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UV-B-induced absorbance changes in the yeast Rhodotorula minuta
J. Photochem. Photobiol. B: BioL, 17 (1993) 127-134
UV-B-induced
absorbance
127
changes in the yeast Rhodotorula minuta’
Megumi Jffada Graduate School of Science and Technology, Kobe Universi& Rokkodai, Kobe 657 (Japan)
Mikiro Tada Department of Biofunction and Genetic Resources, Faculty of Agriculture, Okayama University, Tsushima-naka, Okayama 700 (Japan)
Tohru Hashimoto++ Department of Biology, Faculty of Science and Graduate School of Science and Technology, Kobe University, Rokkodai, Kobe 657
(Japan) (Received
June 15, 1992; accepted
August 25, 1992)
Abstract Short-term effects of UV-B (280-320 nm) irradiation in the range of minutes were recorded spectrophotometrically in the yeast Rhodotonda minuta, which forms carotenoids as additional response to the light treatment. Irradiation at 295 nm of dark-grown cells immediately caused changes in UV absorption of intact cells as well as of the cell wall and membrane. In intact cells, irradiation of a short period (10-30 min) increased the absorbance at 285 nm, and the extension of the irradiation up to 60 min resulted in a decrease at 270 nm as well as an increase at 240 nm. In cell wall fractions a similar increase in absorbance at 28.5 nm was caused by a short period of irradiation, accompanied by a decrease at 247 nm by a long period of irradiation, forming an isosbestic point at about 265 nm. Membrane fractions responded to the irradiation by a striking increase at 240 nm and small changes at several wavelengths between 265 and 300 nm. The remaining soluble fraction showed no appreciable changes in absorbance. Thus the major absorbance changes in intact cells were interpreted as a combination of those changes observed in the cell wall and membrane. The absorbance changes in the cell wall and membranes were both suppressed by the addition to cell culture of tridemorph and fenpropimorph, steroid biosynthesis inhibitors. The absorbance changes observed in the membrane preparations were attributed to a photochemical change of ergosterol in the membrane.
Keywords: Absorbance
change,
ergosterol,
Rhodotonda,
1. Introduction UV light of the B wave band (UV-B) induces a variety of physiological phenomena in higher and lower plants including fungi. In higher plants these phenomena involve the suppression of growth [l], the phototropic curvature of the mesocotyl and coleoptile [2, 31, and the synthesis of anthocyanin and other flavonoids [4, 51; in lower plants it induces the formation of the conidia and conidiophores [6-91 as well as pigment formation. For some of these UV actions, action spectra have been determined, which have the main action peak at 283 nm for conidium formation of Bottytis +This paper is a partial fulfilment quirements at Kobe University. “Author
to whom correspondence
loll-1344/93/$6.00
of M. Hada’s
Ph.D.
should be addressed.
re-
UV-B
cinereas [6], at 285 nm for Altemaria solani [7] and at 300 nm for both Alternaria tomato [8] and Hebninthosporium oryzae [9]. The action peaks for carotenoid synthesis in Rhodotonda minuta has been found at 285 nm [lo], and those for synthesis of flavone glycoside in Petroselinum hortense (parsley) [4, 111 and of anthocyanin in Sorghum bicolor [5] have been reported to be at about 290 nm. The putative photoreceptors for the UV actions are termed UV-B photoreceptors [12, 131. No information is available at present about the nature of the photoreceptors nor their related initial reactions, although substituted pterins have been suggested as a candidate of the UV-B photoreceptor [14], and a novel compound, chromosaponin I, having the absorption at 295 nm has been discovered from Pisum sativum and characterized
P51. 0 1993 - Elsevier
Sequoia.
All rights reserved
M. Hada et al. / W-B-induced
128
These photomorphogenetic actions of UV-B are phenomena which appear after 24-48 h or longer, probably through a variety of intervening biochemical reaction(s) after the light is absorbed by the photoreceptor(s); it is very important to find reaction(s) which occur in a short period after the irradiation. From this viewpoint in the present study we measured the changes observed between the UV absorbance before and the UV absorbance after UV-B irradiation in intact cells of Rh. minutu as well as its cell wall, membrane and soluble fractions.
2. Materials
and methods
2.1. Culture conditions Rh. minutu (Saito) Harrison var. texensk (IF0 1102) was grown in the dark in an asparagine medium at 26 “C. The asparagine medium consisted of 1 1 of deionized water, 30 g of glucose, 1.3 g of asparagine, 1 g of KH2P04, 0.5 g of MgSO,.7H,O, 0.1 g of NaCl, 0.13 g of MnSO, - 7H20, 0.2 mg of FeCl,, 0.1 g of thiamine hydrochloride, 6.0 g of yeast extract and 50 mg ofp-aminobenzoic acid (pH 5.2). Cells were shaken in 500 ml Erlenmeyer flasks with a rotary shaker at 95 cycles min-’ for 48 h unless otherwise stated. Cells were harvested, washed twice and resuspended in deionized water at a final concentration of 0.3 mg dry mass ml- ‘. 2.2. Cell fractionation Cells were homogenized with a Dyno-Mill KDL (WAB Applications, Switzerland) with the outlet of the 200 ml glass container being stoppered. Firstly 150 g of glass beads (average diameter, 0.25-0.5 mm) were placed in the container, then a cell suspension in 10 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sorbitol was poured in and finally the container was filled up with the same buffer. The container was cooled by circulating cooled water at 5 “C. Homogenization of the cells was performed at 3000 rev min-’ for 5 min. The homogenate was filtered through six layers of Miracloth (Calbiochem, San Diego, CA, USA) to remove the glass beads and then centrifuged successively at 45g for 10 min, 270% for 10 min and was 100 OOOg for 60 min. The 45g precipitate discarded, and the 27OOg pellet was used as the cell wall fraction, the 100 OOOgpellet as the cell membrane fraction, and the 100 OOOgsupernatant as the soluble fraction. Each pellet was suspended in deionized water and the supernatant was diluted
absorbance
changes
in Rh. minuta
with deionized water absorbance of 0.15.
