Planta
Planta (1983)159:267 276
9 Springer-Verlag 1983
The action spectrum for chloroplast movements and evidence for blue-light-photoreceptor cycling in the alga Vaucheria* Michael R. Blatt** Carnegie Institution of Washington, Department of Plant Biology, 290 Panama St., Stanford, CA 94305, USA
Abstract. Local stimulation of the coenocytic alga Vaucheria sessilis D.C. by blue light resulted in accumulation of chloroplasts and other organelles. The photoresponse followed a well-defined, wavelength- and fluence-rate-dependent latency period (> 10 s), and could lead to a tenfold decrease in relative cellular transmittance to 675-nm light within 5 min. Light-induced aggregation of chloroplasts was examined at eight wavelengths of light between 385 and 528 nm. A fiber-optic microphotometer was employed and the response was quantitated on the basis of the rate of 675-nm transmittance change after correcting for changes in light scattering. Chloroplast aggregation exhibited a nearly identical quantum-flux-density dependence at all eight wavelengths tested; it showed an action spectrum with a sharp maximum near 470 rim, a trough at 430 nm, and action in the near-ultraviolet spectral region. Light at 454 nm was six times less effective than 473-nm light in stimulating aggregation, a difference which could not be accounted for by chlorophyll screening alone. Beyond the latency period reciprocity did not hold for chloroplast aggregation. Instead, aggregation could be fitted to a kinetic model involving steadystate photoreceptor cycling during continuous irradiation. Chloroplast aggregation in the light was compared with three growth-associated photoresponses in Vaucheria - phototropic bending, branching and apical expansion. Time course and kinetic similarities, and the presence of a cytoplasmic fiber network in growing tips of Vaucheria, indicate that these photoresponses may be related mechanistically. * C.I.W.-D.P.B. Publication No. 823 Botany School, University of Cambridge, Cambridge CB2 3EA, UK ** P r e s e n t a d d r e s s :
Key words: Action spectrum - Blue light - Chloroplast movement - Photoreceptor cycling - Vaucheria - Xanthophyta.
Introduction Of the diverse responses of plants and microorganism to blue light, chloroplast redistributions are among the more rapid and dramatic. These phenomena include local promotion or inhibition of organelle movement within the cell as well as lightinduced alterations of chloroplast orientation and shape (see Haupt and Sch6nbohm 1970; Britz 1979). Chloroplast movements depend both upon the spatial distribution of incident light within the cell and upon its intensity, and probably optimize photosynthetic capacity under adverse lighting conditions (Lechowski 1974; Gabrys-Mizera 1976; see also Britz/979). Photoreceptors for the organelle movements, nonetheless, are not generally associated with the chloroplasts. Action spectra in most instances support the idea that the photosensory pigments are flavoproteins (see Song and Moore 1974; Britz 1979; also discussion by Shropshire 1980). Furthermore, experiments employing irradiations with microbeams or with polarized light (or both) have indicated that the primary photoreceptors are located within the cortical regions of the cells (Fischer-Arnold 1963; Haupt 1970), and recent electrical data from Vaucheria (Blatt et al. 1981) are consistent with a photoreceptor localized at the plasma membrane. Chloroplast movements in coenocytic algae, such as Vaucheria, are particularly striking and can result in extensive organelle accumulation within
268
a small region of the cell. Fischer-Arnold (1963), who first described chloroplast aggregation in Vaucheria in some detail, observed that this behavior resulted from the "trapping" of organelles as they streamed into the light. Aggregation followed a well-defined, wavelength- and intensity-dependent lag period (> 10 s), for which the Bunsen-Roscoe reciprocity law held (I.t= constant), and exhibited maximal sensitivity to blue light (approx. 450 nm) with a minor peak in the near ultra-violet spectral region. Recently, substantial progress has been made toward characterizing the sequence of events between photon absorption and chloroplast aggregation in Vaucheria. Blatt and Briggs (1980) found that aggregation was preceded by and dependent upon formation of a network or reticulum of cytoplasmic fibers, composed of large actin bundles (Blatt et al. 1980). Also associated with aggregation was an outward-directed current, possibly carried by protons, which appeared simultaneously with fiber reticulation (Blatt et al. 1981). In contrast to the Fischer-Arnold's results, however, measurements of the wavelength sensitivity for reticulation (Blatt and Briggs 1980), and early results at two wavelengths for chloroplast aggregation and for the current (Blatt et al. 1981) indicated that all three events were more sensitive to light at 470 nm that at 450 nm. Indeed, the fluence-rate-response data were consistent with a very sharp action maximum near the longer wavelength. Thus, a reinvestigation of the aggregation action spectrum was in order. The action spectrum was of interest, too, because of a possible association of aggregation with three growth-related responses to blue light in Vaucheria, namely