Planta
Planta (1988) 175:471~477
9 Springer-Verlag ~988
Photoprotection of phytochrome Harry Smith, G. Michael Jackson, and Garry C. Whitelam Department of Botany, University of Leicester, Leicester LE1 7RH, U K
Abstract. High-fluence-rate white light is shown to retard the degradation of phytochrome in etiolated seedlings of four different species: Amaranthus caudatus, Phaseolus radiatus (mung bean), Pisum sativurn (garden pea), and Arena sath~a (oat). In Arnaranthus, a high photon fluence rate (approx. 1000 g m o l - m - 2 . s - 1) preserved nearly 50% of the total phytochrome over a period of 5 h; at 3 gmolm - 2 . s - 1, less than 8% remained over the same period. Kinetics of the loss of total phytochrome could be interpreted in terms of two populations, one with rapid, and one with slow, turnover rates. A log-linear relationship between fluence rate and proportion of slowly degrading phytochrome was observed; a similar relationship between fluence rate and the amount of phytoclhrome remaining after a 5-h light treatment was seen. In mung bean, although two populations of differing degradation rates were not resolvable, a similar log-linear relationship between fluence rate and amount remaining after a standard light treatment was evident. Detailed kinetic analyses were not performed with peas and oats, but comparisons of low and high fluence rates demonstrated that photoprotection was similarly effective in these species. In Arnaranthus, transfer from high to low fluence rate was accompanied by a rapid increase in degradation rate, indicating that the retarding effect of highfluence-rate light is not a consequence of the disablement of the degradative machinery. Immunochemical analyses confirmed the existence of photoprotection in all four species, and allowed the extension of the observations to periods of light treatment during which substantial chlorophyll production occurred. Considerable photoprotection was observed in oat seedlings exAbbreviations. Pfr=far-red absorbing form of phytochrome; P o = a m o u n t of phytochrome measured at time zero; P t = amount of phytochrome measured at time t; Ptot = total phytochrome; WL = white light
posed to summer sunlight. These results are interpreted in terms of the accumulation under high fluence rates of photoconversion intermediates not available to the degradative machinery which is specific for the far-red-absorbing form of phytochrome.
Key words: Light and phytochrome - Phytochrome: degradation and photoprotection
Introduction This paper is concerned with the inhibition of phytochrome degradation by high-fluence-rate white light. The investigation was begun with the objective of acquiring information on the potential role of intermediate accumulation during photoconversion in determining phytochrome action in the natural environment. In 1983 Fukshansky and Sch/ifer wrote... "That intermediates play an important r61e.., under bright white light.., simply by saving phytochrome from destruction is obvious" (see p. 91). Photoprotection of phytochrome from the normal process of degradation is here documented for four different species; some characteristics of the phenomenon pose interesting questions with regard to the operation of phytochrome in the light-grown plant. It was shown 15 years ago that photoconversion intermediates accumulated at high fluence rates (Kendrick and Spruit 1972, 1973). The intermediate meta-Rb accumulates in white light because it is the most photostable of all the intermediates, and because the succeeding relaxation step is the slowest in the cycle (Kendrick and Spruit 1977; Rfidiger 1980). Consequently, at daylight levels, phytochrome could become inoperative, either because the accumulation of intermediates leads to the concentration of the far-red-absorbing
472
form (Pfr) being too low, or because individual molecules do not remain as Pfr long enough, to couple to the transduction chain. It is of considerable importance, therefore, to estimate the fluencerate range of white light (WL) over which intermediate accumulation is significant. Direct measurement of the proportion of photoconversion intermediates present requires sophisticated quasi-continuous spectrophotometry (Kendrick and Spruit 1972), a technique not available to us. An indirect way to approach the problem, however, is to make use of the degradative breakdown of etiolated-tissue Pfr (i.e. Type I Pfr) which, for dicotyledonous plants at least, is typically a first-order reaction, the exponential rate constant of Pfr breakdown at any point being proportional to the concentration of Pfr (Kendrick and Frankland 1968). If intermediate accumulation were to increase with fluence rate, then the steady-state concentration of Pfr should decrease; assuming the intermediates are not susceptible to degradation, this would lead to concomitant decreases in the rate of Pfr loss. Kendrick and Spruit (1972) reported a deviation from first-order kinetics of Pfr loss in Amaranthus caudatus at elevated fluence rates, and accounted for this on the basis of intermediate accumulation. We have used this approach to assess the protective effect of WL fluence rates up to those typical of summer daylight using etiolated seedlings of four species. Using immunochemical methods, we have also assessed the protective effect of actual sunlight on Pfr degradation in etiolated oat seedlings. Materials and methods Plant materials and growth conditions. Seeds of Amaranthus caudatus var. viridis (Sinclair McGill plc, Boston, Lincs.) were sown in batches of 100 in 5-cm-deep-form Petri dishes containing two pieces of filter paper (No. 597, Schleicher and Schuell, Dassel, F R G ) moistened with 1.5 cm 3 distilled H20. The dishes were then stacked in a light-tight box containing water-saturated paper towelling and grown for 4 d (89 h) in complete darkness at 25 ~ C. Oat (Arena sativa cv. Dula; Highfield Seeds Ltd, Desford, Leics., U K ) seeds were soaked for 8 h in running water and sown in seed trays containing water-saturated medium-grade vermiculite. An empty seed tray was placed on top and the whole contained within a black polythene bag. Seedlings were grown at 25 ~ C in complete darkness for 4 d (89 h). Pea (Pisum sativum cv. Onward; Asmer Seeds Ltd, Leicester, Leics., U K ) seeds were soaked and sown in the same manner as were the oat seeds. Seedlings were grown in complete darkness at 25 ~ C for 5.5 d (136 h). Mung bean (Phaseolus aureus; acquired from a local store) seeds were sterilised in 0.1% HgCI2 for 10 min, followed by washing for several hours in running tap water. Seeds were sown and grown as were oats, but for 112 h (i.e. 4 d plus overnight).
