Planta
Planta 135, 217-223 (1977)
9 by Springer-Verlag 1977
The Loss of Phytochrome Photoreversibility in vitro II. Properties of Killer and its Reaction with Phytochrome*
L.R. Fox Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA
Abstract. "Killer", a substance extracted from stem tissue of etiolated pea seedlings (Pisum sativum L. v. Alaska), interacts specifically with the far-red-absorbing form of phytochrome (Pfr) in vitro in a temperature-independent, rapid, stoichiometric fashion to cause a loss of phytochrome photoreversibility. The chromatographic, solubility, and spectral properties of partially purified fractions indicate that Killer is a cyclic, unsaturated molecule containing ionizible hydroxyl groups; its molecular weight is unknown, although probably low. Possible mechanisms by which the Killer-phytochrome interaction results in the loss of photoreversibility are discussed. Key words: Killer
Phytochrome - Pisum.
Introduction
The in-vivo phototransformation of the red- to the far-red-absorbing form of phytochrome initiates nonphotochemical reactions (reversion and destruction) which, at least in etiolated tissue, result in decreased levels of assayable far-red-absorbing pigment. The loss of this form of the pigment by the destruction reaction is accompanied by a net decrease in spectrophotometrically detectable phytochrome (Butler and Lane, 1965; Dooskin and Mancinelli, 1968; Furuya, 1968; Hillman, 1967; Kendrick and Frankland, 1969), presumably by converting far-red-absorbing phytochrome to a from which is no longer photoreversible by what appears to be temperature-sensitive, metaldependent, oxidative reaction (Butler et al., 1963; Furuya and Hillman, 1964; Furuya et al., 1965; Kendrick and Frankland, 1969; Manabe and Furuya, 1971; Pratt and Briggs, 1966). The destruction reac*
I : F o x , 1975
tion may account for a loss of up to 80% of the spectrophotometrically detectable phytochrome in etiolated plant parts (Butler and Lane, 1965; Pratt and Briggs, 1966) but essentially nothing is known about its mechanism or product(s) since an in-vitro reaction analogous to that in-vivo has not yet been observed, perhaps because the chemicals normally used to stabilize phytochrome preparations (Briggs and Rice, 1972; Briggs et al., 1968; Butler and Lane, 1965; Furuya, 1968) strongly inhibit the in-vivo destruction reaction (Furuya et al., 1965). A loss of spectrophotometrically detectable phytochrome in vitro has been observed when extracts were exposed to denaturing agents, sulfhydryl reagents and proteolytic enzymes (Butler et al., 1964), to glutaraldehyde and other monoaldehydes (Roux and Hillman, 1969), and to certain metal ions (Lisansky and Galston, 1974). Crude phytochrome extracts from etiolated seedlings contained a substance which reacted with far-red-absorbing phytochrome to cause a loss of photoreversible phytochrome in-vitro (Furuya and Hillman, 1966). This substance, called Killer, was subsequently extracted and partially purified from this tissue (Fox, 1975). The properties of Killer and its interaction with phytochrome in vitro are the subject of this communication. Work on this problem has ceased in this laboratory. It is hoped that the presentation of these observations will excite the curiosity and interest of students of phytochrome behavior in vitro to a point that the identity of Killer and its mechanism of reaction with phytochrome will become known.