so as to give a 350 nm
2.3. Light sources and irradiation Actinic light was obtained from a light source (Super-cure UVF-201S, Sanei Electric, Ltd., Osaka, Japan) equipped with a xenon-mercury lamp through interference filters (Japan Vacuum Optics Co., Tokyo, Japan) having A,, of 315 nm (half-bandwidths, 20 nm), 366 nm (23 nm) and 436 nm (8 nm); longer wavelength actinic light was obtained from another light source (TechnoLight KLS2100R, Kenko Co., Tokyo, Japan) equipped with a halogen lamp through interference filters with A,, of 622 nm (60 nm) and 750 nm (56 nm). These kinds of light were referred to by the A,, values. Green light was supplied from a xenon-mercury lamp source through a layer of blue acrylic resin film (type 63, Ryudensha Ltd., Tokyo, Japan) and two layers of yellow acrylic film (type 109-1-b, Mitsubihsi Rayon, Ltd., Tokyo, Japan). The green light had A,,= 550 nm and h1,2= 68 nm. In experiments to test the effects of carotenoid synthesis inhibitors, another light source, UV310-U340, was used because many samples were required to be irradiated at the same time. The light source consisted of four fluorescent tubes (FL 20S-E, Toshiba, Ltd., Tokyo, Japan) and a UV filter, U340 (Hoya Corporation, Ltd., Tokyo, Japan), and its emission had a maximum of 311 nm and a half-bandwidth of 32 nm. The actinic light beam was introduced through a quartz glass fibre (Fb in Fig. 1) into the sample chamber SC of a spectrophotometer (model 557, Hitachi Ltd., Tokyo, Japan), filtered through a relevant filter Fi, and reflected with the concave mirror CM so that the beam enters the cuvette Cu from the direction of the measuring beam. Filters was installed on the revolver Rv so as to be chosen by rotating it. When the absorption spectra were measured, the mirror CM was removed from the measuring beam path of the spectrophotometer by pulling the mirror stalk MS. The photon irradiance and spectral photon density distribution of the actinic light were determined in front of the revolver with a photon density meter (model HK-1, Riken, Wako-shi, Saitama, Japan) [16] and a spectrophoton densitometer (model HK-3, Riken) respectively. During irradiation and spectral scanning, the temperature of the cuvette holder CH was kept at 5 “C by circulating cooled water through it, and the resulting moisture condensation on the surface
M. Hada et al. / UV-B-induced
(A)
SC
Water . Inlet
MS
Fig. 1. (A) Top view of the irradiation system installed in the sample chamber of the spectrophotometer (557 Hitachi, Ltd.): SC, sample chamber; LS, light source; Fb, quartz glass fibre; Rv, revolver for filters; Cu, cuvette; CM, concave mirror; MS, mirror stalk, PM, photomultiplier tube. (B) Diagram of the filter revolver and the part of the quartz glass fibre: Fi, filter. (C) Side view of the filter revolver.
of the cuvette was blown off by dry air or nitrogen gas. No appreciable settlement of cells occurred and no agitation of the cell suspension was made during irradiation.
absorbance
changes
in Rh. minuta
129
of 1 1 of deionized water, 30 g of glucose, 2.2 g of valine, 1 g of KH2P04, 0.5 g of MgSO,., . 7H20, 0.1 g of NaCl, 0.13 g of MnSO,*7H,O, 0.2 mg of FeCl,, 0.1 g of thiamine hydrochloride, and 50 mg of p-aminobenzoic acid (pH 5.2) [17]. In this medium containing only valine as nitrogen source, cells grew sparingly. After incubation the cells were collected by centrifugation, washed and resuspended in 5 ml deionized water; then the absorption spectrum was recorded from 540 to 460 nm. The difference A&,8-522nm between the absorbance at 498 and 522 nm served as an estimation of carotenoid contents [17]. The cell density of the probes was determined by the turbidity measured at 600 nm. 2.6. Inhibitor experiments The fungicides tridemorph and fenpropimorph were kindly provided by Dr. Ammermann (Crop BASF Aktiengesellschaft, Protection Division, Limburgerhof, Germany). After each fungicide dissolved in acetone was added to culture media, cells were inoculated and grown for 48 h until use. The acetone concentration of all media including the control without the inhibitor was 0.5% by volume. Effects of these fungicides on the growth of the cells were determined by turbidities measured at 600 nm after 24 h incubation
3. Results 2.4. Measurement of UV-induced absorbance change Absorption spectra were determined with a double-beam dual-wavelength spectrophotometer (model 557, Hitachi, Ltd., Tokyo, Japan), operated in the double-beam mode (slit width, 2.0 nm). Before and after actinic irradiation, a 3 ml cell suspension in a cuvette was scanned at 120 nm and post-irradiamin-* to record pre-irradiation tion spectra. To obtain the difference spectrum the pre-irradiation and post-irradiation spectra were digitized and stored in a PC-98 computer (Nippon Electric Co., Tokyo, Japan) and processed with a 557 data management system (System Giken Ltd., Osaka, Japan). The transfer of the data of absorbance was made only at wavelengths of even numbers for a mechanical reason. 2.5. Induction of carotenogenesis and measurement of carotenoid contents After irradiation the cells were collected, resuspended in the valine medium and incubated for 15 h at 26 “C in the dark. The medium consisted
3.1. UV-induced absorbance change in intact cells After irradiation with UV-B (315 nm) at 90 pm01 m-* s-l for 30 min, a slight change in absorption spectrum of dark-grown cell was observed (Fig. 2(a)), but the difference spectrum disclosed various changes in absorbance. It showed a peak at around 285 nm with shoulders at 275 and 297 nm and another peak at 240 nm as well as a trough at 258 nm (Fig. 2(b)). The appearance of the 285 nm peak was observed even after an irradiation for 1 min (data not shown). The peaks increased in height with the extension of irradiation up to 30 min (Fig. 3(a)). As the irradiation was further extended, a trough at 270 nm extending towards long wavelengths was deepened, and this reflected the apparent heights of the peaks, thus deforming the spectra (Fig. 3(b)). On the contrary, the height of the peak at 240 nm increased with the extension of the irradiation. The effects of UV-B as measured by the increase in absorbance at 285 nm followed the Bunsen-Roscoe reciprocity law at least up to 50 mmol
M. Hada et al. / UV-B-induced absorbance changes in Rh. minuta
0.015
-Dark
control
-----lJV-B
treated
./ 0
/ 8
I1
I1
Difference
1
f
I
I1
11
11
spectrum
2 L
I
0
. .
/
10 Photon
Wavelength
[nm]
Fig. 2. Effects of UV-B irradiation on absorption spectrum of intact cells of Rh. minuta: (a) absorption spectra before (-) and after (- - -) a 30 min irradiation; (b) the difference spectrum.
50
x) fluence
[mmol
m-*1
Fig. 4. Effects of UV-B irradiance on the change in absorbance in Rh. minuta: 0, 40 prnol me2 s-‘; 0, 80 pmol m-* SC’. The ordinate shows the difference in the absorbance at 286 nm instead of 285 nm, because the computer recorded only data at wavelengths of even numbers.
Irradiations with near UV (366 nm), blue (436 nm), green (550 nm), red (622 nm) or far-red (750 nm) light were not effective at all in inducing any spectral changes in the wave band from 230 to 350 nm (data not shown).
Wavelength
[nm]
Fig. 3. The difference spectra with intact cells irradiated with UV-B of a photon fluence rate of 90 Frnol mm2 s-’ for (a) 10 min (-), 20 min (-- -) and 30 min (. . . . .) and (b) 30 min (-), 40 min (---), 50 min (. . . .) and 60 min (-.-).
m -’ and were proportional to the logarithms of the applied doses (Fig. 4). UV-B-induced spectral changes at 20 and 5 “C, were compared but no difference was observed. At both temperatures the spectral changes survived equally for at least 30 min. Bubbling with air, N2 or 0, during irradiation did not affect this spectral change (data not shown).
