Planta (BEE.) 107, 341---350 (1972) 9 by Springer-Verlag 1972

Phytochrome Decay in Seedlings Under Continuous Incandescent Light* R. E. K e n d r i e k a n d C. J. P. S p r u i t Laboratory of Plant Physiological Research, Agricultural University Wageningcn, The Netherlands ** Received June 5, 1972

Summary. Under continuous high intensity incandescent light the decay of phytochrome in Amaranthus seedlings deviates from the predicted first order rate characteristic of the Pfr/Ptotal ratio maintained. This deviation takes the form of a slower decay than would be predicted and is only observed at high intensities. Experiments are presented to test the hypothesis that this reduced rate of decay is the result of a high level of phytochrome intermediates maintained under high intensity incandescent light. Accumulation of intermediates under these conditions has been demonstrated using a quasi-continuous measuring speetrophotometer. They are weakly absorbing and their concentration increases with light intensity. Although they form Pfr in darkness, it is proposed that they do not decay. The model predicts that in a sample cuvette, where a light intensity gradient exists, there is more probability of a phytochrome molecule being present as Pfr at the back of the euvette: the region of lowest light intensity. Under conditions which favour phytochrome decay, a preferential loss of phytochrome should result at the back of the cuvette and an increasingly higher proportion of the remaining phytochrome will consequently be measured as intermediate as the experiment progresses. The results confirm the hypothesis and in addition, after 60 min incandescent light, demonstrate an accumulation of intermediates which form Pfr with a longer half-life than at the begining of the experiment. Pisum epieotyl hooks show no such intermediate accumulation or preferential decay at the back of the cuvette, which is in agreement with the observed first order phytoehrome decay under high intensity incandescent light. A scheme is presented explaining the results on the basis of the decay process.

Introduction D e c a y of t h e p h o t o m o r p h o g e n e t i e F i g m e n t p h y t o c h r o m e is its loss of p h o t o r e v e r s i b i l i t y a n d has been s t u d i e d e x t e n s i v e l y in e t i o l a t e d seedlings. I n m a n y d i c o t y l e d o n s d e c a y of Pfr is a first-order r e a c t i o n a n d u n d e r c o n t i n u o u s i l l u m i n a t i o n t h e r a t e of d e c a y of t o t a l p h y t o chrome is p r o p o r t i o n a l to t h e p h o t o s t a t i o n a r y s t a t e (Pfr/Ptotal ratio) m a i n t a i n e d ( K e n d r i e k a n d F r a n k l a n d , 1968, 1969; K e n d r i c k , 1972;

* Abbreviations: F R = f a r - r e d light, R = r e d light, P=phytochrome, P f r = f a r red-absorbing form of P, Pr = red-absorbing form of P. ** 321st communication of this Laboratory.

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R.E. Kendrick and C. J. P. Spruit:

Marm~, 1969). One exception to this rule was found in seedlings of Amaranthus under high-intensity light from incandescent lamps (henceforth simply called incandescent light), where a progressive deviation from first-order d e c a y occurred taking the form of a decrease, with time, of the first order rate constant, predicted on the basis of the Pfr/Ptotal ratio (0.67) maintained (Kendrick, 1972). If low-intensity R and F R light were mixed to maintain the same Pfr/Ptot~l ratio, no deviation from first order decay was observed (Kendriek and Frankland, 1968). The recent observation t h a t high intensity incandescent light maintains high levels of p h y t o e h r o m e intermediates in Amaranthus (Kendriek and Spruit, 1972) offers a possible explanation of this deviation from first order decay. The hypothesis tested here is t h a t high intensity incandescent light maintains a high proportion of the p h y t o e h r o m e as intermediates, which although forming Pfr in darkness, are not sufficiently like Pfr to be reeognised b y the d e c a y process.

Materials and Methods Plant Material. Seeds of Amaranthus caudatus L. ear. viridis were sown on moist filter paper in plastic boxes and were germinated at 25~ in the dark. After 72 h, whole seedlings were used for phytoehrome estimations. Seedlings of Pisum 8ativum L. cv. Krombek were grown using the standard procedure described previously (Spruit and Raven, 1970). Sections of the epicotyl hook were used for phytochrome estimations. Estimations o/ Total Phytochrome. Decay experiments under low- and highintensity incandescent light were carried out using Amaranthus seedlings. The total phytochrome of 10-ram path length samples was measured using a dual wavelength difference spoctrophotometer (Spruit, 1970) with measuring beam wavelengths 735 and 806 nm. Estimations o/ Phytochrome Intermediates. The measurement of phytochrome intermediates was made using a quasi-continuous measuring spectrophotometer with measuring beam wavelengths 738 and 806 nm (Spruit, 1971; Kendrick and Spruit, 1972). This instrument enables measurement at any wavelength while exposing the sample to actinic light, in this case incandescent light from a quartz iodine lamp, filtered through 5 cm of water. The instrument is capable of detecting intermediates with a half-life (t89 in excess of 0.2 ms. This 0.2 ms represents the dead period between actinic and measuring beam flashes and a fraction of the intermediate already decays to Pfr during this period. The values quoted of percent total phytochrome maintained as intermediate should therefore be an underestimate. The intensity of the actinic incandescent light was 7.35 • 10a ~tW cm-2 at the front of the euvette. The average light intensity within the sample is considerably less and probably decreases exponentially during passage through the cuvette (Spruit and Kendrick, 1972). Samples used in this instrument consisted of a 10-ram path length of whole seedlings of Amaranthus or of Pisum epicotyl hooks. Results and Discussion The deviation f r o m first-order deeay of total p h y t o e h r o m e under incandescent light is shown clearly to depend on intensity (Fig. 1). The