Materials and Methods Seeds of Pisum sativum L. cv. Alaska (Asgrow Seed Co., Thomasville, Ga., USA) were sown as previously described (Fox, 1975), and the seedlings were allowed to develop at 26 28~ in darkness
218 for 7 days. Seeds of Arena sativa L. cv. Clintland (Southeastern States Cooperative, Baltimore, Md., USA) were sown dry in polypropylene trays on vermiculite (Zonolite No. 1) saturated with 1/10-strength Hutner's medium (Furuya and Hillman, 1964), were covered to a depth of 2-3 cm with saturated vermiculite, and seedlings were allowed to develop at 26-28~ in darkness for 5 days. Killer was extracted by grinding etiolated pea-stem tissue in absolute methanol (320 ml/100 g fresh weight), filtering the brei, and removing the methanol by flash-evaporation (bath temperature=36~ The resulting aqueous concentrate was clarified by adding CaC12 to a final concentration of 0.01 M and centrifuging for 20 min at 22,000 x g, yielding what is referred to herein as Killer Fraction A. Killer Fraction B was prepared by partitioning Fraction A 6 times against an equal volume of water-saturated n-butanol, removing the n-butanol by flash evaporation, and dissolving the residue in distilled water. All fractions to be tested were adjusted to pH 5.0-6.0 (Fox, 1975). Crude Arena phytochrome extracts were prepared weekly as previously described (Fox, 1975). For the Standard Killer Assay, 1.0 ml of either the fraction to be tested or water was added to 3.0 ml crude Arena phytochrome extract. The assay tubes were warmed to room temperature in the dark for 10 min, exposed to 10 min of red light, and incubated in the dark for 50 rain. After incubation, the tubes were cooled on ice for 10 rain, centrifuged (10 min at 12,000 xg), exposed to 10 rain actinic red light on ice, and the phytochrome content of 1.0-ml aliquots of each tube determined as described by Furuya and Hillman (1964). A fraction was deemed to contain Killer when the addition of that fraction resulted in a decrease in the amount of spectrophot0metrically detectable phytochrome, relative to the addition of water, as determined using a Ratiospect difference spectrophotometer (Agricultural Specialties Co., Beltsville, Md., USA). All manipulations were performed under dim green safelights. The red and green light sources were the same as previously described (Fox, 1975). Far-red light was obtained from one 150-W incandescent flood lamp over 10 12 cm of water and a 0.3-cm thickness of Rohm and Haas FRF-700 Plexiglas (fabricated by Westlake Plastic Co., Lenni Mills, Pa., USA). The following abbreviations are used throughout the text: Pfr=far-red-absorbing form of phytochrome, Pr=red-absorbing form of phytochrome; Ptot (total photoreversible phytochrome) = A (AOD) x 103; AP (Killer activity)=Ptot (water addendum) minus Ptot (fraction tested addendum); R = r e d light; F R = f a r - r e d light; D = d a r k ; ( F R 1 - R t ) = P t o t determined from the first actinic photoreversal; ( F R 2 - R 1 ) = P t o t determined from the second actinic photoreversal. All Ptot values in the Standard Killer Assay represent the mean of 8 determinations, while those in experiments employing first or second actinic photoreversals represent the mean of 4-5 determinations. Student's t-tests were used to determine the statistical significance of observed differences.
Results
Properties of Killer The organic nature of Killer was established by drying a 10-ml sample of Killer Fraction A in a crucible at 98 +_l~ for 24 h. The evaporated sample (0.880 g) was ashed over a Fischer burner for 1 h, and the residue (0.068 g) was redissolved in water, adjusted to pH 5.6, and assayed for Killer activity (Table 1). While there was significant (p0.2). The molecule(s) responsible for Killer activity is relatively stable, as indicated by the data of Table 2. A sample of Killer Fraction B stored in the refrigerator at 2~ underwent no change in Killer activity over a period of 35 days. The relative acid-base solubility of partially purified Killer in aqueous and methanolic solution is shown in Figure 1. Aqueous aliquots of Killer Fraction B were treated as indicated. Precipitates which formed were removed by centrifugation and the pellets redissolved in water using the appropriate pH conditions. To determine Killer behavior in methanolic solution, an aliquot of aqueous Killer Fraction B was dried by flash-evaporation, and the residue was dissolved in absolute methanol and subjected to the acid-base treatments indicated in Figure 1. The resulting methanolic fractions were flash-evaporated to dryness and the residues redissolved in water. The pH of all fractions was adjusted to 5.5-6.0 (Fox, 1975) before the fractions were assayed for Killer activity (Fig. 1). Significant Killer activities were found only in Fractions I, III and VII. The values differ among the active fractions because three different preparations of Killer Fraction B were used. From these results it appeats as if Killer is acid-insoluble in
L.R. Fox: "Killer" and its Reaction with Phytochrome FRACTION
I
219
B
j
H2 0
Me OH
[
J
I
Na OH
pH 12 no ppt.
