mechanism to the tosystem-I1 reactions such as oxygen evolution, DCIP photoactivation of MnZ+catalyst in Mn2+-deficient photoreduction with water as electron donor, fluores- algal cells studied by Cheniae and Martin (1969; 1971; 1972; 1973)and by Radmer and Cheniae (1971). The thermoluminescence from plant materials, which was first Observed by Arnold and his co* Abbreviations: DCIP, 2,6-dichlorophenolindophenol; DPC, diphenylcarbazide. workers (Arnold and Shenvood, 1957; 1959; Arnold, INTRODUCTION
125
126
YORINAOINOUE,TETSUO ICHIKAWA and KAZUOSHIBATA
1966) after the discovery of delayed emission by Strehler and Arnold (1951), has been interpreted as resulting from recombination of electrons and positive holes at the reaction center of photosystem I1 in chloroplasts (Arnold, 1965; Arnold and Azzi, 1968; Rubin and Venediktov, 1969; Shuvalov and Litvin, 1969; Lurk and Bertsch, 1974a, 1974b). Previous measurements (Inoue et al., 1974a) by a sensitive photo-electron counting method of mature leaves excited a t - 60°C showed five thermoluminescence bands emitted at -45, - 10, +25, +40 and +55"C, and these bands were denoted as Z,, A , B , , B 2 and C bands, respectively, according to our previous assignments for designation (Ichikawa et a!., 1975b) based on the nomenclature by Arnold (Arnold and Azzi, 1968) with partial modification. In the present study, we have investigated the thermoluminescence of wheat leaves being greened under continuous and intermittent illumination in order to elucidate the process of development of these bands. The courses of development of various bands were compared with the development of the photosystem-I1 activity measured for similar wheat leaves being activated under the same conditions. MATERIALS AND METHODS
Wheat seeds (Triticum aestiuum L.) were germinated and grown on moist filter paper in darkness at 24 & 1°C. Seven-day old etiolated leaves were harvested and cut into segments 3.5 cm long from apexes and spread on moist filter paper to be illuminated for greening with continuous light from white fluorescent lamps or with intermittent light (I ms light + 5 min dark) from electronic Xe strobes. Intensities of continuous and intermittent light beams were 7.5 J/m2 (750 pW/cm2) and 15 J/m2 (1.5 x lo4 ergs/cm2) per flash, respectively. The greening was continued for 24-28 h. The intermittently illuminated leaves obtained after exposure to 29Cb340 flashes were further subjected to photoactivation of their latent water-splitting system by continuous red light (680 & 5 nm, 0.14 Jim') for 1Cb30 min. Light energy from various light sources were measured with a Kipp & Zonen thermopile (model E-2) or with a Quantronics thermocouple (model 500). The green leaves illuminated under various light conditions were kept in darkness at room temperature for more than 20 min before use. Measurement of thermoluminescence. Thermoluminescence from intact leaves was measured by the method reported previously (Inoue et al., 1974) which is briefly described below. Ten segments of wheat leaves harvested at various stages of greening were arranged close together on moist filter paper and sandwiched between a heater and a glass plate to be mounted on an aluminum plate. The sample leaves were, then, cooled to -60°C in a Dewar bottle with an end of the aluminum holder dipped in cold nitrogen gas. The temperature of the leaves was monitored with a Cu-constantan thermocouple inserted between the sample and the glass plate. The leaves at - 60°C were then illuminated with red light longer than 630 nm (J/m2) for 1 rnin and, then, rapidly cooled down to - 196°C by dipping the holder into liquid N,. In some experiments, leaves were illuminated at a higher temperature of -20°C. The sample was transferred with the Dewar bottle to a housing of a photon-counting device (Jasco model KC-200) equipped with a 30 Hz mechanical chopper and a redsensitive photomultiplier (EM1 9659QB). The leaves were
heated slowly at a rate of 0.5"C/s and the photons emitted from the leaves during heating were counted through a red glass filter (VR-63, Toshiba Kasei Co.). The digital photon-count in every 32 Hz was converted to an analogue signal and recorded against temperature on an X Y recorder. Activity measurements and chlorophyll assay. Chloroplasts for the measurement of the Hill activity were prepared from the sample leaves with 0.5 M Tricine buffer (pH 7.5) containing 0.4 M sucrose and 0.01 M MgCI2. The activity of DCIP photoreduction with water or DPC as electron donor and the chlorophyll concentration in the sample suspension was measured by the procedure described previously (Inoue et al., 1974b).
