Different photoactive states of the red phytochrome form in the cells of etiolated pea and oat seedlings

Journal of Photochemistry and Photobiology, B: Biology, 5 (1990) 197 - 217 197

DIFFERENT PHOTOACTIVE STATES OF THE RED PHYTOCHROME FORM IN THE CELLS OF ETIOLATED PEA AND OAT SEEDLINGS

V. A. SINESHCHEKOV

Biology Faculty, M. V. Lomonosov Moscow State University, Moscow (U.S.S.R.)

A. V. SINESHCHEKOV

K. A. Timiryazev Institute of Plant Physiology of the U.S.S.R. Academy of Sciences, Moscow (U.S.S.R.)

(Received January 12, 1989; accepted July 27, 1989)

Keywords. Phytochrome, in ho, fluorescence, photoconversion, temperature dependence, pea, oat.

Abbreviations

FR, R Pfr, pr

pr1, Pr2, Pr3

J’I - F5

AF variable part of fluorescence

far-red and red light far-red and red form of phytochrome different native states of the red phytochrome form in the cell wavelengths of the actinic, exciting and measured light quantum yield of fluorescence the initial level of P, fluorescence intensity when all the pigment is in the P, state fluorescence levels under various light adaptation conditions

Summary

A study was made of the fluorescence and phototransformations of the red phytochrome form (P,) in etiolated plants in the temperature interval 85 - 293 K, depending on the species (pea, oat), age, site of pigment localization (stems, roots), and the effect of freezing and thawing. These factors had only a slight influence on the spectral properties (position and form of the low-temperature fluorescence spectrum) of the pigment, on its fluorescence quantum yield and the extent of intensity changes connected with the transformation of P, into the far-red form (Pf,).

However, other parameters such as the temperature dependence of the fluorescence yield, the value of the activation energy of its decay and the

Elsevier Sequoia/Printed in The Netherlands

198

extent of reversible and irreversible intensity changes caused by phototransformations of P, into the photoproduct (lumi-R) at low temperature show substantial variation with the above-mentioned factors. Three types of phytochrome centers are present in the cell (Prl, PrZ, Pr3), similar in their spectral properties (isochromic), but differing sharply in the value of the photoreaction activation energy which is 11 f 1, 5 f 0.5 and 38 f 4 kJ mol-‘, respectively. The content of these centers in the cell varies depending on the species, environmental, developmental factors and pretreatment applied this being associated with the transformation of the first two centers into the most stable third one.

1. Introduction

The phytochrome system serves as the principal photoregulatory system in plants, mediating perception of information on the presence or absence of light, its qualitative composition and intensity, and duration of dark and light periods, thus determining the general strategy for light development in plants (photomorphogenesis). Its action is based on the phototransformation of the initial red phytochrome form (Pr) into the physiologically active far-red one (Pf,). It has been suggested that a single type of the pigment molecules initiates all the functional manifestations of the phytochrome system (conception of the multiple phytochrome action [11)-

Of late, however, evidences have been found that the phytochrome system may be much more complicated than formerly considered. Thus, with the aid of the immunochemical method, differences between the phytochrome pools in the etiolated and green plants have been demonstrated. It was also found that in the etiolated seedlings, phytochrome was present in two states-labile and stable. It is supposed that the differences between the phytochrome pools may be connected either with structural changes in the pigment molecule or with its different cellular localization (see ref. 2 for review).

Earlier, our investigations demonstrated the existence of phytochrome fluorescence in the cell [3,4]. A sensitive fluorescence method [5] allowed the detection of three species of P, in the etiolated tissues similar in their fluorescence and excitation (absorption) spectra, but different in their photochemical properties [ 3 - S].

‘In this work, we present results on fluorescence investigations of the three P, centers in the cells of etiolated pea and oat seedlings as examples of dicotyledonous and monocotyledonous plants, respectively. A number of photophysical and photochemical parameters of these centers was determined and the dependence of these parameters on such factors as plant species and age, localization in organs and influence of cold treatment was established. The transformation of two of the P, types into the third one was followed.

199

2. Experimental details

2.1. Materials and methods Etiolated pea (Pisum satiuum L., var. Triumph) and oat (Arena satiua

L., var. the Bug) seedlings aged from 2 days to 2 weeks were used in this work. They were grown in a thermostat in the dark at 26 “C! on filter paper moistened with tap water. Samples for measurement were stems and roots of pea and oat coleoptiles which contained practically no photochlorophyll (-ide) or chlorophyll. The samples were placed into a transparent plastic cuvette, 5 mm thick; of a cryostat - a hollow metal cylinder placed into a transparent glass Dewar flask. The desired sample temperature (within the range of 85 - 293 K) was maintained by pouring liquid nitrogen into the cryostat; the temperature was recorded by a thermocouple with a precision of +2 K.