H. Smith et al. : Photoprotection of phytochrome 5.0
'E
4.0
30 2.0 :a 1.0 000
5;0
6100 7;0 Wavelength (nm3
'800
Fig. 1. Spectral photon distribution of the high-fluence-rate WL. Total photon fluence rate, 400-800 rim, 975 g m o l . m 2. s - l ; 400-700 nm, 811 g m o l . m - 2 - s - 1 ; red: far red, 2.1; Pfr/ Ptot, 0.71
Experimental light source. A light source was set up which would provide a uniform radiation field of approx 80- 20 cm 2, establish a high phytochrome photoequilibrium, and generate fluence rates approaching those of summer daylight. It consisted of three 500-W quartz-iodide lamps (Halogen Floodlight; Philips, Eindhoven, The Netherlands) mounted on a frame and backed by an aluminium reflector. Radiant heat was reduced by a 2-cm deep flowing " w a t e r window", below which was a 1-cm static layer of 1.5% (w/v) CuSO~. 5H20 in water. Dishes and trays of seedlings were placed at varying heights below the source to vary fluence rate; to achieve the lowest fluence rates used required attenuation by means of the a p p r o p r i a t e number of layers of white muslin. The spectral distribution of the radiation is given in Fig. 1. Fluence rates (40~800 rim) and spectral distributions were measured, for every experiment, with a Li-Cor 1800 spectroradiometer (Lincoln Instruments Inc., Lincoln, Neb., USA). Experimental procedure. Dishes or trays of seedlings were wrapped in opaque black polythene for transfer from the darkroom, unwrapped, and placed on the appropriate shelf under the light source. Samples were removed at timed intervals, placed on ice and wrapped in black polythene for transfer to the spectrophotometer room for phytochrome estimations. Measurements of the to levels were made using seedlings directly from the darkroom at the beginning, and the end, of the experiment.
Phytochrome measurements. Total phytochrome in the samples was estimated using a dual-wavelength spectrophotometer (DW2-A; Aminco, Silver Spring, Md., USA) with the measuring beams set at 660 and 730 nm; for oat seedlings, which green-up substantially during the experimental period, measurements were made at 730 and 800 nm. For Amaranthus, a custom-made cuvette was constructed from an aluminium block (45-10.10 m m 3) providing a sample chamber 6 m m diameter with screw-in Perspex windows at each end and a fixed irradiation window on one side. The chamber was just large enough to contain the approx. 100 seedlings from each dish at the 4-d growth stage. For the other seedlings, standard 3.0-cm 3 plastic cuvettes were used, each sample consisting of 2.0 g tissue (5- to 10-mm oat coleoptile tips, pea epicotyl hooks, or mungbean hypocotyl hooks) sliced into small pieces (approx. 2 ram) and packed to a constant depth using a specially prepared plunger.
Immunochemical analyses.Tissue samples treated and harvested as described above were homogenised (1.0 cm3/g tissue) in 50 m M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)HC1, pH7.8, containing 1 0 r a M ethylenediaminetetraacetic acid (EDTA) and 20 m M NazSO3. After clarification, the supernatant was boiled for 2 min with an equal volume of sodium
473
H. Smith et al. : Photoprotection of phytochrome Table 1. Proportion of total phytochrome remaining (Pt/Po) in tissue samples of the stated species after exposure for 5 h to WL of high and low fluence rate
1.0 ~ 0.8
O
0.6
Species
Pt/Po
0.4
975gmol-m 2-s *
3 g m o l . m 2'S 1
0.33
0.057
0.55 0.31 0.124
0.13 0.084 0.024
[
\
9
', ,',,X-,~'~ o ~
-
\ \ \
Amaranthus caudatus Phaseolus aureus Pisum sativum Arena sativa
dodecyl sulphate (SDS) sample buffer and aliquots were separated on 8% SDS-polyacrylamide gels. After electroblotting onto nitrocellulose filters, phytochrome bands were detected with immunoaffinity-isolated rabbit antibodies to etiolated Arena phytochrome, followed by peroxidase-conjugated goat anti-rabbit-immunoglobulin antibodies and staining for bound peroxidase.
Results and discussion
Basic observations with the four species. Seedlings of the four species under investigation were exposed for 5 h (300min) to WL of either 3, or 957 g m o l - m - 2 . s -1 in order to determine the extent of photoprotection, and to decide which species to use for a more-detailed analysis. Table 1 shows that all four species exhibited photoprotection. In all cases, an increase of fluence rate from a value which may be considered as characteristic of that found at the end-of-day (i.e. 3 p m o l . m - 2 . s - t ) , to one representing temperate-zone daylight (957 ~tmol. m - z. s- 1), caused a marked reduction in the amount of total phytochrome lost over a 5-h period. The two most striking effects were seen with Amaranthus and Arena; in the former, a 17-fold loss of phytochrome at the low fluence rate was reduced by the bright WL to a 3-fold loss, whilst in Arena, a 40-fold loss was cut to just 8-fold. Amaranthus was chosen for detailed spectrophotometric studies, partly because earlier observations had been with this species (Kendrick and Spruit 1972, 1973), but also because it gave the largest effect of those investigated. Arena was not further analysed spectrophotometrically because of the detectable "greening" which occurred during the experimental period; immunochemical determinations were, however, carried out and are reported below. For completeness, some further spectrophotometric observations were made with mung-bean seedlings. Amaranthus : kinetics of phytochrome loss at different fluence rates. Figure 2 shows the time courses of phytochrome loss over a 5-h period with W L of various fluence rates. At none of the fluence
Pt PO
\\ \ \ \\ \ k\ \\ \\ ~ \\ \\ \\ \\ \\ \\ ~ \
0.1 0.08 0.06 ~ [ 0.04 ~ i
0
9
9 56
A
3
"\ \ ,, \\ \ \\ \\ \ \
I
I I ! I
o
- --9~7
~o',
I 40
I 80
I 120
f I 160 200 Time [rain)
I 240
I 280
I 320
Fig. 2. Time courses of the loss of total phytochrome in Amaranthus caudatus seedlings exposed to WL of the stated fluence rates. The curves were "peeled" manually (i.e. without a computer) yielding fast (dotted) and slow (solid) log-linear lines, from which half-times were calculated
rates used was the degradation process first order; Kendrick and Frankland (1968) showed log-linear kinetics with a range of wavelengths, but their fluence rates were considerably lower than the higher of the values used here. The shape of the time-course curves presented in Fig. 2 raised the possibility that the phytoc h r o m e in Amaranthus may exist in two populations, one with a slow, and the other with a fast, degradation constant, consistent with the previous observations reported for the same species by Heim et al. (1981), and Brockmann and Schfifer (1982). Manual "peeling" of the curves in Fig. 2 yielded two sets of approximately parallel log-linear lines, indicating that the proportions of the total phytochrome present in the "fast" and the " s l o w " populations varied with fluence rate. Table 2 lists the calculated half-times (tu2) of the proposed " f a s t " and " s l o w " populations for each of the fluence rates shown in Fig. 2; although not identical, the values are clearly closely related, with only those for the lowest fluence rate used showing obvious deviation. Figure 3a shows the relationship between fluence rate and the proportion of the total phytochrome present in the " s l o w " population (incorporating more time-course data than shown in Fig. 2). Between fluence rates of approx. 30 and approx. 1000 t~mol-m -2. s -1, the relationship between fluence rate and the proportion of slowly degrading phytochrome molecules approximates log-linear. Thus, the protective effect of W L
