Planta (Bed.) 102, 286-293 (1972) 9 by Springer-Verlag 1972

Aspects of Phytochrome Decay in Etiolated Seedlings Under Continuous Illumination* I~ICHARD E. KI~NDRICK Biology Department, Brookhaven National Laboratory, Upton, New York Received August 18, 1971 Summary. The rate of total phytochrome decay in the dicotyledons Amaranthus caudatus, Mirabilis jalapa and Pisum sativum under continuous illmnination with red, incandescent, and blue light depends on the PFlC/Ptotal maintained by each source. Amaranthus is an exception to this in that there is a deviation from firstorder decay kinetics under contimmus illumination with incandescent light. This deviation is probably not related to the chlorophyll present in the Amaranthus sample since chlorophyll-rich Pisum buds have the same phytochrome decay rate as epicotyl tissue under continuous incandescent light. Reports of a prolonged lag phase before the onset of first-order decay kinetics of phytochrome in Pisum have not been confirmed and the small lag phase observed in the present work can be accounted for by the time required tm attain the PFR/Pto~alratio characteristic of blue light in a carotenoid rich tissue. In the monocotyledon, Avena sativa, and perhaps monocotyledons in general, decay rate is maximal at a low PFIr concentration and the decay curve is the same under continuous red, incandescent and blue light. This dicotyledon/monocotyledon difference with respect to saturation of phytochrome decay does not correlate with the other dicotyledon/monocotyledon difference, the presence or absence of dark reverions of PF~ to Ply, since the dicotyledons Amaranthus and Mirabilis that lack reversion still show no saturation of decay. Possible growth control by the PFR/Ptotal ratio is discussed in relation to environmental changes in light quality.

Introduction The properties of the photomorphogenic pigment of plants, p h y t o chrome, have been studied extensively in vivo (Itillman, 1967). The PFg form of p h y t o c h r o m e produced from P g b y red light is presumed active and decays (i. e. loses photoreversibility and therefore detectability), (Butler et al., 1963 ; deLint and Spruit, 1963) or reverts via a thermal dark reaction of PI~ (Butler et al., 1963; Hopkins and Itillman, 1965). D e c a y of P ~ is c o m m o n to e~iolated seedlings of both monoco~yledons and dicotyledons whereas reversion is limited to the dicotyledons, the order Centrospermae being an exception in t h a t it lacks reversion (Kendrick and Hfllman, 1971). The d e c a y process is temperature-dependent and * Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission.

Phytochrome Decay in Etiolated Seedlings

287

sensitive to metabolic inhibitors (Buttler and Lane, 1965). PFR decay is a first-order reaction in Amaranthus and the decay of total p h y t o c h r o m e under continuous illumination depends on the proportion maintained in the PF~ form: the PF~/Ptot~l ratio (Kendrick and Frank]and, 1968, 1969b). I n the monocotyledons, Zea (deLint and Spruit, 1963; Butler et al., 1963; P r a t t and Briggs, 1966) and Avena (Chorney and Gordon, t966; Dooskin a n d Maneinelli, 1968) decay appears to be saturated b y low levels of P ~ , above which, zero-order kinetics are followed. U n d e r continuous blue fight, decay has been demonstrated to depend on the P~R/Ptot~I ratio maintained in Amaranthus (Kendriek a n d Frank]and, t969b) and Mirabili8 (Kendrick a n d ttil]man, 1971). ~[owever, a lag phase before the onset of decay under blue fight has been reported b y Smith and Attridge (1970) for P i s u m buds. Chorney and Gordon (1966) have postulated the presence of some other blue-fight absorbing pigment in Avena which reacts with P~ to bring a b o u t decay. A deviation from first-order decay kinetics has been observed under continuous illumination with incandescent fight in Amaranthus (Kendrick and Frank]and, 1969b). I n the present paper an a t t e m p t is made to u n d e r s t a n d these deviations from simple decay kinetics. The decay of p h y t o e h r o m e has been compared under continuous illumination with red, incandescent and blue light in four species: the dicotyledons, Amaranthus caudatus, Mirabills ]alapa, P i s u m sativum, and the m o n o c o t y l e d o n Avcna sativa.