Phytoehrome Decay Under Continuous Light

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level of phytochrome maintained as intermediate has also been demonstrated to be intensity dependent (Kendrick and Spruit, 1972). The intensity of light within the sample cuvette is of some interest in this respect. If we assume a light gradient from the front to the back of the cuvette (Fig. 2) we can make a few predictions on the basis of the levels of intermediates maintained. We would anticipate a higher proportion of the pigment at the front of the cuvette to be maintained as intermediate compared to the pigment at the back. I n other words, there is more probability of a phytoehrome molecule being present as Pfr at the back of the euvette. Therefore under conditions which favour decay, e.g. in air at 22~ we predict a preferential decay of phytoehrome at the back of the euvette, resulting after some time in a concentration gradient between the front and back of the sample. At that moment more phytoehrome will be present at the front part of the sample where the light intensity is highest. This means that the fraction of the remaining phytochrome present as intermediate should increase as phytochrome decay progresses. Fig. 3 shows results of such an experiment with Amaranthus at 22~ in air. Initially at the light intensity used, the

23 Planta(Berl.),Bd.107

344

R.E. Kendriek and C. J. P. Spruit:

CUVETTE

ACTINIC BEAM

SAMPLE

LIGHT INTENSITY

3 Fig. 2. Schematic representation of a euvette demonstrating the light intensity gradient in the sample from front to back

amount of intermediate was 11.8%. After monitoring total phytochrome b y a F R - R - F R cycle at the end of the experiment, the amount of intermediate was 23.7%, double the value at the begining of the experiment, agreeing with the hypothesis. However, the determinations of the amount of intermediates maintained immediately after the 60 min incandescent light and before the FR-I~-FR cycle were much higher (50.0 %) and they decayed to Pfr with a longer t89than initially. Another test of the hypothesis is possible ff phytochrome decay can be prevented. Under such conditions no increase in the percentage of total phytochrome measured as intermediate should occur under incandescent light, since preferential decay at the back of the cuvette cannot occur. The decay process requires oxygen (Butler and Lane, 1965) and therefore can be prevented b y passing nitrogen through the sample in place of air at 22~ Fig. 4 shows t h a t under these conditions there is no increase in the t89 of intermediates or in the percentage of intermediates after 60 min incandescent light. Decay can also be prevented b y keeping the seedlings at 0 ~C. Fig. 5 shows the results of this experiment, and although a higher level of intermediate is maintained, as has to be expected since we arc observing a thermal reaction, there is

Phytochrome Decay Under Continuous Light

345

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no increase in t89 or percentage of total p h y t o c h r o m e maintained as intermediates after 60 rain incandescent light. The results therefore confirm our hypothesis. However, one question remains: w h y is the deviation from first order decay only shown b y Amaranthus (Kendriok, 1972) ~. To s t u d y this point, we have done a similar experiment with Pisum epicotyl hooks at 22~ in air. The results are given in Table 1, and it is clear t h a t in Pisum there is no increase in t89 of intermediates or increase in percentage of total p h y t o c h r o m e maintained as intermediate after 60 min incandescent light. This agrees with the predictions from decay data (Kendrick, 1972). I t is tempting to conclude t h a t values for the 28*

346

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Fig. 4. As Fig. 3, but carried out at 22~ in nitrogen percentage intermediate maintained account for the differences between Amaranthus and Pisum. Although twice as much intermediate was measured in Amaranthus for the same light intensity and same p a t h length (Table 1), our lack of knowledge of the exact internal light distribution within the sample means we cannot be categorical about this point. Also in the case of phytochrome decay experiments whole seedlings or tissue sections are used, where the only light intensity gradient will be within each seedling or piece of tissue. W h a t appears clear is t h a t only Amaranth~s shows accumulation of intermediates with an increased t89 during exposure to incandescent light. This fact we feel

Phytochrome Decay Under Continuous Light I

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Fig. 5. As Fig. 3, but carried out at 0~ Table 1. Percentage intermediate maintained by incandescent light Species

Treatment

Initially

After 60 min incandescent

After 60 min incandescent and FR-R-FR

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is responsible for the p h y t o c h r o m e decay d e v i a t i o n i n Amaranthus, especially since it does n o t occur w h e n decay is prevented. Fig. 6 shows a difference s p e c t r u m for the process intermediate-->Pfr i n whole Amaranthus seedlings. I t is clear t h a t there are no negative