The fluorescence spectra and their intensity were measured on a spec- trofluorimeter assembled on the basis of two double diffraction monochro- mators commonly used for recording Raman spectra - DFS-12 and DFS-24 (aperture 1:5). A xenon arc lamp of 500 W, type DKsSH-500, and a pho- tomultiplier, FEU-79, served as the source and detector of light respectively. The spectral slit width was 2 nm, the intensity of the exciting light being approximately 0.1 W m -2. The following served as the actinic light sources: (1) microscope light sources of 01-28 type with an iodine incandescent lamp of 90 W in combination with interference and cut-off filters and a diffraction monochromatore of the MDR-2 type (aperture 1:2); (2) a He-Ne laser of the LG-56 type (h, = 632.8 nm). The intensity of the actinic light was within the range 10 - 100 W m-2. A more detailed description of the methods is presented in refs. 5 and 6.

2.2. Measurement procedure The main fluorescence parameters characterising the properties of

P, were the position of the maximum in the low-temperature fluorescence spectrum, the temperature dependence of the fluorescence quantum yield (Pi?, and the variation of the fluorescence intensity connected with the phototransformation of P, at room and low temperatures. The procedure for their determination included the following operations: after the sample was placed under dim green light into a cuvette of a cryostat, it was illuminated with saturating far-red (FR) light (X, = 720 nm) at room temperature for 3 - 5 min to transform the trace amounts of Prr, which could have been formed during the sample preparation, into the initial P,; the sample was frozen in darkness to 85 K. In this state the intensity (F, level) and the P, fluorescence spectrum were recorded (Fig. 1, spectrum 1) under monochro- matic light excitation (X, = 650 nm or 610 nm) of very low intensity (0.1 W mw2) which induced practically no photochemical changes in the sample during the measurement (5 min). Following this, the temperature dependence of the fluorescence intensity in the 685 nm maximum (and thus relative quantum yield) was measured point by point during the thawing

-

(4 WAVELENGTH, “m (b) WAVELENGTH, nm

Fig. 1. Low-temperature (85 K) fluorescence spectra of P, in the cells of (a) 7-day-old etiolated pea seedling stems and (b) oat coleoptiles (& = 650 nm, I = 0.1 W m-*). (1) Sample illuminated at 293 K with saturating actinic FR light (ha = 735 nm, 10 - 100 W rnez, 3 min) and frozen to 85 K in darkness; (2) the same as (l), but after illumination with saturating R light (A = 670 nm) at 85 K; (3) the same as (2), but after thawing to 273 K, illumination with saturating R (ha = 650 nm) and freezing in darkness to 85 K; (4) difference spectrum: spectrum 2 normalized to spectrum 1 in the maximum (not presented in the figure) minus spectrum 1.

of the sample, switching on the exciting light for a short time so as to avoid its influence on the sample (Fig. 2a). (It is assumed that (PF is to a first approximation proportional to the P, fluorescence intensity in the maximum, because the width of the fluorescence band did not increase much with temperature, i.e. 1.2 times on heating from 85 to 200 K, and the error connected with this is compensated for by the increase of the absorption in the region of excitation (1, = 650 nm) due to the corresponding widening of the absorption spectrum with temperature.) Without bringing the sample to full thawing (temperature less than or equal to 273 K) it was frozen again to 85 K, and then the fluorescence spectrum was recorded, which coincided with the initial one by its shape and amplitude (F, level) (not presented in Fig. 1). This indicated that the fluorescence spectra and the temperature dependence of the fluorescence intensity were not affected by the exciting light during their recording.

At 85 K the sample was adapted for 3 - 5 min to the saturating R light of A, = 670 nm. As a result, a reduction of 15 - 35% was observed in the fluorescence amplitude at the maximum (Fr level) and a change of the emis-

201

(8)

270 220 170 120 4.2 5.3 6.4 7.4 0.5 9.5

TEMPERATURE, K INVERSE TE~vIPERATUREX~~~

Fig. 2. Temperature dependence of the fluorescence quantum yield (A,= 650 nm, &, = 686 nm) of P, of the 7-day-old etiolated seedlings of (1) pea stems and (2) oat coleoptiles: (A) @ us. temperature; (B) ln(l/k& - 1 - kd) us. l/T (kd = kdkf), where kd is the rate constant of the temperature-independent excitation degradation and kf is the radiative rate constant. Quantum yield, (PF, at 85 K, in accordance with ref. 6, is assumed to be equal to 0.25 and kd for pea and oat is equal to 2 and 3, respectively (B, curves 1 and 2).

sion spectrum appeared i.e. a growth of a fluorescence intensity in the region of 708 - 712 nm (Fig. 1, spectra 2 and 4, and Fig. 3) coincided, as demonstrated earlier [6,7], with the appearance of the photoproduct lumi-R.