Materials and Methods Growth Procedures. All seedlings were grown and manipulations of plant material carried out in a temperature-controlled darkroom at 26 ~ C under a dim green safelight (Hopkins and Hillman, 1965). Amaranthus caudatus L. var. viridis (Love-liesbleeding) seed was obtained from Thompson and Morgan (Ipswich Ltd.), England. 200-seed samples were counted by means of a suctiondevice and sown on two layers of Whatman No. i filter paper in a 5-cm Petri dish moistened with 2 ml of distilled water. Arena sativa L. cv. Garry (oats) seed was obtained from Stanford Seed Co., Buffalo, New York. The seed was soaked for 4 h in distilled water and sown on moistened cellulose inpolyethylenebasins (19 • 29 • 10 cm). Thebasins were covered with cellophane (Saran wrap) to prevent evaporation. M~rabilis ~alapa L. (Four-o'cloeks) seed was obtained from W. Atlee Burpee Co., Philadelphia, Pennsylvania and was sown as previously described (Kendrick and Hillman, 1971). Seed of Pisum sativum L. cv. Alaska was obtained from Asgrow Seed Co., New Haven, Connecticut and was sown using the standard procedures previously described (Fox and Hillman, 1968). At the required age, samples were selected for phytoehrome experiments and these are summarized in Table 1. Apart from the case of Amaranthus in which whole seedlings were used, samples were placed in 5-cm Petri dishes on 2 Whatman No. 1 filter papers moistened with 2 ml of 0.02 M phosphate buffer, pH 6.2. Light Sources. Red light was obtained from three 15-W "Cool White" fluorescent tubes (Sylvania F15T8/CW) in combination with one layer of 3-mm-thiek red (No. 2444) Rohm and Haas plexiglas. With plant material 80 cm beneath this source,

288

R.E. Kendriek: Phytochrome Decay in Etiolated Seedlings Table 1. Summary o/samples and procedures used in phytochrome assay

Plant material

Age Sample (days)

No. of samples per euvette

Diameter of cuvette (ram)

Total phytochrome 103 A (A A)

Amaranthus caudatus

3

Whole seedling

200

13.0

18.8

Avena sativa

4

5-ram coleoptile apex without primary leaf

20

6.0

20.5

Mirabilis ]alapa

7

5-ram hypocotyl hook

12

6.0

35.0

Pisum sativum

7

5-ram apical epicotyl section

12

6.0

35.0

Pisum sativum

7

Bud

12

6.0

31.2

the PF/JPtotal ratio was assumed to be 0.80 (Butler and Lane, 1965). Incandescent light was obtained from four 100-W plus one 60-W incandescent lamps in combination with 10 em of water. With plant material 80 cm beneath this source, a 1)FI~/ Ptot~l ratio of 0.68 was maintained. Blue light was obtained from four 15-W "Blue" fluorescent tubes (Sylvania F15TS/B) in combination with one layer of 3-ram-thick blue (No. 2045) Rohm and Haasplexiglas. The eabinetwas linedwith aluminum foil to prevent fluorescence of the white paint used as a reflecting surface. With the plant material 80 cm beneath this source, a PFR/Ptotal ratio of 0.46 was maintained. Light cabinets were all used in a darkroom maintained at 26~ C and in experiments with continuous incandescent light, dishes were floated on a thermostatically controlled water bath to maintain this temperature. Phytochrome Assays. The total phytochrome of samples (Table 1) was measured throughout in the ASCO Ratiospect (Agricultural Specialities, Inc., Beltsville, Maryland) with wavelengths 735 and 800 nm as previously described (Kendrick and Hillman, 1971). The results are plotted as a proportion of the phytochrome initially present (P/Pc) against time. Experiments with Pisum to determine the time required to attain the PFz~/P~o~I ratio characteristic of blue light were carried out by exposing samples, maintained at 0 C by crushed ice, to vaxious intervals of blue light. The amount of PFR present was determined from the change in absorbance difference after an initial exposure to actinic far-red light.