RESULTS AND DISCUSSION
Activity generation and chlorophyll formation by continuous and intermittent illumination
The curves in Fig. l a show the development of photosystem-I1 activities and the formation of chlorophylls during greening of etiolated wheat leaves under continuous and intermittent illumination. Under continuous illumination with strong light, chlorophylls were rapidly synthesized after a few hours of lag period as shown by curve A. Both activities of DCIP photoreduction with water as electron donor (the Hill activity) and DCIP photoreduction with DPC as the donor became observable after 6 h of illumination and rapidly increased between 6 and 12 h to reach saturation after 18 h (curves B and C). Under intermittent illumination with strong Xe flashes, however, chlorophyll was synthesized at a lower rate (about 30% of the rate under continuous illumination) to yield pale green leaves after 28 h of intermittent illumination (curve D). The activity with DPC as electron donor was generated and increased at a slower rate (curve F) but the Hill activity with water as the donor was not generated a t all (curve E) even after 28 h of the intermittent illumination. When such intermittently illuminated leaves were exposed to continuous light, the Hill activity was rapidly generated, as shown by curve H in Fig. lb. This process of photoactivation proceeded first rapidly and then more slowly to reach saturation after about 60 min of continuous illumination with weak red light (680 & 5 nm, 0.14 J/m2). The activity of DCIP photoreduction with DPC as electron donor was also enhanced about 3 times (curve I), while the chlorophyll content did not increase much during this continuous illumination (curve G). These results are in good agreement with the previous observations (Dujardin et al., 1970; Sironval et al., 1971; Michel and Sironval, 1972; Remy, 1973) that the leaves greened under intermittent illumination are devoid of the water-splitting activity whereas the reaction centers for photosystems I and I1 have been developed almost completely, and that the latent watersplitting system accumulated in the intermittently illuminated leaves is activated by exposure to continuous light.
Thermoluminescence during chloroplast development
-
c
,
I
,
Development of' thermoluminescence batids hy tinuous illumination of etiolated leaves
I
,
a) Greening
b) Photoactivation
Time in h
Time in min
Figure l.(a) Chlorophyll formation (curves A and D) and the development of the activities of DCIP photoreduction with water (curves B and E) and DPC (curves C and F) as electron donor during greening under continuous (solid curves) and intermittent (dotted curves) illumination. Excised leaves of 7-day old etiolated wheat seedlings were exposed to continuous white light (7.5 J/m2; 750 pW/cm2) from fluorescent lamps or to intermittent light (1 ms light + 5 min dark) from electronic Xe flashes (15 J/m2; I .5 x lo4 ergs/cm*/flash). Photosystem-I1 activities measured with chloroplasts isolated from the leaves at various stages of greening are expressed on chlorophyll basis. (b) Generation of the activity of DCIP photoreduction with water (curve H) or DPC (curve I) as electron donor during photoactivation. Intermittently illuminated leaves obtained after exposure to 340 flashes (28 h) were further exposed to continuous red light (680 5 nm, 0.14 Jimz; 14 pW/cm2) in order to activate the latent watersplitting system. The measurements of photosystem-I1 activities were carried out in the same manner as in Fig. la.