3.2. W-induced absorbance change in cell-free fractions In order to locate the site(s) at which the spectral changes occur, spectral change was determined with cell wall and membrane fractions as well as the remaining soluble fraction, which were obtained from dark-cultured cells. These fractions were irradiated with the UV-B, and their absorption spectra were determined before and after actinic irradiation. In the wall fraction the absorbance at 285 nm increased, while that at 245 nm decreased (Fig. 5). The curves in Fig. 5(b) crossed at about 265 nm, suggesting that a single compound was mostly involved. The extent of the changes was proportional to the logarithm of the irradiation periods (Fig. 6). In a cell membrane fraction (Fig. 7) the absorbance was slightly increased at a broad band below 340 nm after a 10 min irradiation, but after 20 or 30 min irradiation it decreased at 270, 281 and 293 nm and markedly increased at 240 nm. The soluble fraction of cells showed only slight absorbance changes, which were not increased even by extended irradiation (Fig. 8). It is known that ergosterol, which is abundant in the cell membrane of baker’s yeast [18], is converted to ergocalciferol non-enzymatically by UV irradiation [19]. So we measured the change
M. Hada et al. I W-B-induced
131
absorbance changes in Rh. minuta
(b)
-... : ll11llI”““‘l 250 Wavelength
[nm]
Fig. 5. Effects of UV-B irradiation on absorption spectrum of a cell wall fraction of Rh. minuta: (a) absorption spectra before (-) and after (---) 30 min irradiation; (b) the difference spectrum after irradiation for 10 min (-), 20 min (---) and 30 min (.... .) with a photon fluence rate of 90 pmol me2 SC’.
‘.,.’ 1 1 1 1 1 1 0 1 1 1 1 1 1 35c 300 250
4.0251
Wavelength [nm]
Fig. 7. Effects of W-B irradiation on the absorption spectrum of the cell membrane fraction of Rh. minuta: (a) Absorption spectra before (-) and after (-- -) 30 min irradiation; (b) the difference spectra after irradiation for 10 min (-), 20 min (---) and 30 min (- . . -). The irradiation conditions were the same as in Fig. 5.
I
’
’
t
’
’
’
’
’
’
’
’
’
’
’
I-
0.4
1
#I!3
10 I
10 I 20 20 40 I,.1 -‘5 Irradiation period [min]
J
40
Fig. 6. Relationship between the irradiation period and the extent of spectral change of a cell wall fraction: (a) increase in absorbance at 280 nm; (b) decrease in absorbance at 246 nm. The experimental conditions were the same as in Fig. 5.
in the absorption spectrum of an ethyl alcohol solution of ergosterol caused by UV-B irradiation (Fig. 9). The pattern of a difference spectrum of the ergosterol solution was similar to that in the cell membrane fraction, i.e. decreases at 270, 281 and 293 nm and increases at 240 and 300 nm. This finding strongly suggests that ergosterol may be responsible for the spectral changes of the membrane fraction. 3.3. Induction of carotenogenesis It is tempting to known whether or not the fluence of the UV-B administered were at the “physiological” levels and not the injurious levels. Also, the actual dosage levels of the UV-B reaching cells in a suspension are difficult to measure because of its absorption and scattering. Hence, the
-0.041 ’ .’
I
250
’
’
’
’
’
’
’
1
3!
Wavelength [nm]
Fig. 8. Effect of UV-B irradiation on the absorption spectrum of the soluble fraction of Rh. minuta: (a) absorption spectra before (-) and after (- - -) 60 min irradiation; (b) the difference spectrum after irradiation for 60 min. The irradiation conditions were the same as in Fig. 5.
cells which served to determine the absorption spectra were transferred to the valine medium and cultured for 15 h to observe the cell population and the synthesis of carotenoids. The cell population was not affected by up to 5 min irradiation (Fig. 10). Carotenoid synthesis occurred, increasing
M. Hada et al. / UV-B-induced
132
absorbance
0.008
changes in Rh. minuta
-
-z : G: $ % 0.007 -
-71
I
/’
3 Irradiation
Wavelength
[nm]
Fig. 9. Effects of UV-B irradiation on the absorption spectrum of 100 PM ergosterol solution in ethyl alcohol: (a) absorption spectra before (-) and after (---) 6 min irradiation; (b) the difference spectra after irradiation for 2 min (-), 4 min (- --) and 6 min (. . . . .). The irradiation conditions were the same as in Fig. 5.
++I
1
21 irradiation
5I
IO 1
20 I
period
[mini
50 I
100 I
Fig. 10. Effects of UV-B irradiation periods on the cell yield after 15 h incubation in the dark. Cells were irradiated to determined the absorption spectra in a cuvette at a density of 0.3 mg dry mass ml-‘. The cells were suspended in the valine medium and incubated in the dark for 15 h. The cell density on the ordinate represents turbidity at 600 nm when the cells were resuspended in 5 ml deionized water. The initial cell density before incubation was 0.081. Each point in the figure represents the mean of six samples+standard error. The irradiation conditions were the same as in Fig. 5. The abscissa is on a logarithmic scale.
with increasing periods of irradiation up to 5 min, where no decrease in cell population was found (Fig. 11). Further extended irradiation suppressed cell population, which may reflect the decline of carotenoid synthesis.
period
5
10
[min]
Fig. 11. Effects of UV-B irradiation periods on carotenoid synthesis. The irradiated cells were incubated as in Fig. 10. The contents of carotenoids were estimated by the difference between the absorbances at 498 and 522 nm. The density of cells at the time of irradiation was 0.3 mg dry mass ml-‘. Each point in the figure represents the mean of six samplesfstandard error. The irradiation conditions were the same as in Fig. 5.
3.4. Effect of sterol inhibitors on Winduced absorbance changes Since tridemorph (2,6-dimethyl-N-tridecylmorpholine) and fenpropimorph (N-[3-(p-teti-butylphenyl)-Zmethylpropyll-cis-2,6-dimethylmorpholine) have been reported to inhibit the biosynthesis of ergosterol in Saccharomyces cerevisiae [20], we examined whether these sterol inhibitors affected the spectral changes and the carotenoid synthesis in Rh. minuta. Cells were grown for 48 h in media containing tridemorph or fenpropimorph at 100 or 1 PM and in the medium without the inhibitors (control) and were tested for UV-B-induced spectral changes and carotenoid synthesis. Both inhibitors at 100 PM inhibited the growth of the cells by about 50% but did not affect it at 1 PM. Treatment with inhibitors abolished the increases in the absorbances at 285 nm and in its neighborhood as well as at 240 nm completely at 100 PM (Figs. 12(a)-12(c)) and reduced them by about 50% at 1 PM (Figs. 12(d)-12(f)). A decrease in absorbance was discerned at 280 nm in 100 PM tridemorph-treated cells and at 260 nm in 100 PM fenpropimorph-treated cells. Carotenoid synthesis was, not inhibited by either inhibitor (Table 1).