Subsequent illumination of the sample with saturating FR light (h, = 696 nm) for 2 min at 85 K in the region of the photoproduct (lumi-R) absorption partially reversed the action of red light with the establishment of a new fluorescence level (F2 level); the irreversible part of the fluorescence change (ri = (F, - F,)/F,, = AF,/F,) reached 25 - 30%, whereas the reversible one (yz = (F, - F,)/F, = AF,/F,) was 5 - 7%, in agreement with our previous results [6 - 81. The sample was then alternatively illuminated with light of 670 and 696 nm and the fluorescence level was recorded as a function of a gradual increase of temperature (Fig. 3, curves 1, 2). In this manner, the temperature dependence of the fluorescence intensity of FR and R light adapted samples was obtained (Fig. 3, curves 1 and 3 respectively). The difference between the curves (yz = AFJF,) reflects the changes in the fluorescence connected with the photoreversible P, phototransformations at each temperature in the interval 85 - 273 K.

After thawing of the sample to less than or equal to 273 K and adaptation at this temperature to saturating R light, X, = 650 nm, the sample was frozen in darkness to 85 K. The intensity (F3 level) and the fluorescence spectrum of P, in the photostationary state with Pf, established under R light at 273 K were measured (Fig. 1, spectrum 3). The spectrum was similar to the initial one but the fluorescence intensity decreased considerably (4 - 5 times), whereas the variable fluorescence (y3 = (F, - F&F,-, = AFJF,)

I

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140 190 240 290 TEMPERATURE, K

(b) TEMPERATURE, K

Fig. 3. Changes in the fluorescence intensity (& = 650 nm, & = 686 nm) of P, in the cells of 7-day-old etiolated seedlings of (a) pea stems and (b) oat coleoptiles connected with its photo-induced transformation under the action of FR (695 nm) and R (670 nm) at 85 K (insert), and in the temperature range 85 - 293 K. Points on curves 1 and 2 were recorded alternatively after the action of FR and R; at temperatures higher than 220 K (1) FR = 725 nm, (2) R = 650 nm.

203

rose to 0.75 - 0.85. Finally, the fluorescence intensity was measured following full thawing of the sample to room temperature, after alternative illumination of the sample with R light (F4 level) and FR light, X, = 735 nm (F, level). The P, variable fluorescence at room temperature (y4 = (F, - F& F5 = AF4/F5) was about 0.6 - 0.7. Since the decrease in fluorescence photo- induced at room temperature, y3, is similar to the extent of P, + Prr transformation in vitro [ 9, lo], and the P, fluorescence spectrum after the transformation into Pr, (Fig. 1, spectrum 4) coincides in shape and position with the P, spectrum before the phototransformation (Fig. 1, spectrum l), it can be concluded that practically all the fluorescence measured in the 686 nm region belongs to P,, and that the fluorescence intensity changes are connected with the P, transformation and with the variation with temperature of L&.

From the temperature dependence of the fluorescence intensity, a determination was made of the fluorescence decay activation parameters, activation energy (E,) and A = In h,, representing it in Arrhenius coordinates (ln(l/q, - 1 - kd) us. l/T) in accordance with the method in ref. 11 where lz,, = k,,/kr is the rate constant of the temperature-dependent dissipation of excitation energy, normalized to the fluorescence (or radiative) rate constant (kf), lzd = k&f - the rate constant of the temperature- independent loss of excitation energy (kd), normalized to the fluorescence rate constant (k+) (Fig. 2b).

A number of fluorescence characteristics were obtained from this evaluation which reflect photophysical and photochemical properties of P, and their changes with various factors (see Table 1). Several (from 3 to 10) series of measurements on different samples were conducted; deviations of experimental parameters from the mean values presented in Table 1 did not exceed +lO% - 15%.

3. Results

3.1. Constant fluorescence The fluorescence of P, in the cells of 5 - 7-day-old etiolated pea and oat

seedlings is characterised by the following parameters. The maximum in the emission spectra at 85 K is at 686 nm in pea and 685 nm in oat (Fig. 1). A temperature increase to 293 K resulted in a decrease of 30 - 40 times in fluorescence intensity at the maximum for both species (Fig. 2, curves 1,2); the intensity ratio at 220 K and 85 K (o = (pF220 K/1&85 K) was 0.17 for pea and 0.16 for oat (Table 1). Assuming a fluorescence quantum yield (PF = 0.25 at 85 K in accordance with evaluations carried out earlier [3 - 51, the plots ln(l/$& - 1 - kd) us. l/T for pea and oat are linear for id = 2.5 and k, = 1.5 respectively with a breaking point in the region 240 - 260 K. It should be noted that the precise determination of (PF is of im- portance for the evaluation of fid and, correspondingly, of A = In &,,; it has, however, no influence on the determination of E, (see ref. 11).

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For pea stems, E, for the fluorescence decay in F. state, when all the pigment is in the P, state, averaged 4.6 and 26.9 kJ mol-’ for the 85 - 220 K and 220 - 293 K intervals, respectively. For oat, E, was, 4.3 and 24.1 kJ mol-‘, respectively for both intervals. A was 5.3 and 17.9, and 5.1 and 15.0, respectively (see Table 1).