474
H. Smith et al. : Photoprotection of phytochrome
Table 2. Effect of WL fluence rate on the half-times (tl/2) of A. caudatus phytochrome populations having fast and slow deg-
radation constants. Data calculated from Fig. 1 Fluence rate (gmol.m Z-s-l)
tl/2 (rain)
3 56 315 957
60
Fast
Slow
30 28 26 26
370 320 302 311
a
'~50
==~ go
c
30
"5
~_ ~ 2 0
~10 ,
b
0
5
110
30 50 100
500 1010
J 10
I 100
10 )0
0.4 o 0,3 co
2 o 0.2 0.1 I
0 C .c 0.6 E oo co 0.4 fl
0.2
0
/ 1[0 1(30 Photon ftuence rate (umol.m2,sq)
1000
Fig. 3a-e. Relationship between WE fluence rate and phytochrome loss. a Amaranthus caudatus, proportion of total phytochrome having slow degradation constant; b A. caudatus, proportion of total phytochromeremainingafter exposureto various fluencerates of WL for 5 h; e as for b, but using Phaseolus aureus. Verticalbars = 4-SE on phytochrome degradation is seen at surprisingly low fluence rates, and has an equally surprising mathematical relationship to higher fluence rates. A log-linear relationship is also seen when measurements are made of the amounts of phytochrome remaining after 5-h exposures to WL of varying fluence rates (Fig. 3 b). A plausible interpretation of these data is that the "fast population" represents the steady-state concentration of Pfr, the component of phytochrome that is specifically degraded by the socalled "destruction" mechanism, involving the rapid sequestration of Pfr into vesicles, or aggregates
(McCurdy and Pratt 1986a, b; Speth et al. 1986), followed by rapid degradation (Butler and Lane 1965), whilst the "slow population" represents the relatively slow, non-specific, degradation to which all proteins are variously susceptible, and which presumably would affect all components of the phytochrome system, including the intermediates if they were to exist for long enough. As fluence rate is increased, the proportion of time each molecule spends as a non-photoconvertible intermediate must increase, and concomitantly, the period of time spent as Pfr must decrease, thereby reducing the proportion of the "fast population" and increasing the proportion of the "slow population". Since the photochemical reactions which yield the intermediates exhibit a log-linear relationship to fluence rate, the data of Fig. 3 a are clearly understandable; with increasing fluence rate, the proportion of intermediates must increase on a loglinear basis, thus causing an equivalent increase in the proportion present in the "slow population". Over an extended time period, the fluencerate-dependent shift in the proportions of fast- and slow-degrading pools of phytochrome molecules is expressed as photoprotection (Fig. 3b), again a log-linear relationship, saving more than 50% of the total phytochrome from degradation over a period of 5 h. An alternative possibility is that a separate light reaction somehow disables the degradative machinery responsible for phytochrome destruction. It is conceivable that such a reaction, if it existed, may display a log-linear relationship with fluence rate and thus produce the relationship of Fig. 3 b. In an attempt to test this possibility, seedlings were exposed to a high fluence rate (315 g m o l . m -2. s-1) for 200 min and then some were transferred to a low fluence rate (3 g m o l ' m - 2 " s - 1 ) (Fig. 4). There is no indication from these data of any impairment of the capacity for Pfr degradation as a result of the exposure to high fluence rates; it consequently seems most likely that the elevated accumulation of intermediates, rather than a decreased capacity for protein degradation, is the basis of photoprotection. M u n g bean: kinetics o f phytochrome loss. Conceptually similar experiments to those using Amaranthus were performed with mung-bean seedlings. In
this case, however, the data could be construed as representing standard first-order degradation time courses (Fig. 5). No evidence of two populations differing in degradation rate constant was obtained, although more variability than expected was observed with the low fluence rate used. With
H. Smith et al. : Photoprotection of phytochrome
475
that is undectable during the time course of the experiments carried out here, to arrive at the kinetics shown in Fig. 5; in other words, the rate of degradation of the "slow population" in mung bean is effectively zero.
1.0 0.8 0.6 315 Pt 0.4 - -
I
\
Po 0'28./l 0,1 0.0 160 240 Time [min)
80
320
400
Fig. 4. Ama ra nthus caudatus: transfer of seedlings from highfluence-rate WL (315~maol.m-2.s -1) to low (3 # m o l m -2" s-1). Time of transfer is indicated by the arrow
0.8-D,6 Pt 0.4 PO
957
0.2
3
0,1 I
80
J
i
160 240 Time (min]
I
320
Fig. 5. Phaseolus aureus: time courses of the loss of total phytochrome at high and low fluenee rates (957 and 3 ~tmol-m 2. s - ~). Vertical bars = + SE
high-fluence-rate WL, a lag of approx. 60 rain was seen before phytochrome loss began, but the variability was such that the lag could simply be due to chance. When seedlings were exposed for a 5-h period to a range of fluence rates, a relationship similar to that found with Amaranthus was revealed, albeit with certain detailed differences (Fig. 3 c). With mung bean, the log-linear relationship between the fluence rate of WL and the proportion of phytochrome saved from degradation, began at rather higher fluence rates (approx. 80 as compared with 3 0 g m o l . m - a . s -a) and saturated at lower fluence rates (approx. 500 ~tmol. m -2"s-1 as opposed to no saturation up to approx. 1000 gmol" m - 2. s- 1 in Amaranthus). Although there is no spectrophotometric evidence for two populations of mung-bean phytochrome with differing degradation rate constants, the situation may, nevertheless, be not very different from that found in Amaranthus. It is only necessary to assume that the non-specific degradation of phytochrome in mung bean proceeds at a rate