348

R.E. Kendrick and C. J. P. Spruit: J

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absorption changes associated with the formation of Pfr. One therefore has to conclude that the intermediates maintained are relatively weakly absorbing compared to Pfr. In Amaranthus seedlings there is a little chlorophyll present and values in the red region of the difference spectrum may be distorted (Grill, in press; Spruit, in press), but similar spectra for Pisum epicotyl-hook tissue, which is very low in chlorophyll, show only small, negative absorption changes. A scheme is proposed in Fig. 7 to account for the results. Pbl denotes the bleached phytochrome intermediate which is maintained under incandescent light and forms Pfr in darkness with a rate constant k 1. Pb] is probably the same intermediate form which has been observed in vitro b y Cross et al. (1968). We propose that in order to decay, Pfr combines with D (this could mean that it binds to a site, metabolite or coeffector) in the presence of oxygen. Once this has been achieved decay can occur (photoreversibility be lost). Before however decay has occurred, we propose that the PfrD complex can be driven into PblD, the intermediate corresponding to Pb]. PblD cannot undergo the decay process itself, but forms Pfr D in darkness with a rate constant k s which is less than k1. For this reason PblD gradually accumulates under highintensity incandescent light. If a short dark period is given after PbID has accumulated then PfrD formed can be driven back to PblD almost quantitatively. A longer dark period would ultimately lead to decay of PfrD. The observation that after a FR-R-FI% cycle, given to determine the total phytochrome at the end of 60 rain incandescent light (Fig. 3), the t89 of intermediate increases while the percentage maintained as intermediate decreases, is explained by one further reaction. This

Phytoehrome Decay Under Continuous Light (N2 or 0~

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reaction is the dissociation of the phytochrome-D complex when it is in the P~D form. This idea agrees with the general concept of the decay process showing recognition only for Pfr. We conclude that accumulation of phytoehrome as an intermediate (PDID), which itself does not decay, accounts for the gradual decrease in the first-order rate constant of phytochrome decay observed under high-intensity incandescent light in Amaranthus. I n Pisum no intermediate accumulates under high-intensity incandescent light and the first-order decay rate remains constant. The difference between Amaranthus and Pisum could be that in the case of Pisum when Pfr combines with D it decays before it can be driven into PD1D. Alternatively, if PD1D is formed, it could be that k2 is about equal to kr The possible effects of a high proportion of total phytochrome being maintained as intermediate under conditions of pigment cycling must now be seriously considered when attempting to interpret physiological experiments under such conditions.

Acknowledgement. R. E. K. was supported by ~ NATO fellowship. References

Butler, W. L., Lane, H.C.: Dark transformations of phytochrome in-vivo. II. Plant Physiol. 40, 13-17 (1965). Cross, D. R., Linschitz, H., Kashe, V., Tenenbaum, J. : Low-temperature studies on phytoehrome: Light and dark reactions in red and far-red transformation and new intermediate forms of phytochrome. Prec. natl. Acad. Sci. (Wash.) 61, 1095-1101 (1968). Grill, R.: The influence of chlorophyll on in-rive difference spectra of phytochrome. Planta (Berl.) (in press). Kendrick, R.E.: Aspects of phytochrome decay in etiolated seedlings under continuous illumination. Planta (Berl.) 102, 286-293 (1972).

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Kendrick and Spruit: Phytochrome Decay Under Continuous Light

Kendrick, R. E., Frankland, B.: Kinetics of phytochrome decay in Amaranthu8 seedlings. Planta (Berl.) 82, 317-320 (1968). Kendrick, R.E., l~rankland, B.: The in-vivo properties of Amaranthus phytochrome. Planta (BEE.) 86, 21-32 (1969). Kendrick, R. E., Spruit, C. J . P . : Light maintains high levels of phytochrome intermediates. Nature (Lond.) New Biol. 287, 281-282 (1972). Marm4, D.: Photometrische Messungen am Phytochromsystem yon Senfkeimlingen (Sinapis alba L.). PIanta (Berl.) 99, 43-57 (1969). Spruit, C. J. P.: Spectrophotometers for the study of phytochrome in-vivo, i~Ieded. Landbouwhogeschool Wageningen 70, No. 14 (1970). Spruit, C. J. P. : Sensitive quasi-continuous measurement of photoinduccd transmission changes. Meded. Landbouwhogeschool Wageningen 71, I~o. 21 (1971). Spruit, C. J. P. : Difference spectrum distortion in non-homogeneous pigment associations: Abnormal phytochrome spectra in-vivo. Bioch. Bioph. Acta (in press). Spruit, C. J.P., Kendriek, R. E. : On kinetics of phytochrome photoconversion in-vivo. Planta (Berl.) 108, 319-326 (1972). Spruit, C. J. P., Raven, C. W.: Regeneration of protochlorophyll in dark grown seedlings following illumination with red and far-red light. Aeta bot. neerl. 19, 165-174 (1970). R. E. Kendrick, C. J. P. Spruit Laboratory of Plant Physiological Research Agricultural University Generaal Foulkesweg 72 Wageningen, The Netherlands