After the adaptation of stems to light of Aa = 670 nm and then ha2 = 696 nm (Fig. 4, curve 2), by which an irreversible reduction of the fluorescence intensity connected with the phototransformations of a part of P, into a photoproduct (F2 level) takes place, a change is observed in the character of the temperature dependence of the fluorescence yield: cr = 0.17 and 0.20 for curves 1 and 2. As follows from Fig. 4, with the rise of temperature from 85 K, curves 1 and 2 approach each other and practically coincide at 170 K. E, and A in the 85 - 220 K interval decreased upon light adaptation causing a transition from level F,, to Fz: E, = 4.8 - 5.1, A = 5.2 - 5.7 for Fo, and E,= 2.7 - 3.7, A = 3.2 - 4.0 for F,.

The variable fluorescence component obtained as a difference of curves 1 and 2, Fig. 4 (AF = F,, - F,) is characterised by a steeper temperature dependence of its intensity than that of the fluorescence in the states F. and F,: at 170 K the fluorescence intensity of AF approaches zero. E, of the variable fluorescence decay calculated from curve 4 in Fig. 4 (insert) is 10.6 kJ mol-‘; A = 12, fid = 2.

inverse temperaturexl0

80 130 180 230 280

TEMPERATURE, K

Fig. 4. Temperature dependence of the fluorescence intensity (h, = 650 nm, A,,,= 686 nm) of P, in the cells of stems of etiolated 7-day-old pea seedlings. (1) Sample adapted to FR light, Aa = 735 nm, at room temperature and frozen in darkness to 85 K; (2) the same sample after illumination at 85 K with saturating R light ha = 670 nm; (3) the difference betw_een curves 1 and 2, multiplied by 2; insert, the same _BS 3, in coordinates ln(l/cpF- 1 - kd) us. l/T (@ is assumed to be equal to 0.25 at 85 K, kd = 2).

206

3.2. Photo-induced fluorescence changes The extent of photo-induced irreversible changes in the fluorescence

intensity, yl, at 85 - 90 K constitutes 0.25 - 0.35 for pea; for oat the values are somewhat lower: 0.13 - 0.16. The reversible changes, however, are comparable for both species and constitute 0.04 - 0.07 and 0.04 respectively at 85 - 90 K. With increasing temperature the extent of reversible changes increases to 0.17 and 0.12, for pea and oat respectively at 130 K. No photoreversible changes in fluorescence within the temperature interval 170 - 220 K are determined for either species: curves 1 and 2 on Fig. 3 in this region coincided. However, changes in P, fluorescence induced by illumination at 170 - 220 K are observed if the fluorescence is recorded after freezing the sample in darkness to 85 K instead of measuring at the temperature at which the photo-adaptation was conducted. The difference in the intensity of the low-temperature (85 K) fluorescence of the sample adapted to FR and R light at 170 - 220 K is 25%. Assuming that there are no changes in the photostationary state between P, and the photoproduct upon cooling the sample down from 170 - 220 to 85 K we can arrive from this experiment at the conclusion (drawn earlier in refs. 6 and 7) that the low-temperature P, fluorescence is non-homogeneous. At temperatures greater than or equal to 170 K the center which is non-photoactive is the one which fluoresces. The emission intensity from the centres capable of low-temperature transformations is very low at these temperatures. This is also evidenced by the differences in the temperature dependence of the fluorescence for the sample in the two states - the one frozen in darkness and the one illuminated at 85 K with actinic R light (Fig. 4).

The extent of changes in fluorescence intensity connected with the P, 2 Pi, phototransformation is similar for pea and oat, 0.80 - 0.85 and 0.65 - 0.77, if the fluorescence is registered at 85 K (ys) and at 293 K (y4) respectively (see Table 1).

3.3. Fluorescence changes with the age of seedling The properties described above reflect the state of phytochrome in

pea and oat seedlings at the maximum concentration which is reached on the 5th to 7th day after sowing. As proved in experiments with pea, some of the measured parameters undergo changes with the seedling age. As follows from Fig. 5a and Table 1, the temperature dependence of the fluorescence intensity displayed by 16day-old seedlings is not as steep ((Y = 0.25) as that of 5 - ‘I-day-old seedlings (a = 0.17). E, within the interval 85 - 220 K is somewhat lower - 4.5 kJ mole1 and 13 kJ mol-l at 220 < T < 293 K for the former. A small decrease in the values of the variable fluorescence of P, at low and room temperatures was observed with age (rI = 0.25; yz = 0.04; y3 = 0.75 - 0.80 for lCday-old seedlings); however, the fluorescence spectrum of P, remained practically unchanged.