lmmunochemical analyses. Photoprotection of phytochrome from degradation is also observable by immunoblot analysis (Fig. 6), providing direct evidence that the spectrophotometric assay is measuring loss ofphytochrome protein. The data in Fig. 6 confirm those in Table 1 for oat, pea, mung bean, and Amaranthus seedlings and show that in all cases high-fluence-rate WL (957 gmol' m - 2. s- 1) leads to substantially less phytochrome degradation than low-fluence-rate WL (3 g m o l . m 2. s-1). Since the immunoblot technique can be applied to tissues containing chlorophyll, it has been possible to assess the protective effect of prolonged exposure to sunlight. For etiolated oat seedlings, transfer to full sunlight ( 1 . 1 - 1 . 7 m m o l ' m - 2 ' s -1 during the exposure) leads to only limited phytochrome loss over an 8-h period, compared with substantial losses consequent upon transfer to filtered sunlight of only 2-3 g m o l . m 2.s-1 for the same period (Fig. 7). The extent of the inhibition of phytochrome degradation in full sunlight is perhaps most clearly seen when comparing immunoblots of extracts obtained from seedlings exposed to sunlight, or maintained in darkness for 8 h, and of extracts of the same tissues given a 5 rain pulse of F R and maintained in darkness for 16 h (Fig. 7). Clearly, only a limited pool of phytochrome is available for degradation in seedlings exposed to bright sunlight. This does not, however, mean that seedlings growing in the natural environment will necessarily possess high levels of phytochrome. Seeds germinating in sub-surface soil layers will be exposed to low fluence rates of light (Mandoli et al. 1982; Bliss and Smith 1985; Tester and Morris 1987), which should allow significant phytochrome degradation prior to seedling emergence. Furthermore, any elevated levels of phytochrome that may result from photoprotection afforded by the exposure of germinating seeds to bright sunlight should be quickly lost at the end of the first day and into the first night. The significance of these observations for plants growing under natural conditions relates to the ability of phytochrome to function at very high fluence rates. These data indicate that the effective Pfr concentration is progressively reduced as fluence rate rises from approx. 50 gmol- m - 2. s- 1 to approx. 1000 gmol. m - 2. s- 1. If phytochrome does operate in natural sunlight, therefore, either
476
H. Smith et al. : Photoprotection of phytochrome
Fig. 6a-d. Immunoblot analyses of phytochrome loss in seedlings exposed to A, high-fluence-rate (957 gmol.m z. s - l ) or B, low-fluence-rate (3 gmol. m-Z.s 1) WL. Etiolated seedlings of a Arena sativa, b Amaranthus caudatus, e Pisum sativum or d Phaseolus aureus
were irradiated for the times shown, after which crude extracts were prepared (1 g of tissue per ml buffer) and aliquots resolved on 8% polyacrylamide gels. Electroblotted proteins were probed with rabbit antiArena phytochrome antibodies (a, b, fl) or rat anti-Pisum phytochrome antibodies (e). k D a = k i l o d a l t o n
H. Smith et al. : Photoprotection of phytochrome
477
Fig. 7. Effect of daylight conditions on loss of immunochemically assayabte phytochrome in Arena sativa. Etiolated seedlings were exposed for various times to either filtered (reduced) sunlight (R) or full sunlight (S). At the indicated times, samples were prepared for immunoblotting as described in the legend for Fig. 6. The right-hand blot shows the effect of exposure to darkness (D), full sunlight (S), low-fluence-rate WL (L) or high-fluence-rate WL (H) for 8 h, followed by 5 min far-red light and a subsequent 16 h incubation in darkness, on immunochemically assayable phytochrome in A. sativa seedlings
the coupling between Pfr and the first element of the transduction chain is much more rapid than coupling to the degradative pathway, or action must require a mechanism not solely dependent on the concentration of Pfr. This view is not altered by the finding that in light-grown plants the predominant phytochrome species is different from that in etiolated plants (Abe et al. 1985; Shimazaki and Pratt 1985; Tokuhisa etal. 1985). Even for a relatively stable phytochrome molecule with a slow turnover, individual Pfr molecules will exist for only a short time under high fluence rates. The senior author (H.S.) is grateful for discussions with Dr. Eberhard Sch/ifer, in whose laboratory in Freiburg the first of the data reported here were obtained, during a brief working visit as long ago as 1984; subsequently, useful discussions were held with Dr. Richard Kendrick at Wageningen, whose advice is also gratefully acknowledged.
References Abe, H., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Characterization of green tissue-specific phytochrome isolated immunochemically from pea seedlings. Plant Cell Physiol. 26, 1387-1399 Bliss, D., Smith, H. (1986) Penetration of light into soil and its role in the control of seed germination. Plant Cell Environ. 8, 475-483 Brockmann, J., Sch/ifer, E. (1982) Analysis of Pfr destruction in Amaranthus caudatus L. - evidence for two pools of phytochrome. Photochem. Photobiol. 35, 555-558 Butler, W.L., Lane, H.C. (1965) Dark transformations of phytochrome in vivo. II. Plant Physiol. 40, 13-17 Fukshansky, L., Sch/ifer, E. (1983) Models in photomorphogenesis. In: Encyclopaedia of plant physiology N.S., vol. 16A: Photomorphogenesis, pp. 69-95, Shropshire, W., Jr., Mohr, E., eds. Springer, Berlin Helm, B., Jabben, M., Schfifer, E. (1981) Phytochrome destruc-
tion in dark- and light-grown Amaranthus caudatus seedlings. Photochem. Photobiol. 34, 89-93 Kendrick, R.E., Frankland, B. (1968) Kinetics of phytochrome decay in Amaranthus seedlings. Planta 82, 317 320 Kendrick, R.E., Spruit, C.J.P. (1972) Light maintains high levels of phytochrome intermediates. Nature 237, 281 282 Kendrick, R.E., Spruit, C.J.P. (1973) Phytochrome intermediates in vivo. I. Effects of temperature, light intensity, wavelength, and oxygen on intermediate accumulation. Photochem. Photobiol. 18, 1349-144 Kendrick, R.E., Spruit, C.J.P. (1977) Phototransformations of phytochrome. Photochem. Photobiol. 26, 201 204 Mandoli, D.F., Waldron, L., Nemson, J.A., Briggs, W.R. (1982) Soil light transmission: implications for pbytochrome-mediated responses. Carnegie Instn. Washington Yearb. 81, 32-34 McCurdy, D.W., Pratt, L.H. (1986a) Kinetics of intracellular redistribution of phytochrome in Arena coleoptiles after its conversion to the active, far-red-absorbing form. Planta 167, 330-336 McCurdy, D.W., Pratt, L.H. (1986b) Immunogold electron microscopy of phytochrome in Arena: Identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability. J. Cell Biol. 103, 2541-2550 Speth, V., Otto, V., Sch/ifer, E. (1986) Intracellular localisation of phytoehrome in oat coleoptiles by electron microscopy. Planta 168, 299-304 R/idiger, W. (1980) Phytochrome, a light receptor of photomorphogenesis. In: Structure and bonding, vol. 40, pp. 101-140, Hemmerich, P., ed. Springer, Berlin Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with rabbit polyclonaI and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Arena shoots. Planta 164, 333-344 Tester, M., Morris, C. (1987) The penetration of light through soil. Plant Cell Environ. 10, 281-286 Tokuhisa, J.G., Daniets, S.M., Quail, P.H. (1985) Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in lightgrown Arena sativa. Planta 164, 321-332 Received 26 November 1987; accepted 14 April 1988