Results Continuous Red Light (Fig. 1). U n d e r c o n t i n u o u s red light first order decay kinetics are followed b y Amaranthus, Mirabilis a n d Pisum, t h e a c t u a l decay c o n s t a n t v a r y i n g from species to species. I n the case of Pisum, p h y t o c h r o m e decay i n buds is the same as t h a t i n epicotyl tissue. I n c o n t r a s t to these dicotyledons, Avena p h y t o c h r o m e decay follows a curve on this logarithmic plot.

CONTINUOUS RED

T

---F--T--T--

f

F-

PISUM o bud

--

AMARANTH US

I.O - ~ o l e 0.8

seedling

0.6 0.4-

0.2 P/Po hO - 8 2 0.8

MIRAB]L]S hypocotyl hook

'k. "~_

0.6 0.4-

0 O.I

. 0

2

I

2

3 0 HOURS

I

2

3

Fig. 1. Decay of total phytoohrome under continuous illumination with red light in Amaranthus, r Mirabilis, and Pisu~z. Plotted as a proportion of total phytochrome remaining on a logarithmic scale (P/Pc) against time. PFB/Ptotal ratio maintained assumed to be 0.80 CONTINUOUS INCANDESCENT 1

I

I

AMARANTHUS

1.0 - ~ w h o ] e 0.80.6 _ ~

0.4

AVENA

seedling

m

"~

9

%

0.2 P/Po

t

I

1.0 _- "-~ 0.8 0.6

I

I

--

M1RABILIS

I

0.4

I

I

/

PISUM o bud

9 epicotyl

0.2 {

0

!

,

[

2

!

!

3 0 HOURS

I

,

f

2

i

3

Fig. 2. Decay of total phytoehrome under continuous illumination with incandescent light in Amaranthus, Avena, Mirabilis, and Pisum. Plotted as a proportion of total phytoehrome remaining on a logarithmic scale (P/Pc) against time. PFtdPtotal ratio maintained 0.68

290

R.E. Kendrick: CONTINUOUS BLUE I

I

I

AMARANTHUS 1.0 O.S 0.6 0.4

0.2 [

I

I

I

I

!

J

I

I

I

AVENA coleopfile

I

I

2

3

1.0 0.8 0.6 PISUM

0.4

o bud 9 epicotyI

0.2

0

I

2

3 0 HOURS

I

Fig. 3. Decay of total phytochrome under continuous illumination with blue light in Amaranthus, Avena, Mirabilis, and Pisum. Plotted as a proportion of total phytochrome remaining on a logarithmic scale (P/Pc) against ~ime. PFR/Ptotal ratio maintained 0.46, except for the Amaranthus data which were taken from Kendrick and Frankland (1969b) where the PFl~/Ptotal ratio maintained was 0.22

Continuous Incandescent Light (Fig. 2). Under continuous incandescent light first-order decay kinetics are followed b y Mirabilis and both buds and epicotyl tissue of Pisum. I t is confirmed t h a t in the case of Amaran. thus a deviation from first-order kinetics occurs (Kendrick and Frankland, 1969b). Arena phytochrome decay follows the same curve observed under red light. Continuous Blue Light (Fig. 3). Under continuous blue light firstorder decay kinetics are followed b y Amaranthus and Mirabilis. Decay in Pisum is the same in both buds and epicotyl tissue, becoming firstorder after a relatively small initial lag phase (Smith and Attridge, 1970). A possible explanation of the small lag phase is t h a t it represents the time required to attain the photostationary state, since Pisum tissue is rich in carotenoids t h a t could act as screening pigments, absorbing strongly in the blue region of the spectrum (B. Frankland, personal communication). That this is the case is shown b y Fig. 4, where a long time is

required to attain the PF~/Ptota] ratio characteristic of blue light, and where phytochrome in buds takes longer than that in epicotyls to reach the PF~/Ptotal ratio. Since the decay process in Pisum is relatively slow, this difference between buds and epicotyls is not reflected in the decay

