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The inactivating effect of visible light on thermosensitized Ophiostoma cells
N. Fries
202
REFERENCES J. A. and CALVIN, M., The Path of Carbon in Photosynthesis. Englewood Cliffs, N.J., 1957.
1. BASSHAM, 2. FRIES,
3. 4. 5. 6. 7.
N., Physiol.
Planf.
Prentice Hall, Inc.,
2, 78 (1949).
__
ibid. 16, 415 (1963). GEZELIUS, K. and RANBY, B. G., Eq~ptl Cell Res. 12, 265 (1957). MURASHIGE, T. and SKOOG, F., Physiol. Plant. 15, 473 (1962). NYMAN, B., Suensk Botan. Tidskr. 55, 129 (1961). SUNDSTR~M, K.-R., Phytopathol. 2. 40, 213 (1960).
THE
INACTIVATING
EFFECT
THERMOSENSITIZED
OF VISIBLE
OPHIOSTOMA
LIGHT
ON
CELLS
N. FRIES Institute of Physiological Botany, University of Uppsala, Sweden Received July 30, 1963
the response of thermosensitized Ophiostoma cells to various chemical and physical agents it was observed that the cells showed an increased sensitivity not only to temperatures above 28°C but also to illumination with visible light. Since this quite unexpected effect of light seemedto deserve someattention a series of experiments was performed, the results of which are summarized in the present communication. Cells from vigorously budding conidial cultures of Ophiostoma multiannulatum (Hedge. & Davids.) were used as material for all the experiments. The cells were produced at 25°C in shake flask cultures with the liquid, synthetic “medium 3” ( =N6, or “Fries medium”), which is commonly used for Ophiostoma [I]. For particulars of the cultivation technique and mode of inducing thermosensitivity reference may be made to an earlier paper [a]. Thermosensitivity was induced (the “heat shock”) by heating the conidia for 2 min at 43°C according to procedure (b) in the paper referred to. Illumination with visible light was provided by the apparatus used by Kihlman [3]. Thus the light source consisted of six Philips “Attralux” lamps, type 13378 E/44, 24 V, 150 W. During illumination the conidia were suspendedin 25 ml glassbottles, 10 ml suspensionin each, through which air or oxygen-free nitrogen was bubbled from 10 min before starting the experiment and during the whole period of treatment. The suspensionwas made in liquid medium 3 and contained IO6 cells/ml. The bottles were immersed in a glass-walled water bath, which was kept at 25°C or 30°C. They were illuminated from two sidesat a distance of 15 cm from the lamps. After this treatment the cells were immediately plated out in medium-3-agar. During this procedure the laboratory was kept as dark as possible. The plates were
IN studying
Experimental
Cell Research 32
Effect
of visible
light
on thermosensitized
Ophiostoma
cells
203
then incubated at 25°C and 30°C. Each experimental series comprised five plates. The number of developing mycelia was scored after four days. If conidia thermosensitized by heat shock were illuminated with white light under the described conditions a decrease in the number of survivors always occurred, as TABLE
I.
The effect of illumination
with white light on preheated Ophiostoma cells,
(thermosensitized)
Control= the cellsgerminatingin agar at 25°Cafter having in darkness with air as the gas phase. mean values given in the table.
The
standard
been heat-shocked and then kept in no case exceeded 25 per cent of the
error
Per cent
Gas phase Air Air N, N, Air Air N* NZ
TABLE
Illumination conditions
Following incubation temp. OC
Dark Light Dark Light
25 25 25 25
Dark Light Dark Light
30 30 30 30
II.
survivors
I Illumination temperature
Expt 1 100 72 92 53 49 33 47 5.3
The effect of illumination (thermosensitized)
Illumination temperature
25°C Expt
Expt 3
2
100
100
71 74 42
56 74 7.1
7.3 3.5 3.6 1.3
Expt
30°C 4
100 53 117 0
16 4.0 6.9 0.2
7.1 5.7 10 0
Expt
5
100 71 118 3.6 2.2 0.8 4.6 0.1
with red and blue light on preheated Ophiostoma cells.
The red filter transmitted the wavelengths above 5900 A and c. 10 per cent of the wavelengths between 4100 and 4600 A; the blue filter transmitted 3500-5000 A and > 6900 A. The filters were made of cellophane by Sateri OY, Valkeakoski, Finland. The illumination was performed at 30°C in nitrogen atmosphere.