phototropic bending, branching and apical expansion (Kataoka 1975a, b, 1981). The present paper deals with this reinvestigation. Additional evidence supports a mechanistic relationship between chloroplast aggregation and the three growth-affiliated photoresponses. Moreover, the kinetic results are fitted to a model involving a photostationary level of excited photoreceptor in the steady state. Material and methods Algal culture and experimental protocol. Axenic cultures of Vaucheria sessilis D.C. were maintained on 0.75 mB1/BBT (a modified Bold's medium with added biotin, vitamin B12 and thiamine; see Blatt and Briggs 1980) agar plates and transferred to liquid 0.75 roB1 medium for experimentation at weekly intervals. Unbranched filaments 2-4 cm in length were mounted in microphotometer chambers (Blatt and Briggs 1977, 1983) in 0.75 mB1 and the chambers placed in the dark 12 h before the beginning of each experiment. All subsequent operations
M.R. Blatt: Action spectrum for Vaucheria chloroplast movements were performed under dim green safelight (see Video analysis below, also Blatt and Briggs 1983). Photometric measurements were carried out as described below. Multiple recordings from single cells were made with the fiber optics repositioned, and test sites were chosen at l-mm intervals along the algal filament. Measurements at the individual sites were repeated, when necessary, only after an intervening l-h dark period. An ambient temperature of 20+ 1~ C was maintained throughout these experiments.
Microphotometry. Chloroplast aggregation was measured with a fiber-optic microphotometer as described previously (Blatt and Briggs 1977, 1983). Individual cells were irradiated locally with light from a single, 55-gin-diameter optical fiber positioned within 10 gm of the cell. Transmitted light was collected end-on at the opposing cell surface with a second optical fiber and conducted to a phototransistor, the output of which was amplified and recorded on a strip-chart recorder. Wavelengths of light at 675 nm (AL675 interference filter, Schott and Gen., Mainz, F R G ; and CS3-66 cut-off filter, Corning Glass, Coming, N.Y., USA) and 743 nm (AL743 interference filter, Schott and Gen. ; and CS2-60 cut-off filter, Corning) were used to measure transmission changes resulting from chlorophyll absorption and to correct for changes in light scattering during chloroplast aggregation, respectively. The measuring beams were chopped alternately, combined with an actinic light beam, and focused on one end of the optical fiber used to irradiate the cells. Transmitted actinic light was removed with two small filter discs (Roscolene 823 and 809, Edmund Scientific, Barrington, N.J., USA) placed in front of the phototransistor. For the present studies, actinic light was provided by a high-pressure mercury arc lamp (PEK 401, PEK Industries, Sunnyvale, Calif., USA), selected interference filters (see Table 1) and an appropriate combination of neutral density filters (Schott and Gen.). Measuring and actinic beams passed through a heat filter (KG-1, 4 ram; Schott and Gen.) to remove wavelengths of light beyond 800 nm. The microphotometer was calibrated and light intensities at the algal cell surface calculated as before (Blatt and Briggs 1980; Blatt et al. 1981; Blatt and Briggs 1983). Measuring-beam energy fluence rates (time averaged) of 100 mW m -z at 675 nm and 10 mW m z at 743 nm were sufficient to follow the transmittance changes during chloroplast
Table 1. Wavelength maxima and halfband widths for Schott (Schott and Gen., Mainz, FRG) DAL and AL, and Balzers (Balzers, Liechtenstein) B-40 series interference filters
Wavelength maximum (nm)
Halfband width (nm)
Filter series
385
11 9 17 18 20 15 9 9 23 20 14 18 22
B-40 DAL DAL DAL DAL DAL B-40 B-40 AL DAL B-40 AL AL
411 431 454 473 491 512 528 554 579 598 675 743
M.R. Blatt: Action spectrum for Vaucheria chloroplast movements
Video analysis and photomicrography. For direct microscopic observation a Leitz Ortholux microscope (Leitz, Wetzlar, FRG) was employed. Non-actinic background lighting (579 nm, 1-3 W m 2) was provided by the microscope illuminator and a 6-mm heat filter (Leitz) plus a DAL579 interference filter (Schott and Gen.). Chloroplast densities (Pc) and scalar streaming velocities (re) were obtained from real time video recordings (Mikroscop Video, Grundig, F/irth, FRG) taken immediately preceding each photometric measurement. A calibrated grid was placed on the video monitor screen and chloroplasts counted in triplicate at 30-s intervals (recording time) over an area corresponding to at least 200 ~tm2. Chloroplast scalar streaming velocities were measured by following the movements of 10-15 organelles during three consecutive 30-s intervals. At first, mean velocities for each interval were recorded separately as a precaution against any photokinetic effects of the background lighting required for video recording. This practice was dropped when no consistent differences in streaming velocities were observed within any set of three intervals. Photomicrographs were taken on a Zeiss Universal microscope equipped with Nomarski Differential Interference Contrast optics (see Blatt and Briggs 1980), and recorded on 25 mm Tri-X Pan film "Eastman Kodak, Rochester, N.Y., USA).