Phytochrome Decay in Etiolated Seedlings i

I

i

I

i

I

I

i

f

I

i

L

291

ol

0.4

0.5 "5

.% o.2 m~ o.i 9 epicolyl

I 0

I

I 50

I

i

i 60 MINUTES

,

,

I 90

,

,

I 120

Fig. 4. Time course of attainment of the PFl~/Ptotal ratio under blue light in bud and epicotyl tissue of Pisum kinetics and a small lag phase of 15-30 rain is found in both cases. Avena phytochrome decay follows the same curve observed under both

continuous red and incandescent light. Discussion The present paper along with previous work (Kendrick and Frankland, 1968, 1969b; Marm@, 1969; Clarkson and Itillman, 1968) demonstrates that decay of total phytochrome in dicotyledons under continuous illumination is only limited by PF~ concentration, and is a first-order reaction after the P~/PtotaI ratio characteristic of the source is attained. In contrast to this, in monocotyledons so far investigated (Butler et al.,

1963; Pratt and Briggs, 1966; Chorney and Gordon, 1966; Dooskin and Mancinelli, 1968) it has been found that decay of P ~ in darkness follows zero order kinetics, and under continuous illumination is independent of the PF~/Ptota] ratio. The present results show identical decay kinetics in Arena under continuous illumination with red, incandescent and blue light: a wide range of PFl~/Ptotut ratios (Figs. 1-3), the decay process being limited by perhaps an essential eofaetor. In contrast to Smith and Attridge (1970), no prolonged lag phase was found in P i s u m before the onset of first-order decay kinetics under continuous blue illumination. This was true for both buds and epicotyl tissue, and the possibility of earotenoids acting as screening pigments, although prolonging the time to reach the PF~/Ptotal ratio in buds (Fig. 4), is not significantly reflected in the phytochrome decay curves (Fig. 3). Chorney and Gordon (1966) observed enhanced decay of phytochrome after exposure to blue light in A r e n a coleoptile tissue containing the primary leaf and suggested a pigment apart from phytochrome, ab-

292

R.E. Kendrick:

sorbing in the blue region of the spectrum, was involved in decay. The present results using coleoptfle tissue only show no evidence of enhanced decay under continuous blue light. Particular attention was paid in the present work to the possibility of blue light causing fluorescence within the light cabinets, either from paint or plastic surfaces. The white enamel paint used as a reflective surface in the light cabinet was found to fluoresce red under blue light (as observed through a red filter), and this was prevented by lining the cabinet with aluminum foil. This fluorescence can contribute appreciably to the PF~/P~otal maintained by blue light, and almost certainly explains the high values (0.55-0.60) which took a long time to attain and the apparent lag phase in decay curves observed previously with Pisum in this laboratory (D. T. Clarkson, personal communication). The deviation from first order decay kinetics observed in Amaranthus incandescent light confirms previous results (Kendrick and Frankland, 1969b). Mirabilis and Avena tissues show no deviations from predicted phytochrome decay curves. Chlorophyll either present or synthesized during the time of the experiment in Amaranthus could act as a screening pigment, preferentially absorbing light in the red region of the spectrum, thereby decreasing the PF~/Ptotal ratio maintained. This possibility was investigated using the chlorophyll-rich buds of Pisum and comparing them to epicotyl tissue. As can be seen from Fig. 2 there is no difference between either tissue, both following first-order kinetics. Apart from the possibility of some other unidentified screening pigment, unique to Amaranthus, this deviation remains unexplained. In conclusion, the decay process in monoeotyledons appears to be saturated by a low concentration of PF~, while in dicotyledons this is not the case and under continuous illumination the decay rate is proportional to the P~/Ptotal maintained. The dark reversion of PF~ to Px is not associated with this distinction since Amaranthus and Mirabilis are both dicotyledons lacking reversion (Kendrick and Frank]and, 1969b; Kendrick and Hillman, 1971). It is hoped that study of phytochrome decay under continuous illumination will give a key to the functional role of phytochrome in the control of plant growth and development. In nature the PFl~/Ptot~lratio maintained by daylight varies on a diurnal basis, as well as with local environment (e. g. beneath a dense leaf cover, where PF~/Ptot~l ratio is lower than in open sunlight [Kasperbauer, 1971]). Variation in the decay rate of phytochrome under continuous illumination is therefore a possible control mechanism for such processes as flowering, seed germination and growth. Control exercised by the PF~/Ptotal ratio has already been postulated for growth (Fox and I-Iillman, 1968) and seed germination (Kendrick and Frankland, 1969a) in laboratory experiments.