3.4. Phy tochrome fluorescence in roots The phytochrome properties depend on its localization in the plant to a

larger extent than on age. As follows from Fig. 5 band Table 1, in pea roots,

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Fig. 5. Low-temperature (85 K) emission spectra (& = 650 nm) (not corrected for spectral sensitivity of the fluorimeter) (insert) and temperature dependence of the fluorescence intensity (& = 650 nm, A, = 686 nm) of P, in the cells of etiolated plants. (a) Stems of (1) 5-day-old and (2) 14-day-old pea seedlings; (b) (1) stems and (2) roots of 5-day-old pea seedlings; (c, d) (1) normal and (2) modified by a freezing-thawing procedure stems of 7-day-old seedlings of (c) pea and (d) oat coleoptiles (the thawed sample remained at room temperature for 5 min). Before freezing all the samples were illuminated for 3 min with saturating FR light (735 min).

an evident change in the character of temperature dependence of the fluorescence yield takes place. The dependence becomes less steep within the low-temperature region (85 - 220 K) (a = 0.43). Within this interval, E, = 1.9 kJ mol-’ is 2 - 2.5 times lower than in the case of P, in stems. On the contrary, in the interval 220 - 293 K the increase of fluorescence intensity with temperature is somewhat steeper, which leads to a larger value of E, = 30.0 kJ mol-r (A = 17.5). There is also a change in the variable fluorescence: the values of the irreversible and reversible fluorescence are much lower, y1 and r2 < 0.04. The extent of the fluorescence changes connected

208

with the phototransformation at room temperature somewhat diminishes, y3 = 0.75. The position (maximum at 684 - 685 nm) and the shape of the fluorescence spectrum undergo minor changes (see Fig. 5a).

3.5. The influence of the freezing-thawing procedure on the fluorescence properties

Effects on the fluorescence properties similar to those connected with the pigment localization (stems and roots) were also observed during the action of freezing and subsequent thawing on pea seedlings. Fig. 5c presents the low-temperature fluorescence spectrum and the temperature dependence of the fluorescence intensity of pea stems frozen once (control sample), and of the same pea stems, which, after freezing, were thawed to 283 K in darkness and then frozen again to 85 K (experimental sample). It is apparent that the procedure of freezing-thawing leads to a slight (1.5 nm) shift in the short wavelength direction without influencing the form of the fluorescence spectra (Fig. 5b). There is only a minor change in the extent of the reversible transformations P, 2 P fr assessed by the value of the variable fluorescence induced by R-FR illumination at room temperature (y3 = 0.81 and 0.74 before and after freezing-thawing, respectively). The total ‘& at 85 K changes only 1.3 times. However, the character of the temperature dependence of pr undergoes considerable changes (Fig. 5b). Thus, in particular in the region of intermediate temperature (170 - 220 K) @ of the experimental and control samples differed by more than twice - (Y = 0.17 and (Y = 0.39. In the plot of ln(l/$$ - 1 - fi,) us. l/T @ dependencies were linear if K, = 2.2 and 2.3 for the experimental and control samples respectively. E, for the two linear regions averaged 4.5 kJ mol-’ and 26.9 kJ mol-’ for the control sample, and 2.5 kJ mol-’ and 36.3 kJ mol-’ for the experimental one, respectively. The values of A were also different for both regions (see Table 1).

There is a sharp fall in the extent of photo-induced irreversible and reversible fluorescence changes at low temperatures (see Table 1). The difference in curves 1 and 2 in Fig. 6 induced by the action of R-FR light is observed only within the temperature ranges approaching the physiological ones (greater than or equal to 220 K). It should be noted that the influence of the freezing-thawing procedure is the stronger the longer the sample is kept at the temperature above 273 K after thawing or the more freezing- thawing cycles were conducted (incomplete thawing (temperatures below or equal to 273 K), as noted, induced no changes in the sample).

Close changes in the fluorescent and photochemical properties to those described above were observed with the action of the freezing-thawing procedure on the coleoptiles of oat seedlings (Fig. 5d). However, as distinct from pea, in oat no increase in E, but rather a reduction of E, from 24.1 to 12.5 kJ mol-’ was observed within the range 220 - 293 K.

The picture obtained with cells of pea roots is different. With the action of freezing-thawing the character of the temperature dependence of the fluorescence remained practically unchanged (o = 0.43 in normal roots and

209

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80 130 180 230 280

TEMPERATURE, K

Fig. 6. Photo-induced changes in the fluorescence intensity (he = 650 nm, hm = 686 nm) of P, in the cells of stems of 7-day-old etiolated pea seedlings subjected to the freezing- thawing procedure (the thawed sample remained at room temperature for 5 min) after the illumination of the sample with (1) FR light (695 nm at 85 - 220 K and 735 nm at temperatures above or equal to 220 K) and (2) R light (670 nm at 85 - 220 K and 650 nm at temperatures above or equal to 220 K). The points on curves 1 and 2 were recorded alternatively in the course of the sample thawing from 85 K to 293 K. Insert: fluorescence spectra (not corrected for spectral sensitivity of the fluorimeter), measured at 85 K after illumination of the sample at room temperature with (1) saturating FR light (735 nm) and (2) R light (650 nm).