4. Discussion In the present study with Rh. minuta cells, we described UV-B-induced absorbance changes occurring at wavelengths shorter than 320 nm. The
M. Hada et al. I UV-B-induced absorbance changes in Rh. minuta
133
100 ulbl Fenpropimorph
1 ut.4 Tridemorph E
0.010
s g 0.005 (0 2 0 a
I,,
-O.o05c,
, ,I
300
250
I I I I I
350
350
250
250
Wavelength [min] Fig. 12. Effects of the steroid inhibitors on IN-induced absorbance changes in Rh. minutu cells. The curves are difference spectra and 20 min (---) and (d)-(f) 5 min (-) and 10 min of intact cells irradiated at 90 pmol m-* s- ’ for (a)-(c) 10 min (-) (---): (a), (d) control cells grown without inhibitors; (b) cells grown with 100 PM tridemorph; (c) cells grown with 100 PM fenpropimorph; (e) cells grown with 1 PM tridemorph; (f) cells grown with 1 PM fenpropimorph. TABLE
1
Effects of the steroid inhibitors, tridemorph and fenpropimorph on UV-B-induced carotenoid synthesis. Cells were grown for 48 h with or without 1 PM tridemorph or fenpropimorph and then irradiated with a photon fluence-rate of 25 pmol m-’ s-’ for 5, 10, 20 min with UV-31OU340. Irradiated cells were incubated in the dark for 15 h. The values in the table are differences in absorbance at 498 and 522 nm, representing contents of carotenoids. Density of cells at the time of irradiation, 5 mg dry weight ml-’ Irradiation
Inhibitors
Absorbance 498-522 nm Exp. 2 Exp. 1
Control
Tridemorph
Fenpropimorph
[l PM]
[l FM]
Dark UV 5 min 10 min 20 min
0.0330 0.0348 0.0335 -
0.0261 0.0360 0.0383 0.0408
Dark UV 5 min 10 min 20 min
0.0350 0.0443 0.0443 -
0.0317 0.0490 0.0513 0.0535
Dark UV 5 min 10 min
0.0348 0.0403 0.0380
-
absorbance changes consisted mostly of two major components: one occurred in the cell wall, and the other in the cell membrane. The absorbance change observed when an ethyl alcohol solution of ergosterol was similarly irradiated with UV-B was quite similar to most of the absorbance changes of the cell membrane (Figs.
7 and 9); the probable abundant existence of ergosterol in the membrane may explain the component of the spectral changes occurring in the cell membrane. The kind of compound that is responsible for the absorbance changes in the cell wall has not been clarified in the present study. However, the difference spectra observed with the cell wall fraction showed not shift in the peak and trough wavelengths and formed an isosbestic point (Fig. 5). In this response the Bunsen-Roscoe law held (Fig. 4). These findings suggest that this absorbance change may involve a single compound, which is converted to a photoproduct having conjugated double bonds. The results of the experiment with the steroid biosynthesis inhibitors suggested that the photoreactant may be a compound related to steroids, or a compound which is produced in the presence of steroids. The UV-B-induced absorbance changes at 285 nm occurred, although slightly, even at the irradiance level of UV-B which does not suppress the cell population but does induce carotenoid synthesis. The photoreaction is very fast and can be observed immediately after the termination of an irradiation of a few minutes at the fluence rate used. However, the possibility is excluded that the absorbance changes may be involved in a primary reaction of the UV-B-induced carotenoid synthesis, because the steroid synthesis inhibitors did not suppress the carotenoid synthesis, although they suppressed the absorbance changes found in this work. Yet the absorption changes occurring in the cell wall and cell membrane are new findings and
134
M. Hada
may contribute to the understanding on Rh. minuta.
et al. / W-B-induced
of UV effects
absorbance
changes
in Rh. minuta
8 T. Kumagai, Action spectra for the blue and near ultraviolet reversible photoreaction in the induction of fungal conidiation, Physiol.
Plant.,
57 (1983)
468471.
9 S. Yamamura, T. Kumagai and Y. Oda, An action spectrum for photoinduced conidiation in Helminthosporium oryzae, Plant Cell Physiol.,
Acknowledgments We thank Dr. tridemorph and Yokohama City puter software oratory for his
Ammermann for kindly providing fenpropimorph, Dr. T. Hisabori, University for his help with comand Dr. M. Uemura of our labhelpful suggestions.
References T. Hashimoto and M. Tajima, Effects of ultraviolet irradiation on growth and pigmentation in seedlings, Plant Cell Physiol., 21 (1980) 1559-1571. G. M. Curry, K. V. Thimann
curvature Physiol.
response Plant.,
of Avena
9 (1956)
and P. M. Ray, The base seedlings to the ultraviolet,
429440.
T. Hashimoto, S. Ito and H. Yatsuhashi, Ultaviolet lightinduced coiling and curvature of broom sorghum first internodes, Physiol. Plant., 61 (1984) 1-7. E. Wellmann, Regulation der Flavonoidbiosyntheses durch ultraviolettes Licht und Phytochrome in Zellkulturen und Keimlingen von Petersilie (Petroselinum hortense Hotfm.), Ber. Dtsch.
Bot. Ges.,
18 (1977)
1163-1166.
10 M. Tada, M. Watanabe and Y. Tada, Mechanism of photoregulated carotenogenesis in Rhodotorula minuta. VII. Action spectrum for photoinduced carotenogenesis, Plant Cell Physiol.,
87 (1974)
267-273.
H. Yatsuhashi, T. Hashimoto and S. Shimizu, Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes, Plant Physiol., 70 (1982) 735-741. Y. Honda and T. Yunoki, Action spectra for photosporogenesis in Botrytis cinereas Per. ex. Fr. Plant Physiol., 61 (1978) 71 l-713. Y. Honda and T. Yunoki, An action spectrum for photoinduced conidium formation in Altemaria solani (Ellis et G. Martin), Soraver, Ann Phytopathol. Sot. Jpn., 47 (1981) 327-334.
31 (1990)
241-246.
flavone glycoside syn11 E. Wellmann, Phytochrome-mediated thesis in cell suspension cultures of Petroselinum hortense after preirradiation with ultraviolet light,Planta, lOl(l971) 283-286. 12 T. Hashimoto and H. Yatsuhashi, Ultraviolet photoreceptors and their interaction in broom sorghum - Analysis of action spectra and fluence-response curves, in H. Senger (ed.), Blue Light Effects in Biological Systems, Springer, Berlin, 1984, pp. 125-136. of fungal devel13 T. Kumagai, Yearly review: photocontrol opment, Photochem. Photobiol., 47 (1988) 889-896. 14 P. Galland and H. Senger, The role of pterins in the photoreception and metabolism of plants, Photochem. Photobiol., 48 (1988)
811-820.
A ypyronyl15 S. Tsurumi, T. Takagi and T. Hashimoto, triterpenoid saponin from Pisum sativum, Phytochemishy 31 (1992)
2435-2438.