Planta (1988) 175:471~477
9 Springer-Verlag ~988
Photoprotection of phytochrome Harry Smith, G. Michael Jackson, and Garry C. Whitelam Department of Botany, University of Leicester, Leicester LE1 7RH, U K
Abstract. High-fluence-rate white light is shown to retard the degradation of phytochrome in etiolated seedlings of four different species: Amaranthus caudatus, Phaseolus radiatus (mung bean), Pisum sativurn (garden pea), and Arena sath~a (oat). In Arnaranthus, a high photon fluence rate (approx. 1000 g m o l - m - 2 . s - 1) preserved nearly 50% of the total phytochrome over a period of 5 h; at 3 gmolm - 2 . s - 1, less than 8% remained over the same period. Kinetics of the loss of total phytochrome could be interpreted in terms of two populations, one with rapid, and one with slow, turnover rates. A log-linear relationship between fluence rate and proportion of slowly degrading phytochrome was observed; a similar relationship between fluence rate and the amount of phytoclhrome remaining after a 5-h light treatment was seen. In mung bean, although two populations of differing degradation rates were not resolvable, a similar log-linear relationship between fluence rate and amount remaining after a standard light treatment was evident. Detailed kinetic analyses were not performed with peas and oats, but comparisons of low and high fluence rates demonstrated that photoprotection was similarly effective in these species. In Arnaranthus, transfer from high to low fluence rate was accompanied by a rapid increase in degradation rate, indicating that the retarding effect of highfluence-rate light is not a consequence of the disablement of the degradative machinery. Immunochemical analyses confirmed the existence of photoprotection in all four species, and allowed the extension of the observations to periods of light treatment during which substantial chlorophyll production occurred. Considerable photoprotection was observed in oat seedlings exAbbreviations. Pfr=far-red absorbing form of phytochrome; P o = a m o u n t of phytochrome measured at time zero; P t = amount of phytochrome measured at time t; Ptot = total phytochrome; WL = white light
posed to summer sunlight. These results are interpreted in terms of the accumulation under high fluence rates of photoconversion intermediates not available to the degradative machinery which is specific for the far-red-absorbing form of phytochrome.
Key words: Light and phytochrome - Phytochrome: degradation and photoprotection
Introduction This paper is concerned with the inhibition of phytochrome degradation by high-fluence-rate white light. The investigation was begun with the objective of acquiring information on the potential role of intermediate accumulation during photoconversion in determining phytochrome action in the natural environment. In 1983 Fukshansky and Sch/ifer wrote... "That intermediates play an important r61e.., under bright white light.., simply by saving phytochrome from destruction is obvious" (see p. 91). Photoprotection of phytochrome from the normal process of degradation is here documented for four different species; some characteristics of the phenomenon pose interesting questions with regard to the operation of phytochrome in the light-grown plant. It was shown 15 years ago that photoconversion intermediates accumulated at high fluence rates (Kendrick and Spruit 1972, 1973). The intermediate meta-Rb accumulates in white light because it is the most photostable of all the intermediates, and because the succeeding relaxation step is the slowest in the cycle (Kendrick and Spruit 1977; Rfidiger 1980). Consequently, at daylight levels, phytochrome could become inoperative, either because the accumulation of intermediates leads to the concentration of the far-red-absorbing
472
form (Pfr) being too low, or because individual molecules do not remain as Pfr long enough, to couple to the transduction chain. It is of considerable importance, therefore, to estimate the fluencerate range of white light (WL) over which intermediate accumulation is significant. Direct measurement of the proportion of photoconversion intermediates present requires sophisticated quasi-continuous spectrophotometry (Kendrick and Spruit 1972), a technique not available to us. An indirect way to approach the problem, however, is to make use of the degradative breakdown of etiolated-tissue Pfr (i.e. Type I Pfr) which, for dicotyledonous plants at least, is typically a first-order reaction, the exponential rate constant of Pfr breakdown at any point being proportional to the concentration of Pfr (Kendrick and Frankland 1968). If intermediate accumulation were to increase with fluence rate, then the steady-state concentration of Pfr should decrease; assuming the intermediates are not susceptible to degradation, this would lead to concomitant decreases in the rate of Pfr loss. Kendrick and Spruit (1972) reported a deviation from first-order kinetics of Pfr loss in Amaranthus caudatus at elevated fluence rates, and accounted for this on the basis of intermediate accumulation. We have used this approach to assess the protective effect of WL fluence rates up to those typical of summer daylight using etiolated seedlings of four species. Using immunochemical methods, we have also assessed the protective effect of actual sunlight on Pfr degradation in etiolated oat seedlings. Materials and methods Plant materials and growth conditions. Seeds of Amaranthus caudatus var. viridis (Sinclair McGill plc, Boston, Lincs.) were sown in batches of 100 in 5-cm-deep-form Petri dishes containing two pieces of filter paper (No. 597, Schleicher and Schuell, Dassel, F R G ) moistened with 1.5 cm 3 distilled H20. The dishes were then stacked in a light-tight box containing water-saturated paper towelling and grown for 4 d (89 h) in complete darkness at 25 ~ C. Oat (Arena sativa cv. Dula; Highfield Seeds Ltd, Desford, Leics., U K ) seeds were soaked for 8 h in running water and sown in seed trays containing water-saturated medium-grade vermiculite. An empty seed tray was placed on top and the whole contained within a black polythene bag. Seedlings were grown at 25 ~ C in complete darkness for 4 d (89 h). Pea (Pisum sativum cv. Onward; Asmer Seeds Ltd, Leicester, Leics., U K ) seeds were soaked and sown in the same manner as were the oat seeds. Seedlings were grown in complete darkness at 25 ~ C for 5.5 d (136 h). Mung bean (Phaseolus aureus; acquired from a local store) seeds were sterilised in 0.1% HgCI2 for 10 min, followed by washing for several hours in running tap water. Seeds were sown and grown as were oats, but for 112 h (i.e. 4 d plus overnight).