aggregation. Control experiments (data not shown) were performed in which the measuring-beam pulses (nominally 0.75 s each, frequency 0.2 s - j ) were reduced to a frequency of 0.03 s- 1. The 579-nm interference filter in front of the microscope illuminator (see below) was replaced by a second AL675/ CS3-66 or AL743/CS2-60 filter combination, or by a red cut-off filter (CS2-60; Corning), but no measurable effect of light at these wavelengths on aggregation was observed, even at fluence rates of 1 W m - 2. During chloroplast aggregation, relative cell transmittance to 675- and 743-nm light decreased exponentially. Time points at 15-s intervals following the onset of aggregation and initial decline in 675 nm transmittance (time t = 0 ) were selected and the scatter-corrected relative transmittance to 675 nm light, Toorr, t, calculated as ln(T~ ..... ) =ln(T675.t)--~,ln(T74.3. t)
(1)
where T675, ~ and Tv43, t are the relative transmittances to 675and 743-nm light at time t normalized to their zero-time values, and ~'r is the scattering-coefficient ratio for the two wavelengths and equals 1.32 (Blatt and Briggs 1983). Values of Toom t during the first 3 rain of aggregation were subject to log-linear leastsquares regression analysis (regression coefficients > 0.990) and the slope, -Aln(Too,r ' t)/At taken as a measure of the chloroplast aggregation rate. The rate of aggregation, rather than its extent at any given time, provided a satisfactory index of the photoresponse for two reasons. First, data scatter was minimized, since rate measurement effectively averaged transmittance changes over a 3-min period. Second, the photoresponse itself was independent of the organelles entering the light beam (see Fig. 2 and Results, also Fischer-Arnold 1963; Blatt and Briggs 1980, 1983) and, thus, could be viewed as a light-mediated "trapping potential" proportional to the photometric aggregation rate.
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0.80
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269
Results
Before examining the wavelength and fluence-rate dependence of chloroplast aggregation, it was necessary to eliminate, or at least account for, other factors which might influence the photoresponse as measured photometrically. Fischer-Arnold
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g "~ 0.992) at all eight wavelengths (see Table 3), suggesting that a single photoreceptor mediates the response. The action spectrum for aggregation at the 0.63 unit (50%) response level is shown in Fig. 4. Chloroplast aggregation showed a sharp action maximum at 473 rim, a trough between 410 and 450 nm and action in the near-UV. Light at 598 nm resulted in only small changes in corrected transmittance over 10-15 min, even at the highest fluence
272
M.R. Btatt: Action spectrum for Vaucheria chloroplast movements
Table 3. Summary of regression analyses for data in Fig. 3. Data points for each wavelength were subjected to log-linear least-squares fitting. Response thresholds were estimated by extrapolation of the resultant regression lines to the zero response level
which might be attributed also to a change in chloroplast shape (Fischer-Arnold 1963; Blatt and Briggs 1983).