+
I27 COIZ-
Figure 2a shows the change of thermoluminescence profile of etiolated wheat leaves during greening under continuous illumination. The temperature for actinic illumination of leaves for the measurement was -60°C. The profile of etiolated leaves (curve A) showed a single band around +55"C. Unlike othcr bands, the C band was observable without actinic illumination for all of tthe sample leaves as shown by broken curves in Fig. 2. The height of the C band fluctuated considerably depending on freezing conditions, but the height without actinic illurnination was generally higher than that after the illumination. Considering these, it seems likely that this band is not concerned with photosynthetic activities. The profile of fully greened leaves (curve G) showed three distinct luminescence peaks. The peaks around -45 and +55"C were denoted previously as Z, and C band, respectively, and the middle peak around + 30°C with a shoulder around 40°C are a composite of B1 and B, bands. The assignment for designation of these thermoluminescence bands used in the present study was mainly based on the nomenclature by Arnold (Arnold and Azzi, 1968) and the correspondence between these bands is summarized in Table 1. together with the data obtained by previous investigators. The emission temperature of the Z,. band was found to be variable depending on the illumination temperature, whereas those of the other bands were constant, being independent of the illumination temperature. The Z , band is a newly found one and some of its characteristics including its response to the
+
Table 1. Correspondence of thermoluminescence bands in the present study to those reported previously by various investigators Author
Thermoluminescence bands
z
Arnold L A z z i (-
(1968)
155 o c )
B (t3OoC)
A (-6°C)
C (f550C) n
Shuvalov
&
Litvin
(1969)
Component I1 (-160OC) 1, (Component I)
Component I11 (-15'C)
Sane et al.
peak
(19n)-
(118'K)
peak
Component V
(t2OOC)
peak 1 (-10°C)
Lurie L Bertsch (1974a)
Component Iv
*
(254OK)
peak 2 (+3OoC)
peak
*
(257'K)
peak 3 (+40°C)
__
peak
peak
(290°K:
(320'Kj
pi
Inoue g &. (present paper) L
1
Ichikawa et al. (1975b)- -
Zt
(-160'C)
+
t
zv (variable)
r a l t e r n a t i v e-
L
A (-1OOC)
B1 (t25'C)
B2 (t4OoC)
C (+550C)
* These temperatures were read on the glow curve in Sane's paper. t The relationship between the Z and the Z , bands has been discussed previously (Ichikawa et al., 1975b). 5 Either the A band or the group of B, and B2 bands was observed in wheat leaves depending on the excitation temperature. I/ This component is a delayed emission with life time of 5 ms observed below 0°C. 71 Another component of delayed emission with life time of 1@15 s observed above 0°C.
YORINAO INOUE, TETSUOICHIKAWAand KAZUOSHIBATA
128
the A band and the group of B , and B, bands will be discussed later. Curves B, C, D, E and F show light the process of development of these luminescence bands during continuous illumination. The B , and B2 bands were progressively enhanced until 28 h to be the strong bands shown on curve G, whereas the Z , band was not much intensified. The time courses of the appearance of these luminescence bands are summarized in Fig. 3a. Curves B and C show the development of the €3, and B, bands, respectively, which proceeded approxi(A ) mately in parallel with the development of the Hill 4 -50 0 +50 activity shown by curve B in Fig. la. On the other Temperature in "C hand, curve A showing the development of the Z,, band increased to a much slighter extent. Figure 2. Development of thermoluminescence bands durThe above profiles were measured for the leaves ing greening under continuous (Fig. 2a) and intermittent (Fig. 2b) illumination. Sample leaves obtained at various excited by the illumination at -60°C. The profiles stages of greening in the experiment of Fig. l a were illu- obtained for similar samples excited at higher temminated at - 60°C for excitation with red light (above 630 peratures are considerably different. Curves A and B nm, 6 J/mz; 600 pW/cm2) for 1 min before the measure- in Fig. 4,which are typical examples for comparison, ment of thermoluminescence. Curves A, B, C, D, E, F and G were the profiles obtained for leaves after continuous are the profiles of mature leaves excited at -60°C illumination for 3, 5, 10, 15 and 28 h, respectively, and and -2o"C, respectively. When the leaves were curves H, I and J are those obtained after intermittent excited at -20"C, a strong A band with weaker B , illumination for 5, 12 and 28 h, respectively. The broken and B, bands were observed, but the