16 T. Hashimoto, H. Yatsuhashi and H. Kato, Self-corrected, convertible handy irradiance meter with a photodiode, in Matsumoto (ed.), Abstracts, Annu. Meet. of Japanese Society of Plant Physiology, 1982, 1982, p. 38. 17 M. Tada and M. Shiroishi, Mechanism of photoregulated carotenogenesis in Rhodotonda minuta. I. Photocontrol of carotenoid production, Plant Cell Physiol., 23 (1982) 541-547. 18 H. Suomalainen and T. Nurminen, Structure and function of the yeast cell envelope, in J. R. Villanueva, I. GarciaAcha, S. Gascon and F. Uruburu (eds.), Yeast, Mould and Plant Protoplasts, Academic Press, London, 1973, pp. 167-186. 19 A. L. Hirsch, Vitamin D, in M. Grayson (ed.), Encyclopedia of Chemical Technology, 3rd edn., Vol. 24, Wiley-Interscience, New York, 1984, pp. 186-213. 20 R. I. Baloch, E. I. Mercer, T. E. Wiggins and B. C. Baldwin, Inhibition of ergosterol biosynthesis in Saccharomyces cerevisiae and Ustilago maydis by tridemorph, fenpropimorph and fenpropidin, Phytochemistry, 23 (1984) 2219-2226.
UV-B-induced
absorbance
127
changes in the yeast Rhodotorula minuta’
Megumi Jffada Graduate School of Science and Technology, Kobe Universi& Rokkodai, Kobe 657 (Japan)
Mikiro Tada Department of Biofunction and Genetic Resources, Faculty of Agriculture, Okayama University, Tsushima-naka, Okayama 700 (Japan)
Tohru Hashimoto++ Department of Biology, Faculty of Science and Graduate School of Science and Technology, Kobe University, Rokkodai, Kobe 657
(Japan) (Received
June 15, 1992; accepted
August 25, 1992)
Abstract Short-term effects of UV-B (280-320 nm) irradiation in the range of minutes were recorded spectrophotometrically in the yeast Rhodotonda minuta, which forms carotenoids as additional response to the light treatment. Irradiation at 295 nm of dark-grown cells immediately caused changes in UV absorption of intact cells as well as of the cell wall and membrane. In intact cells, irradiation of a short period (10-30 min) increased the absorbance at 285 nm, and the extension of the irradiation up to 60 min resulted in a decrease at 270 nm as well as an increase at 240 nm. In cell wall fractions a similar increase in absorbance at 28.5 nm was caused by a short period of irradiation, accompanied by a decrease at 247 nm by a long period of irradiation, forming an isosbestic point at about 265 nm. Membrane fractions responded to the irradiation by a striking increase at 240 nm and small changes at several wavelengths between 265 and 300 nm. The remaining soluble fraction showed no appreciable changes in absorbance. Thus the major absorbance changes in intact cells were interpreted as a combination of those changes observed in the cell wall and membrane. The absorbance changes in the cell wall and membranes were both suppressed by the addition to cell culture of tridemorph and fenpropimorph, steroid biosynthesis inhibitors. The absorbance changes observed in the membrane preparations were attributed to a photochemical change of ergosterol in the membrane.
Keywords: Absorbance
change,
ergosterol,
Rhodotonda,
1. Introduction UV light of the B wave band (UV-B) induces a variety of physiological phenomena in higher and lower plants including fungi. In higher plants these phenomena involve the suppression of growth [l], the phototropic curvature of the mesocotyl and coleoptile [2, 31, and the synthesis of anthocyanin and other flavonoids [4, 51; in lower plants it induces the formation of the conidia and conidiophores [6-91 as well as pigment formation. For some of these UV actions, action spectra have been determined, which have the main action peak at 283 nm for conidium formation of Bottytis +This paper is a partial fulfilment quirements at Kobe University. “Author
to whom correspondence
loll-1344/93/$6.00
of M. Hada’s
Ph.D.
should be addressed.
re-
UV-B
cinereas [6], at 285 nm for Altemaria solani [7] and at 300 nm for both Alternaria tomato [8] and Hebninthosporium oryzae [9]. The action peaks for carotenoid synthesis in Rhodotonda minuta has been found at 285 nm [lo], and those for synthesis of flavone glycoside in Petroselinum hortense (parsley) [4, 111 and of anthocyanin in Sorghum bicolor [5] have been reported to be at about 290 nm. The putative photoreceptors for the UV actions are termed UV-B photoreceptors [12, 131. No information is available at present about the nature of the photoreceptors nor their related initial reactions, although substituted pterins have been suggested as a candidate of the UV-B photoreceptor [14], and a novel compound, chromosaponin I, having the absorption at 295 nm has been discovered from Pisum sativum and characterized
P51. 0 1993 - Elsevier
Sequoia.
All rights reserved
M. Hada et al. / W-B-induced
128
These photomorphogenetic actions of UV-B are phenomena which appear after 24-48 h or longer, probably through a variety of intervening biochemical reaction(s) after the light is absorbed by the photoreceptor(s); it is very important to find reaction(s) which occur in a short period after the irradiation. From this viewpoint in the present study we measured the changes observed between the UV absorbance before and the UV absorbance after UV-B irradiation in intact cells of Rh. minutu as well as its cell wall, membrane and soluble fractions.
2. Materials
and methods
2.1. Culture conditions Rh. minutu (Saito) Harrison var. texensk (IF0 1102) was grown in the dark in an asparagine medium at 26 “C. The asparagine medium consisted of 1 1 of deionized water, 30 g of glucose, 1.3 g of asparagine, 1 g of KH2P04, 0.5 g of MgSO,.7H,O, 0.1 g of NaCl, 0.13 g of MnSO, - 7H20, 0.2 mg of FeCl,, 0.1 g of thiamine hydrochloride, 6.0 g of yeast extract and 50 mg ofp-aminobenzoic acid (pH 5.2). Cells were shaken in 500 ml Erlenmeyer flasks with a rotary shaker at 95 cycles min-’ for 48 h unless otherwise stated. Cells were harvested, washed twice and resuspended in deionized water at a final concentration of 0.3 mg dry mass ml- ‘. 2.2. Cell fractionation Cells were homogenized with a Dyno-Mill KDL (WAB Applications, Switzerland) with the outlet of the 200 ml glass container being stoppered. Firstly 150 g of glass beads (average diameter, 0.25-0.5 mm) were placed in the container, then a cell suspension in 10 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sorbitol was poured in and finally the container was filled up with the same buffer. The container was cooled by circulating cooled water at 5 “C. Homogenization of the cells was performed at 3000 rev min-’ for 5 min. The homogenate was filtered through six layers of Miracloth (Calbiochem, San Diego, CA, USA) to remove the glass beads and then centrifuged successively at 45g for 10 min, 270% for 10 min and was 100 OOOg for 60 min. The 45g precipitate discarded, and the 27OOg pellet was used as the cell wall fraction, the 100 OOOgpellet as the cell membrane fraction, and the 100 OOOgsupernatant as the soluble fraction. Each pellet was suspended in deionized water and the supernatant was diluted
absorbance
changes
in Rh. minuta
with deionized water absorbance of 0.15.