Illumination conditions
Following incubation temp. “C
Per cent Expt
6
No light Red light Blue light White light
25 25 25 25
100 33 7.0 1.4
No light Red light Blue light White light
30 30 30 30
7.0 2.4 0.7 0.3
survivors Expt
7
100 90 5.0 6.6 -
Experimental
Cell Research 32
N. Fries can be seen from Table I. This decrease was evident both in the plates incubated at 25°C and in those incubated at 30°C. In the latter case, however, all figures were lower because of the inactivating influence of the higher temperatures on the thermosensitized cells. Table I also shows that the effect of the white light was considerably stronger in the absence than in the presence of oxygen and perhaps somewhat stronger when the illumination had been performed at 30°C than at 25°C. In order to obtain at least an idea of which wavelengths were most effective, experiments were performed with three illumination chambers, one of which was illuminated with white, one with red and one with blue light. The gas phase consisted of nitrogen in all chambers including the unilluminated control chamber. As shown in Table II, the blue light had about the same inactivating effect as the white light. Some effect was also obtained with the red light; it must be remembered, however, that the red filter also transmitted a certain amount of blue light. The effect of visible light here described resembles photoreactivation in so far as it is independent of oxygen and seems to be due to a blue-absorbing photoreceptor, but differs by being an inactivation instead of a reactivation process. Unlike the phenomenon of photodynamic action, on the other hand, this photoinactivation of thermosensitized cells is independent of oxygen and occurs without an added absorbing pigment. An elucidation of the biochemical mechanism behind the photoinactivation described in the present paper must probably run parallel with an analysis of the cellular changes caused by the induction of thermosensitivity. This analysis is in progress. The assistance of Mrs. Harriette Cedervall and Miss Gunilla Ahlen is acknowledged with gratitude. This investigation was supported by research grant RG-8157 from the National Institutes of Health, Public Health Service, U.S.A.
REFERENCES N., Physiol. Plant. 2, 78 (1949). 2. __ ibid. 16, 415 (1963). 3. KIHLMAN, B. A., Exptl Cell Res. 17, 590 (1959). 1. FRIES,
Experimental
Cell Research
32
202
REFERENCES J. A. and CALVIN, M., The Path of Carbon in Photosynthesis. Englewood Cliffs, N.J., 1957.
1. BASSHAM, 2. FRIES,
3. 4. 5. 6. 7.
N., Physiol.
Planf.
Prentice Hall, Inc.,
2, 78 (1949).
__
ibid. 16, 415 (1963). GEZELIUS, K. and RANBY, B. G., Eq~ptl Cell Res. 12, 265 (1957). MURASHIGE, T. and SKOOG, F., Physiol. Plant. 15, 473 (1962). NYMAN, B., Suensk Botan. Tidskr. 55, 129 (1961). SUNDSTR~M, K.-R., Phytopathol. 2. 40, 213 (1960).
THE
INACTIVATING
EFFECT
THERMOSENSITIZED
OF VISIBLE
OPHIOSTOMA
LIGHT
ON
CELLS
N. FRIES Institute of Physiological Botany, University of Uppsala, Sweden Received July 30, 1963
the response of thermosensitized Ophiostoma cells to various chemical and physical agents it was observed that the cells showed an increased sensitivity not only to temperatures above 28°C but also to illumination with visible light. Since this quite unexpected effect of light seemedto deserve someattention a series of experiments was performed, the results of which are summarized in the present communication. Cells from vigorously budding conidial cultures of Ophiostoma multiannulatum (Hedge. & Davids.) were used as material for all the experiments. The cells were produced at 25°C in shake flask cultures with the liquid, synthetic “medium 3” ( =N6, or “Fries medium”), which is commonly used for Ophiostoma [I]. For particulars of the cultivation technique and mode of inducing thermosensitivity reference may be made to an earlier paper [a]. Thermosensitivity was induced (the “heat shock”) by heating the conidia for 2 min at 43°C according to procedure (b) in the paper referred to. Illumination with visible light was provided by the apparatus used by Kihlman [3]. Thus the light source consisted of six Philips “Attralux” lamps, type 13378 E/44, 24 V, 150 W. During illumination the conidia were suspendedin 25 ml glassbottles, 10 ml suspensionin each, through which air or oxygen-free nitrogen was bubbled from 10 min before starting the experiment and during the whole period of treatment. The suspensionwas made in liquid medium 3 and contained IO6 cells/ml. The bottles were immersed in a glass-walled water bath, which was kept at 25°C or 30°C. They were illuminated from two sidesat a distance of 15 cm from the lamps. After this treatment the cells were immediately plated out in medium-3-agar. During this procedure the laboratory was kept as dark as possible. The plates were
IN studying
Experimental
Cell Research 32
Effect
of visible
light
on thermosensitized
Ophiostoma
cells
203
then incubated at 25°C and 30°C. Each experimental series comprised five plates. The number of developing mycelia was scored after four days. If conidia thermosensitized by heat shock were illuminated with white light under the described conditions a decrease in the number of survivors always occurred, as TABLE
I.