NJoH pH 12
HCt pH 2
I
]
HC[ pH 2
I
ii
i
• -34.8
i~ + 0.2
ii
i
J1 I -42.1
J..Ev
v
* 0.2
-1.1
a q u e o u s solution and acid-soluble in methanolic solution. F u r t h e r m o r e , extremes o f pH, at least for short periods o f time, did not appear to alter Killer activity appreciably. C h r o m a t o g r a p h y o f crude pea p h y t o c h r o m e preparations on Sephadex G-50 was used to separate p h y t o c h r o m e (which was excluded) f r o m Killer (Furuya and Hillman, 1966). C o n t i n u e d elution of the c o l u m n yielded a fraction which, when added to column-purified p h y t o c h r o m e , caused a loss o f photoreversibility. Thus, Killer appeared to be retained on Sephadex G-50 colums. Aliquots (20 ml) o f Killer Fraction B were placed on a 5 c m • 34 cm column o f Sephadex G-25 (medium grade) which had been p a c k e d and washed with distilled water. The column was eluted with distilled water at a flow rate of 0.6-0.8 ml/min, 9.4-9.5-ml fractions collected, the optical density at 270 n m o f all fractions determined, and peak fractions assayed for Killer activity. A typical elution profile is presented in Figure 2. Killer activity eluted with the first peak at a position which
Fig. 1. Killer acid-base solubility in aqueous and methanolJc solution. Methanolic fractions were flash-evaporated to dryness and redissolved in distilled I HC[ water. All fractions were adjusted to pH pH2 5.5-6.0 before assay in a Standard Killer Assay. Numbers beneath fraction I i designations represent the Killer activity l] f_2.1 v V__L (AP) and are the means of 8 determinations -13.2 on 2 samples -0.4
c o r r e s p o n d e d to the exclusion volume o f the column, as determined by previous calibration with 0.5% (w/v) Blue D e x t r a n 2000 (Pharmacia Fine Chemicals). The fraction purified on Sephadex G-25 had a fairly b r o a d ultraviolet absorption m a x i m u m in aqueous solution (pH 5.4) centered a r o u n d 2 9 2 n m (Fig. 3, curve A), and the addition o f a few drops o f 1 N K O H caused a b a t h o c h r o m i c shift o f 2 0 - 2 2 n m (Fig. 3, curve B). Acid precipitation o f this fraction yielded an active Killer fraction which had an ultraviolet absorption m a x i m u m in aqueous solution ( p H 5 . 6 ) between 2 8 5 n m and 2 8 7 n m (Fig. 3, curve C), although thin-layer c h r o m a t o g r a p h y indicated that this fraction was not homogeneous. Acid precipitation did not alter the Sephydex G-25 elution profile of Killer.
0,8
>_ 0 . 6 o0 z w c~
2.0
! "~
j
1.5
%
v
x o_
z~
0.4
? v
LO
0.2
50
~J 0.5
ooL.-.--
o
0.0 0
50 FRACTION NUMBER
IO0 ( 9 . 4 - 9 . 5 m[/FRACTJON)
Z:
150
Fig. 2. Sephadex G-25 chromatography of Killer Fraction B. A 20-ml aliquot was eluted from a 5 cm x 34 cm column in distilled water at a flow rate of 0.6-0.8 mI/min. Solid bars on the abscissa represent Killer activity of fractions as determined in a Standard Killer Assay
2;o
~o WAVELENGTH
~;o
~io
'
~o
{nm)
Fig. 3. Ultra-violet absorption spectra of Sephadex G-25 purified Killer. Curve A, spectrum of pooled active fractions from column in aqueous solution at pH 5.4. Curve B, spectrum of A. after the addition of a few drops of 1 N KOH. Curve C, spectrum in aqueous solution (pH 5.6) following acid precipitation of pooled active fractions from column
220
L.R. Fox: " K i l l e r " and its Reaction with Phytochrome
The Killer-Phytochrome Interaction
.