(x = 0.45 in roots subjected to freezing and subsequent thawing) as well as the activation parameters of the fluorescence decay and the values that characterise the variable fluorescence.

4. Discussion

The data obtained indicate heterogeneity of the low-temperature fluorescence of P, which could reflect the existence of at least three types of the native pigment centres (Prl, Pr2, Pr3) in the cells of etiolated seedlings. Within the framework of this concept [6 - 81 it is also possible to interpret the variations in the P, fluorescence connected with changes in the physiological conditions of the seedlings. Results of this work permit a more complete characterization of the P, states, and also the determination of their relative content in the cell as follows.

4.1. P,, This state is characterised by the irreversible (or very slowly reversible)

phototransformation into lumi-RI at low (less than or equal to 85 K) temperature. (Our preliminary estimations show that at 85 K the quantum yield of the back phototransformation from lumi-R to P, is approximately

210

20 - 40 times lower for P,i than that for P,& The fluorescence yield of P,, depends more on temperature than the total (Pi? for P, (see Fig. 4). The activation parameters of P,, fluorescence decay are (in pea) E,, = 11 SC 1 kJ mol-‘, A = 12. The extent of the P,i + Pf, transformation is apparently about 0.80 - 0.85, since for the samples containing considerable P,, propor- tion y3 = 0.80 - 0.85 and it decreases when P,, and Pr2 content decreases (in roots and after freezing-thawing) (see below).

4.2. P,, P,, is a state capable of the low temperature photoreversible photo-

transformations. The amplitude of the photoreversible fluorescence changes (yZ) which we refer to P,, constitutes 4% - 7% at 85 - 90 K and it increases with the increase of temperature. The extent of the P,, transformation into Pf, constitutes 0.8 - 0.85, as in the case of P,, (y3 = 0.8 - 0.85 in conditions of P,, fluorescence prevalence).

After the adaptation of the sample to R light (X, = 670 nm) at 85 K the P,, state is transformed into the photoproduct lumi-Ri, whereas the contribution of the P,, state to the fluorescence of native samples is relatively small and the intensity of P,, fluorescence does not change much with the temperature at or below 220 K (see ref. 7 and below). In this case the temperature dependence of the fluorescence would primarily reflect the properties of the P,, state and Ea and A are estimated to be 4.5 - 5.5 kJ mol-’ and 5 - 5.5 respectively (Table 1). The P,, parameters for oat are similar to those for pea.

4.3. Pr3

Fluorescence of Pr3 dominates at temperatures above or equal to 200 K. In normal conditions (5 - 7-day-old pea stems and oat coleoptiles) E, = 26.9 kJ mol-’ for pea and 24.0 kJ mol-’ for oat. At the same time, E, is found to be somewhat larger in the roots of pea and in the stems subjected to the freezing-thawing procedure where the Pr3 content suffers a significant increase (see below). Assessments of E, give a somewhat larger value, 30.0 and 36.3 kJ mol-‘, respectively. E, for the interval 85 - 220 K was 1.9 - 2.5 kJ mol-‘. Taking these data into account, in addition to the results of simulation of the experimental curves of the P, fluorescence intensity changes with temperature (see below), E, = 38 f 4 kJ mol-’ for Pr3 and A = 20 - 22. Photoactivity of Pr3 manifests itself at temperatures greater than or equal to 220 K, with the maximum extent of transformation into Pf, about 0.7 (since at room temperature and temperatures close to it, when P,, fluoresces most, the extent of fluorescence changes, y4, is about 0.7 and also in the case of P, in the roots and in the stems after freezing-thawing, when the share of P,, (see below) increases, y3 = 0.70 - 0.75). At temperatures less than or equal to 220 K, phototransformations are practically absent. These characteristics of the three P, states are in good agreement with those obtained earlier in [ 6, 71.

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I c f ISOMERI SATION

Fig. 7. Hypothetical scheme of energy levels and transitions between them in the molecules of pigments of photochromic nature. (A) initial state; (B) the first stable photoproduct; A-B the shortliving unstable intermediate; k, rate constants of fluorescence (kf), temperature-independent degradation of excitation; (kd), primary photoreaction (kp ), phototransformation into the product (kab), and return to the initial state (kba);

E, and Ea’, activation energies of the temperature-dependent dilrect and reverse primary photoreactions.

The properties of the P, states can be considered within the framework of the scheme [ 12,131 which was suggested on the basis of the similarity in the properties of the excited states of (bacterio)rhodopsin, phytochrome and phycobilins for the description of the primary protoprocesses in the photochromic pigments (Fig. 7). The following are the main features of the scheme.

(1) Structural changes of the chromophore already in the excited state (transition 2-3) in which it acquires a more flexible conformation. This is pointed to by the violation of the mirror symmetry rule, the emission spectrum being more diffuse than the excitation (absorption) one.