The effect of illumination
with white light on preheated Ophiostoma cells,
(thermosensitized)
Control= the cellsgerminatingin agar at 25°Cafter having in darkness with air as the gas phase. mean values given in the table.
The
standard
been heat-shocked and then kept in no case exceeded 25 per cent of the
error
Per cent
Gas phase Air Air N, N, Air Air N* NZ
TABLE
Illumination conditions
Following incubation temp. OC
Dark Light Dark Light
25 25 25 25
Dark Light Dark Light
30 30 30 30
II.
survivors
I Illumination temperature
Expt 1 100 72 92 53 49 33 47 5.3
The effect of illumination (thermosensitized)
Illumination temperature
25°C Expt
Expt 3
2
100
100
71 74 42
56 74 7.1
7.3 3.5 3.6 1.3
Expt
30°C 4
100 53 117 0
16 4.0 6.9 0.2
7.1 5.7 10 0
Expt
5
100 71 118 3.6 2.2 0.8 4.6 0.1
with red and blue light on preheated Ophiostoma cells.
The red filter transmitted the wavelengths above 5900 A and c. 10 per cent of the wavelengths between 4100 and 4600 A; the blue filter transmitted 3500-5000 A and > 6900 A. The filters were made of cellophane by Sateri OY, Valkeakoski, Finland. The illumination was performed at 30°C in nitrogen atmosphere.
Illumination conditions
Following incubation temp. “C
Per cent Expt
6
No light Red light Blue light White light
25 25 25 25
100 33 7.0 1.4
No light Red light Blue light White light
30 30 30 30
7.0 2.4 0.7 0.3
survivors Expt
7
100 90 5.0 6.6 -
Experimental
Cell Research 32
N. Fries can be seen from Table I. This decrease was evident both in the plates incubated at 25°C and in those incubated at 30°C. In the latter case, however, all figures were lower because of the inactivating influence of the higher temperatures on the thermosensitized cells. Table I also shows that the effect of the white light was considerably stronger in the absence than in the presence of oxygen and perhaps somewhat stronger when the illumination had been performed at 30°C than at 25°C. In order to obtain at least an idea of which wavelengths were most effective, experiments were performed with three illumination chambers, one of which was illuminated with white, one with red and one with blue light. The gas phase consisted of nitrogen in all chambers including the unilluminated control chamber. As shown in Table II, the blue light had about the same inactivating effect as the white light. Some effect was also obtained with the red light; it must be remembered, however, that the red filter also transmitted a certain amount of blue light. The effect of visible light here described resembles photoreactivation in so far as it is independent of oxygen and seems to be due to a blue-absorbing photoreceptor, but differs by being an inactivation instead of a reactivation process. Unlike the phenomenon of photodynamic action, on the other hand, this photoinactivation of thermosensitized cells is independent of oxygen and occurs without an added absorbing pigment. An elucidation of the biochemical mechanism behind the photoinactivation described in the present paper must probably run parallel with an analysis of the cellular changes caused by the induction of thermosensitivity. This analysis is in progress. The assistance of Mrs. Harriette Cedervall and Miss Gunilla Ahlen is acknowledged with gratitude. This investigation was supported by research grant RG-8157 from the National Institutes of Health, Public Health Service, U.S.A.
REFERENCES N., Physiol. Plant. 2, 78 (1949). 2. __ ibid. 16, 415 (1963). 3. KIHLMAN, B. A., Exptl Cell Res. 17, 590 (1959). 1. FRIES,
Experimental
Cell Research
32