I~
23 ~ C
4-0
Aliquots of serially diluted samples of Killer Fractions A and B were added to samples of crude Arena phytochrome preparations and assayed for activity using the Standard Killer Assay (Fig. 4). There appeared to be a linear relationship between the amount of Killer added and the loss of phytochrome photoreversibility in vitro, indicating a specific interaction between phytochrome and Killer. The regression lines were calculated by the least squares method, and a test of significance of the regression line yielded a p
Planta 135, 217-223 (1977)
9 by Springer-Verlag 1977
The Loss of Phytochrome Photoreversibility in vitro II. Properties of Killer and its Reaction with Phytochrome*
L.R. Fox Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA
Abstract. "Killer", a substance extracted from stem tissue of etiolated pea seedlings (Pisum sativum L. v. Alaska), interacts specifically with the far-red-absorbing form of phytochrome (Pfr) in vitro in a temperature-independent, rapid, stoichiometric fashion to cause a loss of phytochrome photoreversibility. The chromatographic, solubility, and spectral properties of partially purified fractions indicate that Killer is a cyclic, unsaturated molecule containing ionizible hydroxyl groups; its molecular weight is unknown, although probably low. Possible mechanisms by which the Killer-phytochrome interaction results in the loss of photoreversibility are discussed. Key words: Killer
Phytochrome - Pisum.
Introduction
The in-vivo phototransformation of the red- to the far-red-absorbing form of phytochrome initiates nonphotochemical reactions (reversion and destruction) which, at least in etiolated tissue, result in decreased levels of assayable far-red-absorbing pigment. The loss of this form of the pigment by the destruction reaction is accompanied by a net decrease in spectrophotometrically detectable phytochrome (Butler and Lane, 1965; Dooskin and Mancinelli, 1968; Furuya, 1968; Hillman, 1967; Kendrick and Frankland, 1969), presumably by converting far-red-absorbing phytochrome to a from which is no longer photoreversible by what appears to be temperature-sensitive, metaldependent, oxidative reaction (Butler et al., 1963; Furuya and Hillman, 1964; Furuya et al., 1965; Kendrick and Frankland, 1969; Manabe and Furuya, 1971; Pratt and Briggs, 1966). The destruction reac*
I : F o x , 1975
tion may account for a loss of up to 80% of the spectrophotometrically detectable phytochrome in etiolated plant parts (Butler and Lane, 1965; Pratt and Briggs, 1966) but essentially nothing is known about its mechanism or product(s) since an in-vitro reaction analogous to that in-vivo has not yet been observed, perhaps because the chemicals normally used to stabilize phytochrome preparations (Briggs and Rice, 1972; Briggs et al., 1968; Butler and Lane, 1965; Furuya, 1968) strongly inhibit the in-vivo destruction reaction (Furuya et al., 1965). A loss of spectrophotometrically detectable phytochrome in vitro has been observed when extracts were exposed to denaturing agents, sulfhydryl reagents and proteolytic enzymes (Butler et al., 1964), to glutaraldehyde and other monoaldehydes (Roux and Hillman, 1969), and to certain metal ions (Lisansky and Galston, 1974). Crude phytochrome extracts from etiolated seedlings contained a substance which reacted with far-red-absorbing phytochrome to cause a loss of photoreversible phytochrome in-vitro (Furuya and Hillman, 1966). This substance, called Killer, was subsequently extracted and partially purified from this tissue (Fox, 1975). The properties of Killer and its interaction with phytochrome in vitro are the subject of this communication. Work on this problem has ceased in this laboratory. It is hoped that the presentation of these observations will excite the curiosity and interest of students of phytochrome behavior in vitro to a point that the identity of Killer and its mechanism of reaction with phytochrome will become known.