(2) Three parallel routes of the excited state deactivation - fluorescence (with the radiative rate constant lzf), temperature-independent degradation into heat (kd) and temperature-dependent relaxation initiating the isomerization process (kp). Such an inverse correlation (competition) between the fluorescence and photoactivity is observed in the experiment: with the fall of the fluorescence quantum yield of P,, and Pr3 the extent of their photochemical transformation increases (see above and ref. 7). It should also be noted that E, of the fluorescence decay of P,, obtained in refs. 7 and 18 and that from this work (Ea = 4.5 - 5.5 kJ mol-l) is close to E, of lumi-R formation determined in vitro in ref. 14 using absorption spectroscopy (E, = 3.6 f 0.5 kJ molll) and in our own experiments

212

(unpublished results) on the temperature dependence of the initial rate of the decrease of the P, fluorescence intensity following the P, + lumi-R phototransformation in pea stems (&is approximately 4.5 kJ mol-‘).

(3) An intermediate (level 5-5’) unstable at low temperatures is formed with excess energy between the initial state and photoproduct. At this point a branching of the photochemical transformation takes place -. back to the initial state (uncompleted photoreaction) or to the photoproduct (com- pleted photoreaction) (Fig. 7). Such a transitional state, prelumi-R, was observed in ref. 15 between P, and lumi-R.

In accordance with this scheme some assessments can be made with respect to the yield of the primary photoprocesses in various P, states and their changes with temperature. The fluorescence quantum yield is determined as

kf ‘F = kf + kd + k,, epEaIRT (1)

Since (PF of all the three states at 85 K or less (when the photochemical way of the energy dissipation is practically closed, k,, eeEaIRT + 0) do not differ much, the kd values should correspondingly be close to each other. The yield of deactivation along the photochemical route 3-5 is

k e--EataT PO

” = kf + kd + k,, epEaIRT (2)

and (PAB, the yield of the phototransformation of the initial state (A) into the photoproduct (B), is equal to the product (pp (Pa,,, where (Pab is the proba- bility of the transition from the level 5 to the ground level of the photoproduct (&b = kab/(kab + kba)). It is assumed that the symmetrical scheme of the transitions (see Fig. 7) and similar correlations are true for the reverse phototransformation of the photoproduct (lumi-R) into the initial state, and that the back and direct photoreactions go via one and the same intermediate.

Figure 8 presents theoretical curves of changes of $$ and (pp for the three states which were calculated using the activation parameters obtained in the experiment (see Table 2). As follows from the figure, at temperatures near physiological ones, (pp approaches unity for all the states and the yield of the photoproduct formation cp AB shall be determined by p&. Since for all the P, states the extent of transformation into Pi, is, judging by y3, approximately the same (0.7 - 0.8) and in the first approximation the states are isochromic, (Pab for P,,, Prz, P,, should be equal or close to one another (apparently about 0.07 - 0.12 in accordance with the evaluation of the P, -j Pi, photoreaction yield in refs. 16 and 17). One cannot exclude, however, that the value of (Pab may be higher, because in Heihoff et al. [18] the quantum yield of the phototransformation of P, into the first set of the intermediates is determined to be 0.5 or more.

At low temperatures in the interval 85 - 200 K, when the photoproduct is stable, the extent and reversibility of the P, phototransformation into the

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TEMPERATURE, K Fig. 8. Theoretical temperature curves of the changes in the quantum fluorescence yield (@) (1 - 3) and of the primary photoreaction (a) (1’ - 3’) (transition 3 + 5, see scheme on Fig. 7 and the text) of the three states of the red phytochrome form. Activation parameters, see Table 2: P,r (l,l’), P,a (2,2’), Pr3 (3,3’).

TABLE 2

Characteristics of the three P, native states in the cells of etiolated pea seedlings

State Activation parameters Rate constant of Relative concentration of of fluorescence decay temperature-independent the P, states (C) and photoreaction excit@ion dissipation

Stems Roots Stems after E,, kJ mol-’

kd = kdkf s-’ freezing-

A = In kg&-l thawing

P l.1 10 - 12 12-13 lOa 0.3 0.05 0 P r2 4.5 - 5.5 5 -5.5 10s 0.6 0.45 0.65 P l-3 34-42 20 -22 10s 0.1 0.5 0.35

According to ref. 22 kf = 0.5 x ~O’S-~ for Pr in vitro.

photoproduct depends on the ratio of the quantum yields of the back and direct photoreactions and eventually on the E,’ and E, values. For Prl, in which the photostationary equilibrium is strongly shifted in the direction of the photoproduct and the back reaction is much slower than the direct one, E, <E,‘. In the case of Prz, E, and E,’ are apparently comparable and the reaction goes in both directions.