Z , band could curve on each profile shows the luminescence observed without actinic illumination. The heights of the C bands not be recognized. The A band was developed during in the profiles of the leaves at early stages of greening greening of etiolated leaves by continuous illuminaunder continuous illumination (curves A to E) or under tion. Either the A band or the group of B , and B, intermittent illumination (curves H to J) were the averages bands was observed as a major band@), depending taken from the data of repeated measurements. on the excitation temperature. It is likely that the energy given by light for excitation is stored in differtreatments with photosynthetic inhibitors have been ent sites in a structure developed by the continuous discussed in the previous paper (Ichikawa et al., illumination. 1975b). The alternative characteristics found between Development of thermoluminescence bands by intermittent illumination of etiolated leaves a) Greening ' b) PAotoac+;vation' ' b)' ' ' Intermittent light
.-
=
(u
I
~
I
c
/
3
Time in h
p9
Figure 2b shows the change of thermoluminescence profile during intermittent illumination of etiolated leaves at intervals of 5 min. In the measurements,
Time in min
Figure 3. Time courses of the development of various thermoluminescence bands during greening under continuous and intermittent illumination (Fig. 3a) and during photoactivation of the intermittently illuminated leaves by continuous red light (Fig. 3b). Solid curves A, B and C indicate the development of the Z,, B , and B2 bands, respectively, during continuous illumination of etiolated leaves with white light, and dotted curves D and E show the development of the 2, band and the group of B1 and B z bands, respectively, during intermittent illumination of etiolated leaves. Curves F, G and H in Fig. 3b show the change in height of the Z,, B I and BZ bands, respectively, during the illumination of intermittently illuminated leaves with continuous red light (680 5 nm, 0.14 J/m2; 14 pW/cm2).
-50
0 +50
-50 0 +50 Temperature in "C
Figure 4. Thermoluminescence profiles of mature (curves A and B) and intermittently illuminated wheat leaves (curves C and D) illuminated for excitation of thermoluminescence at -60°C (curves A and C) and at -20°C (curves B and D), respectively. Broken curve on each profile shows the luminescence observed without actinic illumination, and the heights of the C bands were the averages taken from the data of repeated measurements.
129
Thermoluminescence during chloroplast development
the sample leaves were excited by 1-min illumination at -60°C. As seen from curves H, I and J, the Z , band instead of the B , and B2 bands was developed by intermittent illumination. The Z , band appeared at 5 h and was enhanced during prolonged intermittent illumination up to 28 h. The height of the Z, band on curve J at the end of this intermittent illumination was slightly higher than that in the profile of leaves greened under continuous illumination (curve GI. It is remarkable that the B , and B, bands were not developed at all even after 28 h of the exposure to about 340 flashes, so that the profiles of intermittently illuminated leaves are composed of only two bands (2, and C bands). Curve E in Fig. la shows that the Hill activity was not generated during this intermittent illumination, and curve E in Fig. 3a shows that the B , and B , bands were not developed at all during the illumination. Curve D in the same figure shows the gradual intensification of the Z,, band. The absence of these B , and B, bands in the profile of intermittently illuminated leaves is consistent with the previous observation that the B, and B 2 bands were absent in the profile of the photosystem-I1 particles prepared with Triton X-100 which are devoid of the oxygen-evolving activity (Ichikawa et al., 1975b). The profile of intermittently illuminated leaves measured by excitation at -20°C shows only the C band (curve D in Fig. 4), being different from the profile (curve C) of the same sample measured by excitation at -60°C. This would be expected from the absence of the Z,, band in the profile (curve B) of mature leaves measured by excitation at -20°C. Deuelopmmt of thermaluminescence hands by continuous illurnination of the leaves greened under intermittent illumination