so as to give a 350 nm
2.3. Light sources and irradiation Actinic light was obtained from a light source (Super-cure UVF-201S, Sanei Electric, Ltd., Osaka, Japan) equipped with a xenon-mercury lamp through interference filters (Japan Vacuum Optics Co., Tokyo, Japan) having A,, of 315 nm (half-bandwidths, 20 nm), 366 nm (23 nm) and 436 nm (8 nm); longer wavelength actinic light was obtained from another light source (TechnoLight KLS2100R, Kenko Co., Tokyo, Japan) equipped with a halogen lamp through interference filters with A,, of 622 nm (60 nm) and 750 nm (56 nm). These kinds of light were referred to by the A,, values. Green light was supplied from a xenon-mercury lamp source through a layer of blue acrylic resin film (type 63, Ryudensha Ltd., Tokyo, Japan) and two layers of yellow acrylic film (type 109-1-b, Mitsubihsi Rayon, Ltd., Tokyo, Japan). The green light had A,,= 550 nm and h1,2= 68 nm. In experiments to test the effects of carotenoid synthesis inhibitors, another light source, UV310-U340, was used because many samples were required to be irradiated at the same time. The light source consisted of four fluorescent tubes (FL 20S-E, Toshiba, Ltd., Tokyo, Japan) and a UV filter, U340 (Hoya Corporation, Ltd., Tokyo, Japan), and its emission had a maximum of 311 nm and a half-bandwidth of 32 nm. The actinic light beam was introduced through a quartz glass fibre (Fb in Fig. 1) into the sample chamber SC of a spectrophotometer (model 557, Hitachi Ltd., Tokyo, Japan), filtered through a relevant filter Fi, and reflected with the concave mirror CM so that the beam enters the cuvette Cu from the direction of the measuring beam. Filters was installed on the revolver Rv so as to be chosen by rotating it. When the absorption spectra were measured, the mirror CM was removed from the measuring beam path of the spectrophotometer by pulling the mirror stalk MS. The photon irradiance and spectral photon density distribution of the actinic light were determined in front of the revolver with a photon density meter (model HK-1, Riken, Wako-shi, Saitama, Japan) [16] and a spectrophoton densitometer (model HK-3, Riken) respectively. During irradiation and spectral scanning, the temperature of the cuvette holder CH was kept at 5 “C by circulating cooled water through it, and the resulting moisture condensation on the surface
M. Hada et al. / UV-B-induced
(A)
SC
Water . Inlet
MS
Fig. 1. (A) Top view of the irradiation system installed in the sample chamber of the spectrophotometer (557 Hitachi, Ltd.): SC, sample chamber; LS, light source; Fb, quartz glass fibre; Rv, revolver for filters; Cu, cuvette; CM, concave mirror; MS, mirror stalk, PM, photomultiplier tube. (B) Diagram of the filter revolver and the part of the quartz glass fibre: Fi, filter. (C) Side view of the filter revolver.
of the cuvette was blown off by dry air or nitrogen gas. No appreciable settlement of cells occurred and no agitation of the cell suspension was made during irradiation.
absorbance
changes
in Rh. minuta
129
of 1 1 of deionized water, 30 g of glucose, 2.2 g of valine, 1 g of KH2P04, 0.5 g of MgSO,., . 7H20, 0.1 g of NaCl, 0.13 g of MnSO,*7H,O, 0.2 mg of FeCl,, 0.1 g of thiamine hydrochloride, and 50 mg of p-aminobenzoic acid (pH 5.2) [17]. In this medium containing only valine as nitrogen source, cells grew sparingly. After incubation the cells were collected by centrifugation, washed and resuspended in 5 ml deionized water; then the absorption spectrum was recorded from 540 to 460 nm. The difference A&,8-522nm between the absorbance at 498 and 522 nm served as an estimation of carotenoid contents [17]. The cell density of the probes was determined by the turbidity measured at 600 nm. 2.6. Inhibitor experiments The fungicides tridemorph and fenpropimorph were kindly provided by Dr. Ammermann (Crop BASF Aktiengesellschaft, Protection Division, Limburgerhof, Germany). After each fungicide dissolved in acetone was added to culture media, cells were inoculated and grown for 48 h until use. The acetone concentration of all media including the control without the inhibitor was 0.5% by volume. Effects of these fungicides on the growth of the cells were determined by turbidities measured at 600 nm after 24 h incubation
3. Results 2.4. Measurement of UV-induced absorbance change Absorption spectra were determined with a double-beam dual-wavelength spectrophotometer (model 557, Hitachi, Ltd., Tokyo, Japan), operated in the double-beam mode (slit width, 2.0 nm). Before and after actinic irradiation, a 3 ml cell suspension in a cuvette was scanned at 120 nm and post-irradiamin-* to record pre-irradiation tion spectra. To obtain the difference spectrum the pre-irradiation and post-irradiation spectra were digitized and stored in a PC-98 computer (Nippon Electric Co., Tokyo, Japan) and processed with a 557 data management system (System Giken Ltd., Osaka, Japan). The transfer of the data of absorbance was made only at wavelengths of even numbers for a mechanical reason. 2.5. Induction of carotenogenesis and measurement of carotenoid contents After irradiation the cells were collected, resuspended in the valine medium and incubated for 15 h at 26 “C in the dark. The medium consisted
3.1. UV-induced absorbance change in intact cells After irradiation with UV-B (315 nm) at 90 pm01 m-* s-l for 30 min, a slight change in absorption spectrum of dark-grown cell was observed (Fig. 2(a)), but the difference spectrum disclosed various changes in absorbance. It showed a peak at around 285 nm with shoulders at 275 and 297 nm and another peak at 240 nm as well as a trough at 258 nm (Fig. 2(b)). The appearance of the 285 nm peak was observed even after an irradiation for 1 min (data not shown). The peaks increased in height with the extension of irradiation up to 30 min (Fig. 3(a)). As the irradiation was further extended, a trough at 270 nm extending towards long wavelengths was deepened, and this reflected the apparent heights of the peaks, thus deforming the spectra (Fig. 3(b)). On the contrary, the height of the peak at 240 nm increased with the extension of the irradiation. The effects of UV-B as measured by the increase in absorbance at 285 nm followed the Bunsen-Roscoe reciprocity law at least up to 50 mmol
M. Hada et al. / UV-B-induced absorbance changes in Rh. minuta
0.015
-Dark
control
-----lJV-B
treated
./ 0
/ 8
I1
I1
Difference
1
f
I
I1
11
11
spectrum
2 L
I
0
. .
/
10 Photon
Wavelength
[nm]
Fig. 2. Effects of UV-B irradiation on absorption spectrum of intact cells of Rh. minuta: (a) absorption spectra before (-) and after (- - -) a 30 min irradiation; (b) the difference spectrum.