Materials and Methods Seeds of Pisum sativum L. cv. Alaska (Asgrow Seed Co., Thomasville, Ga., USA) were sown as previously described (Fox, 1975), and the seedlings were allowed to develop at 26 28~ in darkness
218 for 7 days. Seeds of Arena sativa L. cv. Clintland (Southeastern States Cooperative, Baltimore, Md., USA) were sown dry in polypropylene trays on vermiculite (Zonolite No. 1) saturated with 1/10-strength Hutner's medium (Furuya and Hillman, 1964), were covered to a depth of 2-3 cm with saturated vermiculite, and seedlings were allowed to develop at 26-28~ in darkness for 5 days. Killer was extracted by grinding etiolated pea-stem tissue in absolute methanol (320 ml/100 g fresh weight), filtering the brei, and removing the methanol by flash-evaporation (bath temperature=36~ The resulting aqueous concentrate was clarified by adding CaC12 to a final concentration of 0.01 M and centrifuging for 20 min at 22,000 x g, yielding what is referred to herein as Killer Fraction A. Killer Fraction B was prepared by partitioning Fraction A 6 times against an equal volume of water-saturated n-butanol, removing the n-butanol by flash evaporation, and dissolving the residue in distilled water. All fractions to be tested were adjusted to pH 5.0-6.0 (Fox, 1975). Crude Arena phytochrome extracts were prepared weekly as previously described (Fox, 1975). For the Standard Killer Assay, 1.0 ml of either the fraction to be tested or water was added to 3.0 ml crude Arena phytochrome extract. The assay tubes were warmed to room temperature in the dark for 10 min, exposed to 10 min of red light, and incubated in the dark for 50 rain. After incubation, the tubes were cooled on ice for 10 rain, centrifuged (10 min at 12,000 xg), exposed to 10 rain actinic red light on ice, and the phytochrome content of 1.0-ml aliquots of each tube determined as described by Furuya and Hillman (1964). A fraction was deemed to contain Killer when the addition of that fraction resulted in a decrease in the amount of spectrophot0metrically detectable phytochrome, relative to the addition of water, as determined using a Ratiospect difference spectrophotometer (Agricultural Specialties Co., Beltsville, Md., USA). All manipulations were performed under dim green safelights. The red and green light sources were the same as previously described (Fox, 1975). Far-red light was obtained from one 150-W incandescent flood lamp over 10 12 cm of water and a 0.3-cm thickness of Rohm and Haas FRF-700 Plexiglas (fabricated by Westlake Plastic Co., Lenni Mills, Pa., USA). The following abbreviations are used throughout the text: Pfr=far-red-absorbing form of phytochrome, Pr=red-absorbing form of phytochrome; Ptot (total photoreversible phytochrome) = A (AOD) x 103; AP (Killer activity)=Ptot (water addendum) minus Ptot (fraction tested addendum); R = r e d light; F R = f a r - r e d light; D = d a r k ; ( F R 1 - R t ) = P t o t determined from the first actinic photoreversal; ( F R 2 - R 1 ) = P t o t determined from the second actinic photoreversal. All Ptot values in the Standard Killer Assay represent the mean of 8 determinations, while those in experiments employing first or second actinic photoreversals represent the mean of 4-5 determinations. Student's t-tests were used to determine the statistical significance of observed differences.
Results
Properties of Killer The organic nature of Killer was established by drying a 10-ml sample of Killer Fraction A in a crucible at 98 +_l~ for 24 h. The evaporated sample (0.880 g) was ashed over a Fischer burner for 1 h, and the residue (0.068 g) was redissolved in water, adjusted to pH 5.6, and assayed for Killer activity (Table 1). While there was significant (p0.2). The molecule(s) responsible for Killer activity is relatively stable, as indicated by the data of Table 2. A sample of Killer Fraction B stored in the refrigerator at 2~ underwent no change in Killer activity over a period of 35 days. The relative acid-base solubility of partially purified Killer in aqueous and methanolic solution is shown in Figure 1. Aqueous aliquots of Killer Fraction B were treated as indicated. Precipitates which formed were removed by centrifugation and the pellets redissolved in water using the appropriate pH conditions. To determine Killer behavior in methanolic solution, an aliquot of aqueous Killer Fraction B was dried by flash-evaporation, and the residue was dissolved in absolute methanol and subjected to the acid-base treatments indicated in Figure 1. The resulting methanolic fractions were flash-evaporated to dryness and the residues redissolved in water. The pH of all fractions was adjusted to 5.5-6.0 (Fox, 1975) before the fractions were assayed for Killer activity (Fig. 1). Significant Killer activities were found only in Fractions I, III and VII. The values differ among the active fractions because three different preparations of Killer Fraction B were used. From these results it appeats as if Killer is acid-insoluble in
L.R. Fox: "Killer" and its Reaction with Phytochrome FRACTION
I
219
B
j
H2 0
Me OH
[
J
I
Na OH
pH 12 no ppt.