H. Smith et al. : Photoprotection of phytochrome 5.0
'E
4.0
30 2.0 :a 1.0 000
5;0
6100 7;0 Wavelength (nm3
'800
Fig. 1. Spectral photon distribution of the high-fluence-rate WL. Total photon fluence rate, 400-800 rim, 975 g m o l . m 2. s - l ; 400-700 nm, 811 g m o l . m - 2 - s - 1 ; red: far red, 2.1; Pfr/ Ptot, 0.71
Experimental light source. A light source was set up which would provide a uniform radiation field of approx 80- 20 cm 2, establish a high phytochrome photoequilibrium, and generate fluence rates approaching those of summer daylight. It consisted of three 500-W quartz-iodide lamps (Halogen Floodlight; Philips, Eindhoven, The Netherlands) mounted on a frame and backed by an aluminium reflector. Radiant heat was reduced by a 2-cm deep flowing " w a t e r window", below which was a 1-cm static layer of 1.5% (w/v) CuSO~. 5H20 in water. Dishes and trays of seedlings were placed at varying heights below the source to vary fluence rate; to achieve the lowest fluence rates used required attenuation by means of the a p p r o p r i a t e number of layers of white muslin. The spectral distribution of the radiation is given in Fig. 1. Fluence rates (40~800 rim) and spectral distributions were measured, for every experiment, with a Li-Cor 1800 spectroradiometer (Lincoln Instruments Inc., Lincoln, Neb., USA). Experimental procedure. Dishes or trays of seedlings were wrapped in opaque black polythene for transfer from the darkroom, unwrapped, and placed on the appropriate shelf under the light source. Samples were removed at timed intervals, placed on ice and wrapped in black polythene for transfer to the spectrophotometer room for phytochrome estimations. Measurements of the to levels were made using seedlings directly from the darkroom at the beginning, and the end, of the experiment.
Phytochrome measurements. Total phytochrome in the samples was estimated using a dual-wavelength spectrophotometer (DW2-A; Aminco, Silver Spring, Md., USA) with the measuring beams set at 660 and 730 nm; for oat seedlings, which green-up substantially during the experimental period, measurements were made at 730 and 800 nm. For Amaranthus, a custom-made cuvette was constructed from an aluminium block (45-10.10 m m 3) providing a sample chamber 6 m m diameter with screw-in Perspex windows at each end and a fixed irradiation window on one side. The chamber was just large enough to contain the approx. 100 seedlings from each dish at the 4-d growth stage. For the other seedlings, standard 3.0-cm 3 plastic cuvettes were used, each sample consisting of 2.0 g tissue (5- to 10-mm oat coleoptile tips, pea epicotyl hooks, or mungbean hypocotyl hooks) sliced into small pieces (approx. 2 ram) and packed to a constant depth using a specially prepared plunger.
Immunochemical analyses.Tissue samples treated and harvested as described above were homogenised (1.0 cm3/g tissue) in 50 m M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)HC1, pH7.8, containing 1 0 r a M ethylenediaminetetraacetic acid (EDTA) and 20 m M NazSO3. After clarification, the supernatant was boiled for 2 min with an equal volume of sodium
473
H. Smith et al. : Photoprotection of phytochrome Table 1. Proportion of total phytochrome remaining (Pt/Po) in tissue samples of the stated species after exposure for 5 h to WL of high and low fluence rate
1.0 ~ 0.8
O
0.6
Species
Pt/Po
0.4
975gmol-m 2-s *
3 g m o l . m 2'S 1
0.33
0.057
0.55 0.31 0.124
0.13 0.084 0.024
[
\
9
', ,',,X-,~'~ o ~
-
\ \ \
Amaranthus caudatus Phaseolus aureus Pisum sativum Arena sativa
dodecyl sulphate (SDS) sample buffer and aliquots were separated on 8% SDS-polyacrylamide gels. After electroblotting onto nitrocellulose filters, phytochrome bands were detected with immunoaffinity-isolated rabbit antibodies to etiolated Arena phytochrome, followed by peroxidase-conjugated goat anti-rabbit-immunoglobulin antibodies and staining for bound peroxidase.
Results and discussion
Basic observations with the four species. Seedlings of the four species under investigation were exposed for 5 h (300min) to WL of either 3, or 957 g m o l - m - 2 . s -1 in order to determine the extent of photoprotection, and to decide which species to use for a more-detailed analysis. Table 1 shows that all four species exhibited photoprotection. In all cases, an increase of fluence rate from a value which may be considered as characteristic of that found at the end-of-day (i.e. 3 p m o l . m - 2 . s - t ) , to one representing temperate-zone daylight (957 ~tmol. m - z. s- 1), caused a marked reduction in the amount of total phytochrome lost over a 5-h period. The two most striking effects were seen with Amaranthus and Arena; in the former, a 17-fold loss of phytochrome at the low fluence rate was reduced by the bright WL to a 3-fold loss, whilst in Arena, a 40-fold loss was cut to just 8-fold. Amaranthus was chosen for detailed spectrophotometric studies, partly because earlier observations had been with this species (Kendrick and Spruit 1972, 1973), but also because it gave the largest effect of those investigated. Arena was not further analysed spectrophotometrically because of the detectable "greening" which occurred during the experimental period; immunochemical determinations were, however, carried out and are reported below. For completeness, some further spectrophotometric observations were made with mung-bean seedlings. Amaranthus : kinetics of phytochrome loss at different fluence rates. Figure 2 shows the time courses of phytochrome loss over a 5-h period with W L of various fluence rates. At none of the fluence
Pt PO
\\ \ \ \\ \ k\ \\ \\ ~ \\ \\ \\ \\ \\ \\ ~ \
0.1 0.08 0.06 ~ [ 0.04 ~ i
0
9
9 56
A
3
"\ \ ,, \\ \ \\ \\ \ \
I
I I ! I
o
- --9~7
~o',
I 40
I 80
I 120
f I 160 200 Time [rain)
I 240
I 280
I 320
Fig. 2. Time courses of the loss of total phytochrome in Amaranthus caudatus seedlings exposed to WL of the stated fluence rates. The curves were "peeled" manually (i.e. without a computer) yielding fast (dotted) and slow (solid) log-linear lines, from which half-times were calculated
rates used was the degradation process first order; Kendrick and Frankland (1968) showed log-linear kinetics with a range of wavelengths, but their fluence rates were considerably lower than the higher of the values used here. The shape of the time-course curves presented in Fig. 2 raised the possibility that the phytoc h r o m e in Amaranthus may exist in two populations, one with a slow, and the other with a fast, degradation constant, consistent with the previous observations reported for the same species by Heim et al. (1981), and Brockmann and Schfifer (1982). Manual "peeling" of the curves in Fig. 2 yielded two sets of approximately parallel log-linear lines, indicating that the proportions of the total phytochrome present in the "fast" and the " s l o w " populations varied with fluence rate. Table 2 lists the calculated half-times (tu2) of the proposed " f a s t " and " s l o w " populations for each of the fluence rates shown in Fig. 2; although not identical, the values are clearly closely related, with only those for the lowest fluence rate used showing obvious deviation. Figure 3a shows the relationship between fluence rate and the proportion of the total phytochrome present in the " s l o w " population (incorporating more time-course data than shown in Fig. 2). Between fluence rates of approx. 30 and approx. 1000 t~mol-m -2. s -1, the relationship between fluence rate and the proportion of slowly degrading phytochrome molecules approximates log-linear. Thus, the protective effect of W L