50
x) fluence
[mmol
m-*1
Fig. 4. Effects of UV-B irradiance on the change in absorbance in Rh. minuta: 0, 40 prnol me2 s-‘; 0, 80 pmol m-* SC’. The ordinate shows the difference in the absorbance at 286 nm instead of 285 nm, because the computer recorded only data at wavelengths of even numbers.
Irradiations with near UV (366 nm), blue (436 nm), green (550 nm), red (622 nm) or far-red (750 nm) light were not effective at all in inducing any spectral changes in the wave band from 230 to 350 nm (data not shown).
Wavelength
[nm]
Fig. 3. The difference spectra with intact cells irradiated with UV-B of a photon fluence rate of 90 Frnol mm2 s-’ for (a) 10 min (-), 20 min (-- -) and 30 min (. . . . .) and (b) 30 min (-), 40 min (---), 50 min (. . . .) and 60 min (-.-).
m -’ and were proportional to the logarithms of the applied doses (Fig. 4). UV-B-induced spectral changes at 20 and 5 “C, were compared but no difference was observed. At both temperatures the spectral changes survived equally for at least 30 min. Bubbling with air, N2 or 0, during irradiation did not affect this spectral change (data not shown).
3.2. W-induced absorbance change in cell-free fractions In order to locate the site(s) at which the spectral changes occur, spectral change was determined with cell wall and membrane fractions as well as the remaining soluble fraction, which were obtained from dark-cultured cells. These fractions were irradiated with the UV-B, and their absorption spectra were determined before and after actinic irradiation. In the wall fraction the absorbance at 285 nm increased, while that at 245 nm decreased (Fig. 5). The curves in Fig. 5(b) crossed at about 265 nm, suggesting that a single compound was mostly involved. The extent of the changes was proportional to the logarithm of the irradiation periods (Fig. 6). In a cell membrane fraction (Fig. 7) the absorbance was slightly increased at a broad band below 340 nm after a 10 min irradiation, but after 20 or 30 min irradiation it decreased at 270, 281 and 293 nm and markedly increased at 240 nm. The soluble fraction of cells showed only slight absorbance changes, which were not increased even by extended irradiation (Fig. 8). It is known that ergosterol, which is abundant in the cell membrane of baker’s yeast [18], is converted to ergocalciferol non-enzymatically by UV irradiation [19]. So we measured the change
M. Hada et al. I W-B-induced
131
absorbance changes in Rh. minuta
(b)
-... : ll11llI”““‘l 250 Wavelength
[nm]
Fig. 5. Effects of UV-B irradiation on absorption spectrum of a cell wall fraction of Rh. minuta: (a) absorption spectra before (-) and after (---) 30 min irradiation; (b) the difference spectrum after irradiation for 10 min (-), 20 min (---) and 30 min (.... .) with a photon fluence rate of 90 pmol me2 SC’.
‘.,.’ 1 1 1 1 1 1 0 1 1 1 1 1 1 35c 300 250
4.0251
Wavelength [nm]
Fig. 7. Effects of W-B irradiation on the absorption spectrum of the cell membrane fraction of Rh. minuta: (a) Absorption spectra before (-) and after (-- -) 30 min irradiation; (b) the difference spectra after irradiation for 10 min (-), 20 min (---) and 30 min (- . . -). The irradiation conditions were the same as in Fig. 5.
I
’
’
t
’
’
’
’
’
’
’
’
’
’
’
I-
0.4
1
#I!3
10 I
10 I 20 20 40 I,.1 -‘5 Irradiation period [min]
J
40
Fig. 6. Relationship between the irradiation period and the extent of spectral change of a cell wall fraction: (a) increase in absorbance at 280 nm; (b) decrease in absorbance at 246 nm. The experimental conditions were the same as in Fig. 5.
in the absorption spectrum of an ethyl alcohol solution of ergosterol caused by UV-B irradiation (Fig. 9). The pattern of a difference spectrum of the ergosterol solution was similar to that in the cell membrane fraction, i.e. decreases at 270, 281 and 293 nm and increases at 240 and 300 nm. This finding strongly suggests that ergosterol may be responsible for the spectral changes of the membrane fraction. 3.3. Induction of carotenogenesis It is tempting to known whether or not the fluence of the UV-B administered were at the “physiological” levels and not the injurious levels. Also, the actual dosage levels of the UV-B reaching cells in a suspension are difficult to measure because of its absorption and scattering. Hence, the
-0.041 ’ .’
I
250
’
’
’
’
’
’
’
1
3!
Wavelength [nm]
Fig. 8. Effect of UV-B irradiation on the absorption spectrum of the soluble fraction of Rh. minuta: (a) absorption spectra before (-) and after (- - -) 60 min irradiation; (b) the difference spectrum after irradiation for 60 min. The irradiation conditions were the same as in Fig. 5.
cells which served to determine the absorption spectra were transferred to the valine medium and cultured for 15 h to observe the cell population and the synthesis of carotenoids. The cell population was not affected by up to 5 min irradiation (Fig. 10). Carotenoid synthesis occurred, increasing
M. Hada et al. / UV-B-induced
132
absorbance
0.008
changes in Rh. minuta
-
-z : G: $ % 0.007 -
-71
I
/’
3 Irradiation
Wavelength
[nm]
Fig. 9. Effects of UV-B irradiation on the absorption spectrum of 100 PM ergosterol solution in ethyl alcohol: (a) absorption spectra before (-) and after (---) 6 min irradiation; (b) the difference spectra after irradiation for 2 min (-), 4 min (- --) and 6 min (. . . . .). The irradiation conditions were the same as in Fig. 5.
++I
1
21 irradiation
5I
IO 1
20 I
period
[mini
50 I
100 I
Fig. 10. Effects of UV-B irradiation periods on the cell yield after 15 h incubation in the dark. Cells were irradiated to determined the absorption spectra in a cuvette at a density of 0.3 mg dry mass ml-‘. The cells were suspended in the valine medium and incubated in the dark for 15 h. The cell density on the ordinate represents turbidity at 600 nm when the cells were resuspended in 5 ml deionized water. The initial cell density before incubation was 0.081. Each point in the figure represents the mean of six samples+standard error. The irradiation conditions were the same as in Fig. 5. The abscissa is on a logarithmic scale.
with increasing periods of irradiation up to 5 min, where no decrease in cell population was found (Fig. 11). Further extended irradiation suppressed cell population, which may reflect the decline of carotenoid synthesis.
period
5
10
[min]
Fig. 11. Effects of UV-B irradiation periods on carotenoid synthesis. The irradiated cells were incubated as in Fig. 10. The contents of carotenoids were estimated by the difference between the absorbances at 498 and 522 nm. The density of cells at the time of irradiation was 0.3 mg dry mass ml-‘. Each point in the figure represents the mean of six samplesfstandard error. The irradiation conditions were the same as in Fig. 5.