Rough estimates of the relative concentrations of the three P, species can be made from the low-temperature fluorescence belonging to the centers, taking into account that their quantum yields at low temperatures (85 K and below) do not differ much. Furthermore, P,, fluorescence yield reaches saturation already at 200 - 220 K, at which temperature the P,, and P,, fluorescence yields are still relatively low (see Fig. 8). In the stems

214

of native 5 - 7-day-old pea seedlings, P,, is about 0.25 - 0.3 of the total phytochrome content and 0.16 in the coleoptiles of oat seedlings, this being evidenced by the value of the irreversible component of the variable fluorescence (ri). The relative Pr3 content is not very large in both cases, apparently not over 0.10. The main share falls on P,*: in pea, 0.5 - 0.6, and somewhat greater in oat, 0.6 - 0.7. In pea roots and also in stems after freezing- thawing, the reduction of the photo-induced fluorescence changes at temperatures of 220 K and below, and increase of the relative fluorescence intensity at 220 K and above indicate that the Pr3 share increases (up to 0.4 - 0.5) at the expense of the other two states.

The adequacy of the suggested model (of the description of the P, state in the cell with the aid of the three centers) and correctness of determination of the activation parameters of their fluorescence and relative concentrations are confirmed by the satisfactory simulation of the experimental temperature curves of the fluorescence intensity changes with the theoretical ones. These are drawn in accordance with the expression

F(T) = 10 Lx Ctot ~(CM, + C2~2 + C3 93)

. . where IO is the light mtensity, EAex the absorbance at the wavelength of the exciting light (assumed to be equal for all three states), I the optical path- way, Ctot the concentration of phytochrome in the P, state, and C1, C2, C3 and pl cp2 p3 are relative concentration and fluorescence quantum yields of Prl, Pr2 and Pr3 (Fig. 9).

The observed changes of the relative concentration of the P, states upon freezing-thawing and in roots may be due to limited proteolysis of the P,, and P,, transforming them into the most stable Pr3. This is primarily indicated by the well known fact that P, is unstable in homogenates and by the fact that the extent of the fluorescence changes in the case of the action of freezing-thawing is greater - the longer the thawed sample remains at room temperature. Secondly, the temperature curves of the fluorescence yield obtained on the native [ 191 and “large” [ 201 phytochrome are similar in form to those of Pr2 and Pr3, respectively. Thirdly, more profound changes in the P, properties were observed in oat, in which, according to ref. 21, protease activity was more pronounced. This assumption, however, needs a direct experimental verification.

5. Conclusion

Investigation of the temperature dependence of (PF and the extent of photo-induced changes of fluorescence of the red phytochrome form in the cells of etiolated pea and oat seedlings reveals the existence in viuo of three P, states (Prl, Pr2, Pr3) confirming earlier conclusions [6 - 81. Their main difference is the value of E, for the fluorescence decay which is 10 - 12,4.5 - 5.5 and 34 - 42 kJ mol-‘, respectively. The data obtained earlier and in the present work suggest that the same parameter (E,) also determines the

215

25-

100 150 200 250 TEMPERATURE, K

Fig. 9. (1) Experimental and (2) theoretical temperature curves of the changes in the fluorescence intensity of Pr of etiolated pea seedlings. Theoretical curves were obtained by summing individual theoretical curves for P rr, Pr2 and Pr3 (see Fig. 8) and multiplying by the proportionality coefficients, corresponding to the share of the center (Cl, Cs, Ca) of the total pigment content (&et) in the Pr state (see text). (a) Stems adapted to FR light (720 nm) and frozen in darkness to 85 K, Cr = 0.3, Cz = 0.6, Ca = 0.1; (b) the same as (a), but after additional adaptation to R light (670 nm) at 85 K, Cr = 0, Ca = 0.82, Cs = 0.15; (c) stems frozen to 85 K in darkness after preliminary freezing and thawing and illumination with FR light (720 nm), C1 = 0, Cz = 0.65, C3 = 0.35.

temperature dependence of the rate constant and the yield of the photochemical reaction which serves as the main and, possibly, the only temperature-dependent channel for the deactivation of the excited P, state.

Activation parameters of the fluorescence decay and photoreaction of the three P, states prove to be close for pea and oat. The relative content of the centres is also close in both species: in native 5 - ‘I-day-old pea stems and oat coleoptiles there is a prevalence of Prs state (50% - 70%), whereas the P,, and Pr3 share is much lower (15% - 30% and 10% - 15%, respectively).

216

With the change of physiological state of the cells under study (their age, localization in the plant and preliminary cold treatment) changes take place in the character of the temperature curve of the P, fluorescence intensity and in the extent of its photo-induced low-temperature changes. This reflects reduction in the Prl and P,, content and increase on their account of Pr3 (up to 40% - 50% in the roots and also in the stems of pea following their cold treatment and thawing). It is suggested that P,i and P,, transformation into Pr3 in the cells might be caused by limited proteolysis of the pigment molecules.

Acknowledgment

The authors express their gratitude to Professor S. E. Braslavsky for her thorough editing of the paper.

References

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Different photoactive states of the red phytochrome form in the cells of etiolated pea and oat seedlings