NJoH pH 12
HCt pH 2
I
]
HC[ pH 2
I
ii
i
• -34.8
i~ + 0.2
ii
i
J1 I -42.1
J..Ev
v
* 0.2
-1.1
a q u e o u s solution and acid-soluble in methanolic solution. F u r t h e r m o r e , extremes o f pH, at least for short periods o f time, did not appear to alter Killer activity appreciably. C h r o m a t o g r a p h y o f crude pea p h y t o c h r o m e preparations on Sephadex G-50 was used to separate p h y t o c h r o m e (which was excluded) f r o m Killer (Furuya and Hillman, 1966). C o n t i n u e d elution of the c o l u m n yielded a fraction which, when added to column-purified p h y t o c h r o m e , caused a loss o f photoreversibility. Thus, Killer appeared to be retained on Sephadex G-50 colums. Aliquots (20 ml) o f Killer Fraction B were placed on a 5 c m • 34 cm column o f Sephadex G-25 (medium grade) which had been p a c k e d and washed with distilled water. The column was eluted with distilled water at a flow rate of 0.6-0.8 ml/min, 9.4-9.5-ml fractions collected, the optical density at 270 n m o f all fractions determined, and peak fractions assayed for Killer activity. A typical elution profile is presented in Figure 2. Killer activity eluted with the first peak at a position which
Fig. 1. Killer acid-base solubility in aqueous and methanolJc solution. Methanolic fractions were flash-evaporated to dryness and redissolved in distilled I HC[ water. All fractions were adjusted to pH pH2 5.5-6.0 before assay in a Standard Killer Assay. Numbers beneath fraction I i designations represent the Killer activity l] f_2.1 v V__L (AP) and are the means of 8 determinations -13.2 on 2 samples -0.4
c o r r e s p o n d e d to the exclusion volume o f the column, as determined by previous calibration with 0.5% (w/v) Blue D e x t r a n 2000 (Pharmacia Fine Chemicals). The fraction purified on Sephadex G-25 had a fairly b r o a d ultraviolet absorption m a x i m u m in aqueous solution (pH 5.4) centered a r o u n d 2 9 2 n m (Fig. 3, curve A), and the addition o f a few drops o f 1 N K O H caused a b a t h o c h r o m i c shift o f 2 0 - 2 2 n m (Fig. 3, curve B). Acid precipitation o f this fraction yielded an active Killer fraction which had an ultraviolet absorption m a x i m u m in aqueous solution ( p H 5 . 6 ) between 2 8 5 n m and 2 8 7 n m (Fig. 3, curve C), although thin-layer c h r o m a t o g r a p h y indicated that this fraction was not homogeneous. Acid precipitation did not alter the Sephydex G-25 elution profile of Killer.
0,8
>_ 0 . 6 o0 z w c~
2.0
! "~
j
1.5
%
v
x o_
z~
0.4
? v
LO
0.2
50
~J 0.5
ooL.-.--
o
0.0 0
50 FRACTION NUMBER
IO0 ( 9 . 4 - 9 . 5 m[/FRACTJON)
Z:
150
Fig. 2. Sephadex G-25 chromatography of Killer Fraction B. A 20-ml aliquot was eluted from a 5 cm x 34 cm column in distilled water at a flow rate of 0.6-0.8 mI/min. Solid bars on the abscissa represent Killer activity of fractions as determined in a Standard Killer Assay
2;o
~o WAVELENGTH
~;o
~io
'
~o
{nm)
Fig. 3. Ultra-violet absorption spectra of Sephadex G-25 purified Killer. Curve A, spectrum of pooled active fractions from column in aqueous solution at pH 5.4. Curve B, spectrum of A. after the addition of a few drops of 1 N KOH. Curve C, spectrum in aqueous solution (pH 5.6) following acid precipitation of pooled active fractions from column
220
L.R. Fox: " K i l l e r " and its Reaction with Phytochrome
The Killer-Phytochrome Interaction
.
I~
23 ~ C
4-0
Aliquots of serially diluted samples of Killer Fractions A and B were added to samples of crude Arena phytochrome preparations and assayed for activity using the Standard Killer Assay (Fig. 4). There appeared to be a linear relationship between the amount of Killer added and the loss of phytochrome photoreversibility in vitro, indicating a specific interaction between phytochrome and Killer. The regression lines were calculated by the least squares method, and a test of significance of the regression line yielded a p