3.4. Effect of sterol inhibitors on Winduced absorbance changes Since tridemorph (2,6-dimethyl-N-tridecylmorpholine) and fenpropimorph (N-[3-(p-teti-butylphenyl)-Zmethylpropyll-cis-2,6-dimethylmorpholine) have been reported to inhibit the biosynthesis of ergosterol in Saccharomyces cerevisiae [20], we examined whether these sterol inhibitors affected the spectral changes and the carotenoid synthesis in Rh. minuta. Cells were grown for 48 h in media containing tridemorph or fenpropimorph at 100 or 1 PM and in the medium without the inhibitors (control) and were tested for UV-B-induced spectral changes and carotenoid synthesis. Both inhibitors at 100 PM inhibited the growth of the cells by about 50% but did not affect it at 1 PM. Treatment with inhibitors abolished the increases in the absorbances at 285 nm and in its neighborhood as well as at 240 nm completely at 100 PM (Figs. 12(a)-12(c)) and reduced them by about 50% at 1 PM (Figs. 12(d)-12(f)). A decrease in absorbance was discerned at 280 nm in 100 PM tridemorph-treated cells and at 260 nm in 100 PM fenpropimorph-treated cells. Carotenoid synthesis was, not inhibited by either inhibitor (Table 1).
4. Discussion In the present study with Rh. minuta cells, we described UV-B-induced absorbance changes occurring at wavelengths shorter than 320 nm. The
M. Hada et al. I UV-B-induced absorbance changes in Rh. minuta
133
100 ulbl Fenpropimorph
1 ut.4 Tridemorph E
0.010
s g 0.005 (0 2 0 a
I,,
-O.o05c,
, ,I
300
250
I I I I I
350
350
250
250
Wavelength [min] Fig. 12. Effects of the steroid inhibitors on IN-induced absorbance changes in Rh. minutu cells. The curves are difference spectra and 20 min (---) and (d)-(f) 5 min (-) and 10 min of intact cells irradiated at 90 pmol m-* s- ’ for (a)-(c) 10 min (-) (---): (a), (d) control cells grown without inhibitors; (b) cells grown with 100 PM tridemorph; (c) cells grown with 100 PM fenpropimorph; (e) cells grown with 1 PM tridemorph; (f) cells grown with 1 PM fenpropimorph. TABLE
1
Effects of the steroid inhibitors, tridemorph and fenpropimorph on UV-B-induced carotenoid synthesis. Cells were grown for 48 h with or without 1 PM tridemorph or fenpropimorph and then irradiated with a photon fluence-rate of 25 pmol m-’ s-’ for 5, 10, 20 min with UV-31OU340. Irradiated cells were incubated in the dark for 15 h. The values in the table are differences in absorbance at 498 and 522 nm, representing contents of carotenoids. Density of cells at the time of irradiation, 5 mg dry weight ml-’ Irradiation
Inhibitors
Absorbance 498-522 nm Exp. 2 Exp. 1
Control
Tridemorph
Fenpropimorph
[l PM]
[l FM]
Dark UV 5 min 10 min 20 min
0.0330 0.0348 0.0335 -
0.0261 0.0360 0.0383 0.0408
Dark UV 5 min 10 min 20 min
0.0350 0.0443 0.0443 -
0.0317 0.0490 0.0513 0.0535
Dark UV 5 min 10 min
0.0348 0.0403 0.0380
-
absorbance changes consisted mostly of two major components: one occurred in the cell wall, and the other in the cell membrane. The absorbance change observed when an ethyl alcohol solution of ergosterol was similarly irradiated with UV-B was quite similar to most of the absorbance changes of the cell membrane (Figs.
7 and 9); the probable abundant existence of ergosterol in the membrane may explain the component of the spectral changes occurring in the cell membrane. The kind of compound that is responsible for the absorbance changes in the cell wall has not been clarified in the present study. However, the difference spectra observed with the cell wall fraction showed not shift in the peak and trough wavelengths and formed an isosbestic point (Fig. 5). In this response the Bunsen-Roscoe law held (Fig. 4). These findings suggest that this absorbance change may involve a single compound, which is converted to a photoproduct having conjugated double bonds. The results of the experiment with the steroid biosynthesis inhibitors suggested that the photoreactant may be a compound related to steroids, or a compound which is produced in the presence of steroids. The UV-B-induced absorbance changes at 285 nm occurred, although slightly, even at the irradiance level of UV-B which does not suppress the cell population but does induce carotenoid synthesis. The photoreaction is very fast and can be observed immediately after the termination of an irradiation of a few minutes at the fluence rate used. However, the possibility is excluded that the absorbance changes may be involved in a primary reaction of the UV-B-induced carotenoid synthesis, because the steroid synthesis inhibitors did not suppress the carotenoid synthesis, although they suppressed the absorbance changes found in this work. Yet the absorption changes occurring in the cell wall and cell membrane are new findings and
134
M. Hada
may contribute to the understanding on Rh. minuta.
et al. / W-B-induced
of UV effects
absorbance
changes
in Rh. minuta
8 T. Kumagai, Action spectra for the blue and near ultraviolet reversible photoreaction in the induction of fungal conidiation, Physiol.
Plant.,
57 (1983)
468471.
9 S. Yamamura, T. Kumagai and Y. Oda, An action spectrum for photoinduced conidiation in Helminthosporium oryzae, Plant Cell Physiol.,
Acknowledgments We thank Dr. tridemorph and Yokohama City puter software oratory for his
Ammermann for kindly providing fenpropimorph, Dr. T. Hisabori, University for his help with comand Dr. M. Uemura of our labhelpful suggestions.
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A ypyronyl15 S. Tsurumi, T. Takagi and T. Hashimoto, triterpenoid saponin from Pisum sativum, Phytochemishy 31 (1992)
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16 T. Hashimoto, H. Yatsuhashi and H. Kato, Self-corrected, convertible handy irradiance meter with a photodiode, in Matsumoto (ed.), Abstracts, Annu. Meet. of Japanese Society of Plant Physiology, 1982, 1982, p. 38. 17 M. Tada and M. Shiroishi, Mechanism of photoregulated carotenogenesis in Rhodotonda minuta. I. Photocontrol of carotenoid production, Plant Cell Physiol., 23 (1982) 541-547. 18 H. Suomalainen and T. Nurminen, Structure and function of the yeast cell envelope, in J. R. Villanueva, I. GarciaAcha, S. Gascon and F. Uruburu (eds.), Yeast, Mould and Plant Protoplasts, Academic Press, London, 1973, pp. 167-186. 19 A. L. Hirsch, Vitamin D, in M. Grayson (ed.), Encyclopedia of Chemical Technology, 3rd edn., Vol. 24, Wiley-Interscience, New York, 1984, pp. 186-213. 20 R. I. Baloch, E. I. Mercer, T. E. Wiggins and B. C. Baldwin, Inhibition of ergosterol biosynthesis in Saccharomyces cerevisiae and Ustilago maydis by tridemorph, fenpropimorph and fenpropidin, Phytochemistry, 23 (1984) 2219-2226.