474
H. Smith et al. : Photoprotection of phytochrome
Table 2. Effect of WL fluence rate on the half-times (tl/2) of A. caudatus phytochrome populations having fast and slow deg-
radation constants. Data calculated from Fig. 1 Fluence rate (gmol.m Z-s-l)
tl/2 (rain)
3 56 315 957
60
Fast
Slow
30 28 26 26
370 320 302 311
a
'~50
==~ go
c
30
"5
~_ ~ 2 0
~10 ,
b
0
5
110
30 50 100
500 1010
J 10
I 100
10 )0
0.4 o 0,3 co
2 o 0.2 0.1 I
0 C .c 0.6 E oo co 0.4 fl
0.2
0
/ 1[0 1(30 Photon ftuence rate (umol.m2,sq)
1000
Fig. 3a-e. Relationship between WE fluence rate and phytochrome loss. a Amaranthus caudatus, proportion of total phytochrome having slow degradation constant; b A. caudatus, proportion of total phytochromeremainingafter exposureto various fluencerates of WL for 5 h; e as for b, but using Phaseolus aureus. Verticalbars = 4-SE on phytochrome degradation is seen at surprisingly low fluence rates, and has an equally surprising mathematical relationship to higher fluence rates. A log-linear relationship is also seen when measurements are made of the amounts of phytochrome remaining after 5-h exposures to WL of varying fluence rates (Fig. 3 b). A plausible interpretation of these data is that the "fast population" represents the steady-state concentration of Pfr, the component of phytochrome that is specifically degraded by the socalled "destruction" mechanism, involving the rapid sequestration of Pfr into vesicles, or aggregates
(McCurdy and Pratt 1986a, b; Speth et al. 1986), followed by rapid degradation (Butler and Lane 1965), whilst the "slow population" represents the relatively slow, non-specific, degradation to which all proteins are variously susceptible, and which presumably would affect all components of the phytochrome system, including the intermediates if they were to exist for long enough. As fluence rate is increased, the proportion of time each molecule spends as a non-photoconvertible intermediate must increase, and concomitantly, the period of time spent as Pfr must decrease, thereby reducing the proportion of the "fast population" and increasing the proportion of the "slow population". Since the photochemical reactions which yield the intermediates exhibit a log-linear relationship to fluence rate, the data of Fig. 3 a are clearly understandable; with increasing fluence rate, the proportion of intermediates must increase on a loglinear basis, thus causing an equivalent increase in the proportion present in the "slow population". Over an extended time period, the fluencerate-dependent shift in the proportions of fast- and slow-degrading pools of phytochrome molecules is expressed as photoprotection (Fig. 3b), again a log-linear relationship, saving more than 50% of the total phytochrome from degradation over a period of 5 h. An alternative possibility is that a separate light reaction somehow disables the degradative machinery responsible for phytochrome destruction. It is conceivable that such a reaction, if it existed, may display a log-linear relationship with fluence rate and thus produce the relationship of Fig. 3 b. In an attempt to test this possibility, seedlings were exposed to a high fluence rate (315 g m o l . m -2. s-1) for 200 min and then some were transferred to a low fluence rate (3 g m o l ' m - 2 " s - 1 ) (Fig. 4). There is no indication from these data of any impairment of the capacity for Pfr degradation as a result of the exposure to high fluence rates; it consequently seems most likely that the elevated accumulation of intermediates, rather than a decreased capacity for protein degradation, is the basis of photoprotection. M u n g bean: kinetics o f phytochrome loss. Conceptually similar experiments to those using Amaranthus were performed with mung-bean seedlings. In
this case, however, the data could be construed as representing standard first-order degradation time courses (Fig. 5). No evidence of two populations differing in degradation rate constant was obtained, although more variability than expected was observed with the low fluence rate used. With
H. Smith et al. : Photoprotection of phytochrome
475
that is undectable during the time course of the experiments carried out here, to arrive at the kinetics shown in Fig. 5; in other words, the rate of degradation of the "slow population" in mung bean is effectively zero.
1.0 0.8 0.6 315 Pt 0.4 - -
I
\
Po 0'28./l 0,1 0.0 160 240 Time [min)
80
320
400
Fig. 4. Ama ra nthus caudatus: transfer of seedlings from highfluence-rate WL (315~maol.m-2.s -1) to low (3 # m o l m -2" s-1). Time of transfer is indicated by the arrow
0.8-D,6 Pt 0.4 PO
957
0.2
3
0,1 I
80
J
i
160 240 Time (min]
I
320
Fig. 5. Phaseolus aureus: time courses of the loss of total phytochrome at high and low fluenee rates (957 and 3 ~tmol-m 2. s - ~). Vertical bars = + SE
high-fluence-rate WL, a lag of approx. 60 rain was seen before phytochrome loss began, but the variability was such that the lag could simply be due to chance. When seedlings were exposed for a 5-h period to a range of fluence rates, a relationship similar to that found with Amaranthus was revealed, albeit with certain detailed differences (Fig. 3 c). With mung bean, the log-linear relationship between the fluence rate of WL and the proportion of phytochrome saved from degradation, began at rather higher fluence rates (approx. 80 as compared with 3 0 g m o l . m - a . s -a) and saturated at lower fluence rates (approx. 500 ~tmol. m -2"s-1 as opposed to no saturation up to approx. 1000 gmol" m - 2. s- 1 in Amaranthus). Although there is no spectrophotometric evidence for two populations of mung-bean phytochrome with differing degradation rate constants, the situation may, nevertheless, be not very different from that found in Amaranthus. It is only necessary to assume that the non-specific degradation of phytochrome in mung bean proceeds at a rate