The changes in the profile during continuous illumination of the leaves greened under intermittent illumination are shown in Fig. 5. The B , and B, bands were developed by a brief illumination with weak continuous red light (3 min, 680 t 5 nm, 0.14 J/m2, curve B) and the profile obtained after 10 min of continuous illumination with such light (curve D) showed the B , and B, bands more strongly and distinctly at +25 and +4o”C, respectively. These bands were enhanced during the prolonged illumination with red light up to 20 min to form the strong bands shown on curve E. Further illumination with strong white light (7.5 J/m2) for 120 min did not change the profile appreciably as seen from curve F. The Z, and C bands in the profile of such fully photoactivated leaves were about @80% in height of the same bands found in the profile of mature leaves. This difference in height may arise from the lower chlorophyll content in the intermittently illuminated leaves. The Z,, band, on the contrary, decreased slightly as the B , and B, bands were intensified. The C band in the profiles in the early stage of photoactivation by continuous illumination was considerably higher than
Temperature in
“C
Figure 5. Development of thermoluminescence bands during photoactivation of the latent water-splitting system in intermittently illuminated wheat leaves by continuous light. Curves A, B, C, D and E show the profiles obtained for the leaves after illumination with continuous red light (680 f 5 nm, 0.14 J/m2; 14 pW/cm2) for 0, 3, 5, 10 and 20 min, respectively. Curve F shows the profile obtained after exposure of the similar sample leaves used for the measurement of curve E to strong white light (7.5 J/m2; 750 pW/cm2) for further 120 min. Broken curve on each profile shows the luminescence observed without actinic illumination, and the heights of the C bands were the averages taken from the data of repeated measurements.
that in the profiles of mature leaves, and was lowered in the later stage of the continuous illumination. It may be deduced from this result that the C band is emitted from some defects in pigment proteins or membrane such as “primary thylakoid” developed by intermittent illumination (Sironval et al., 1971). The development of the B1 and B2 bands during continuous illumination proceeded as shown by curves G and H in Fig. 3b, which are similar to the development of the Hill activity shown by curve H in Fig. lb. The fully photoactivated leaves as well as the leaves during photoactivation when excited at - 20°C showed a strong A band with lower B1 and B2 bands as found on the continuous illumination of etiolated leaves (Fig. 4). The Z , band was also lacking in the profiles. It was demonstrated in the present study that the development of the B, and B, bands proceeds in parallel with the development of the Hill activity during the continuous illumination of etiolated leaves or intermittently illuminated leaves. The close correlation between these bands and the Hill activity is also seen from the fact that these bands and the Hill activity were not developed in the leaves during greening under intermittent illumination (curve E in Fig. l a and curve E in Fig. 3a). It may be concluded from these data that there are at least two different mechanisms for the energy storage in photosystem I1 to emit thermoluminescence; one is the mechanism for the emission of the B , and B , bands or the A band, and the other is the mechanism for the Z , band. The structure required for the former mechanism is developed under continuous illumination but not under inter-
130
YORINAOINOUE,TETSUOICHIKAWAand KAZUOSHIBATA
mittent illumination at intervals of minutes. Considering that the activity of DCIP photoreduction with DPC as the electron donor is developed under such intermittent illumination, this structure responsible for the B , and B, bands or the A band may be closely associated with the oxygen-evolving system. The Z,, band which is developed under intermittent illumination therefore, may not be associated with the system for oxygen evolution. Considering that the photoactivation of the latent oxygen-evolution system in algal cells is the activation of the Mn2+ catalyst (Cheniae
and Martin, 1969; 1971; 1972; 1973), the oxidation of the activated Mn2+ catalyst in the oxygen-evolution system to yield its cation for energy storage may be one of the possible mechanisms involved in the luminescence of the B1 and B2 bands or the A band. Acknowledgements-The present study was supported by a research grant on “Photosynthetic reaction centers” from the Ministry of Education and by a grant for the study of “Life sciences” at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho). The technical assistance of Mr. S. Furuta is gratefully acknowledged.
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