Phytochrome Decay in Etiolated Seedlings

293

I would like to thank Dr. W. S. Hillman for helpful discussions throughout the work and during the preparation of the manuscript. I am most grateful to Helen Kelly and Rosemarie Dearing for excellent technical assistance. References Butler, W. L., Lane, H. C. : Dark transformations on phytochromc in vivo. II. Plant Physiol. 40, 13-17 (1965). - - - - Siegelman, H. W.: Nonphotoehemical transformations of phytochrome in vivo. Plant Physiol. 38, 514-519 (1963). Chorney, W., Gordon, S. A. : Action spectrum and characteristics of light activated disappearance of phytochrome in oat seedlings. Plant Physiol. 41, 891-896 (1966). Clarkson, D. T., Hillman, W. S. : Stable concentrations of phytochrome under continuous illumination with red light. Plant Physiol. 43, 88-92 (1968). Dooskin, R. H., Mancinelli, A. L.: Phytochrome decay and eoleoptile elongation in Avena following various light treatments. Bull.Torrey bot. Club 95, 474-487 (1968). Fox, L. R., Hillman, W. S.: Response of tissue with different phytochrome contents to various initial photostationary states. Plant Physiol. 43, 823-826 (1968). Hillman, W. S.: The physiology of phytochrome. Ann. Rev. Plant Physiol. 18, 301-324 (1967). Hopkins, W. G. : Correlation of phytochrome transformation with photocontrol of Avena coleoptile segment elongation. Canad. J. Bot. 49, 467-470 (1971). - - Hillman, W. S.: Phytochrome changes in tissues of dark grown seedlings representing various photoperiodic classes. Amer. J. Bot. 52, 427-432 (1965). Kasperbauer, M. T.: Spectral distribution of light in a tobaceo canopy and effects of end-of-day light quality on growth and development. Plant Physiol 47, 775-778 (1971). Kendrick, R. E., Frankland, B.: Kinetics of phytochrome decay in Amaranthus seedlings. Planta (Berl.) 82, 317-320 (1968). - - - - Photocontrol of germination in Amaranthus caudatus. Planta (Berl.) 85, 326-339 (1969a). - - - - The in vivo properties of Amaranthus phytochrome. Planta (Berl.) 86, 21-32 (1969b). -Hillman, W. S. : Absence of phytochrome dark reversion in seedlings of the Centrospermae. Amer. J. Bot. 58, 424-428 (1971). Lint, P. J. A. L. de, Spruit, C. J. P. : Phytochrome destruction following illumination of mesocotyls of Zea mays L. Meded. Landbouwhogeschool Wageningen 6 3 , 1-7 (1963). Marm6, D. : Photometrische Messungen am Phytochromsystem yon Senfkeimlingen (Sinapis alba L.). Planta (Berl.) 99, 43-57 (1969). Pratt, L.M., Briggs, W. R.: Photochemical and nonphotochemical reactions of phytoehrome in vivo. Plant Physiol. 41, 467-474 (1966). Smith, H., Attridge, T.: Increased phenylalanine ammonia-lyase activity due to light treatment and its significance for the mode of action of phytochrome. Phytochemistry 9, 487-495 (1970). Dr. R. E. Kendrick Laboratorium voor Plantenphysiologisch Onderzoek Landbouwhogeschool Generaal Foulkesweg 72 Wageningen, The Netherlands 21 Planta (Berl.),Bd. 102