lmmunochemical analyses. Photoprotection of phytochrome from degradation is also observable by immunoblot analysis (Fig. 6), providing direct evidence that the spectrophotometric assay is measuring loss ofphytochrome protein. The data in Fig. 6 confirm those in Table 1 for oat, pea, mung bean, and Amaranthus seedlings and show that in all cases high-fluence-rate WL (957 gmol' m - 2. s- 1) leads to substantially less phytochrome degradation than low-fluence-rate WL (3 g m o l . m 2. s-1). Since the immunoblot technique can be applied to tissues containing chlorophyll, it has been possible to assess the protective effect of prolonged exposure to sunlight. For etiolated oat seedlings, transfer to full sunlight ( 1 . 1 - 1 . 7 m m o l ' m - 2 ' s -1 during the exposure) leads to only limited phytochrome loss over an 8-h period, compared with substantial losses consequent upon transfer to filtered sunlight of only 2-3 g m o l . m 2.s-1 for the same period (Fig. 7). The extent of the inhibition of phytochrome degradation in full sunlight is perhaps most clearly seen when comparing immunoblots of extracts obtained from seedlings exposed to sunlight, or maintained in darkness for 8 h, and of extracts of the same tissues given a 5 rain pulse of F R and maintained in darkness for 16 h (Fig. 7). Clearly, only a limited pool of phytochrome is available for degradation in seedlings exposed to bright sunlight. This does not, however, mean that seedlings growing in the natural environment will necessarily possess high levels of phytochrome. Seeds germinating in sub-surface soil layers will be exposed to low fluence rates of light (Mandoli et al. 1982; Bliss and Smith 1985; Tester and Morris 1987), which should allow significant phytochrome degradation prior to seedling emergence. Furthermore, any elevated levels of phytochrome that may result from photoprotection afforded by the exposure of germinating seeds to bright sunlight should be quickly lost at the end of the first day and into the first night. The significance of these observations for plants growing under natural conditions relates to the ability of phytochrome to function at very high fluence rates. These data indicate that the effective Pfr concentration is progressively reduced as fluence rate rises from approx. 50 gmol- m - 2. s- 1 to approx. 1000 gmol. m - 2. s- 1. If phytochrome does operate in natural sunlight, therefore, either
476
H. Smith et al. : Photoprotection of phytochrome
Fig. 6a-d. Immunoblot analyses of phytochrome loss in seedlings exposed to A, high-fluence-rate (957 gmol.m z. s - l ) or B, low-fluence-rate (3 gmol. m-Z.s 1) WL. Etiolated seedlings of a Arena sativa, b Amaranthus caudatus, e Pisum sativum or d Phaseolus aureus
were irradiated for the times shown, after which crude extracts were prepared (1 g of tissue per ml buffer) and aliquots resolved on 8% polyacrylamide gels. Electroblotted proteins were probed with rabbit antiArena phytochrome antibodies (a, b, fl) or rat anti-Pisum phytochrome antibodies (e). k D a = k i l o d a l t o n
H. Smith et al. : Photoprotection of phytochrome
477
Fig. 7. Effect of daylight conditions on loss of immunochemically assayabte phytochrome in Arena sativa. Etiolated seedlings were exposed for various times to either filtered (reduced) sunlight (R) or full sunlight (S). At the indicated times, samples were prepared for immunoblotting as described in the legend for Fig. 6. The right-hand blot shows the effect of exposure to darkness (D), full sunlight (S), low-fluence-rate WL (L) or high-fluence-rate WL (H) for 8 h, followed by 5 min far-red light and a subsequent 16 h incubation in darkness, on immunochemically assayable phytochrome in A. sativa seedlings
the coupling between Pfr and the first element of the transduction chain is much more rapid than coupling to the degradative pathway, or action must require a mechanism not solely dependent on the concentration of Pfr. This view is not altered by the finding that in light-grown plants the predominant phytochrome species is different from that in etiolated plants (Abe et al. 1985; Shimazaki and Pratt 1985; Tokuhisa etal. 1985). Even for a relatively stable phytochrome molecule with a slow turnover, individual Pfr molecules will exist for only a short time under high fluence rates. The senior author (H.S.) is grateful for discussions with Dr. Eberhard Sch/ifer, in whose laboratory in Freiburg the first of the data reported here were obtained, during a brief working visit as long ago as 1984; subsequently, useful discussions were held with Dr. Richard Kendrick at Wageningen, whose advice is also gratefully acknowledged.
References Abe, H., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Characterization of green tissue-specific phytochrome isolated immunochemically from pea seedlings. Plant Cell Physiol. 26, 1387-1399 Bliss, D., Smith, H. (1986) Penetration of light into soil and its role in the control of seed germination. Plant Cell Environ. 8, 475-483 Brockmann, J., Sch/ifer, E. (1982) Analysis of Pfr destruction in Amaranthus caudatus L. - evidence for two pools of phytochrome. Photochem. Photobiol. 35, 555-558 Butler, W.L., Lane, H.C. (1965) Dark transformations of phytochrome in vivo. II. Plant Physiol. 40, 13-17 Fukshansky, L., Sch/ifer, E. (1983) Models in photomorphogenesis. In: Encyclopaedia of plant physiology N.S., vol. 16A: Photomorphogenesis, pp. 69-95, Shropshire, W., Jr., Mohr, E., eds. Springer, Berlin Helm, B., Jabben, M., Schfifer, E. (1981) Phytochrome destruc-
tion in dark- and light-grown Amaranthus caudatus seedlings. Photochem. Photobiol. 34, 89-93 Kendrick, R.E., Frankland, B. (1968) Kinetics of phytochrome decay in Amaranthus seedlings. Planta 82, 317 320 Kendrick, R.E., Spruit, C.J.P. (1972) Light maintains high levels of phytochrome intermediates. Nature 237, 281 282 Kendrick, R.E., Spruit, C.J.P. (1973) Phytochrome intermediates in vivo. I. Effects of temperature, light intensity, wavelength, and oxygen on intermediate accumulation. Photochem. Photobiol. 18, 1349-144 Kendrick, R.E., Spruit, C.J.P. (1977) Phototransformations of phytochrome. Photochem. Photobiol. 26, 201 204 Mandoli, D.F., Waldron, L., Nemson, J.A., Briggs, W.R. (1982) Soil light transmission: implications for pbytochrome-mediated responses. Carnegie Instn. Washington Yearb. 81, 32-34 McCurdy, D.W., Pratt, L.H. (1986a) Kinetics of intracellular redistribution of phytochrome in Arena coleoptiles after its conversion to the active, far-red-absorbing form. Planta 167, 330-336 McCurdy, D.W., Pratt, L.H. (1986b) Immunogold electron microscopy of phytochrome in Arena: Identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability. J. Cell Biol. 103, 2541-2550 Speth, V., Otto, V., Sch/ifer, E. (1986) Intracellular localisation of phytoehrome in oat coleoptiles by electron microscopy. Planta 168, 299-304 R/idiger, W. (1980) Phytochrome, a light receptor of photomorphogenesis. In: Structure and bonding, vol. 40, pp. 101-140, Hemmerich, P., ed. Springer, Berlin Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with rabbit polyclonaI and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Arena shoots. Planta 164, 333-344 Tester, M., Morris, C. (1987) The penetration of light through soil. Plant Cell Environ. 10, 281-286 Tokuhisa, J.G., Daniets, S.M., Quail, P.H. (1985) Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in lightgrown Arena sativa. Planta 164, 321-332 Received 26 November 1987; accepted 14 April 1988