Wavelength (nm)
Slope
Discussion
385 411 431 454 473 491 512 528
0.200 0.217 0.196 0.220 0.217 0.200 0.192 0.191
1.0
_
Response threshold (pmol c m - z s-
1)
32 160 250 110 19 32 270 33,000
Regression coefficient
0.998 0.992 0.998 0.997 0.996 0.992 0.993 0.998
l
0.8
-~
~)
:
--
6
i
~:
0.6
400
-
i
450
I
500
W a v e L e n g t h , nm
E
Z o
0,4
0.2
l__L '400
"~
I 450
500
Wavelength, nm Fig. 4. Action spectrum for chloroplast aggregation in Vaucheria at the 50% response level. Data from Fig. 3. Quantum flux densities giving half-maximal response were determined according to Eq. (4) and Fig. 2 (legend). Inset:Action spectrum plotted logarithmically
rates attainable (
Planta (1983)159:267 276
9 Springer-Verlag 1983
The action spectrum for chloroplast movements and evidence for blue-light-photoreceptor cycling in the alga Vaucheria* Michael R. Blatt** Carnegie Institution of Washington, Department of Plant Biology, 290 Panama St., Stanford, CA 94305, USA
Abstract. Local stimulation of the coenocytic alga Vaucheria sessilis D.C. by blue light resulted in accumulation of chloroplasts and other organelles. The photoresponse followed a well-defined, wavelength- and fluence-rate-dependent latency period (> 10 s), and could lead to a tenfold decrease in relative cellular transmittance to 675-nm light within 5 min. Light-induced aggregation of chloroplasts was examined at eight wavelengths of light between 385 and 528 nm. A fiber-optic microphotometer was employed and the response was quantitated on the basis of the rate of 675-nm transmittance change after correcting for changes in light scattering. Chloroplast aggregation exhibited a nearly identical quantum-flux-density dependence at all eight wavelengths tested; it showed an action spectrum with a sharp maximum near 470 rim, a trough at 430 nm, and action in the near-ultraviolet spectral region. Light at 454 nm was six times less effective than 473-nm light in stimulating aggregation, a difference which could not be accounted for by chlorophyll screening alone. Beyond the latency period reciprocity did not hold for chloroplast aggregation. Instead, aggregation could be fitted to a kinetic model involving steadystate photoreceptor cycling during continuous irradiation. Chloroplast aggregation in the light was compared with three growth-associated photoresponses in Vaucheria - phototropic bending, branching and apical expansion. Time course and kinetic similarities, and the presence of a cytoplasmic fiber network in growing tips of Vaucheria, indicate that these photoresponses may be related mechanistically. * C.I.W.-D.P.B. Publication No. 823 Botany School, University of Cambridge, Cambridge CB2 3EA, UK ** P r e s e n t a d d r e s s :
Key words: Action spectrum - Blue light - Chloroplast movement - Photoreceptor cycling - Vaucheria - Xanthophyta.
Introduction Of the diverse responses of plants and microorganism to blue light, chloroplast redistributions are among the more rapid and dramatic. These phenomena include local promotion or inhibition of organelle movement within the cell as well as lightinduced alterations of chloroplast orientation and shape (see Haupt and Sch6nbohm 1970; Britz 1979). Chloroplast movements depend both upon the spatial distribution of incident light within the cell and upon its intensity, and probably optimize photosynthetic capacity under adverse lighting conditions (Lechowski 1974; Gabrys-Mizera 1976; see also Britz/979). Photoreceptors for the organelle movements, nonetheless, are not generally associated with the chloroplasts. Action spectra in most instances support the idea that the photosensory pigments are flavoproteins (see Song and Moore 1974; Britz 1979; also discussion by Shropshire 1980). Furthermore, experiments employing irradiations with microbeams or with polarized light (or both) have indicated that the primary photoreceptors are located within the cortical regions of the cells (Fischer-Arnold 1963; Haupt 1970), and recent electrical data from Vaucheria (Blatt et al. 1981) are consistent with a photoreceptor localized at the plasma membrane. Chloroplast movements in coenocytic algae, such as Vaucheria, are particularly striking and can result in extensive organelle accumulation within
268
a small region of the cell. Fischer-Arnold (1963), who first described chloroplast aggregation in Vaucheria in some detail, observed that this behavior resulted from the "trapping" of organelles as they streamed into the light. Aggregation followed a well-defined, wavelength- and intensity-dependent lag period (> 10 s), for which the Bunsen-Roscoe reciprocity law held (I.t= constant), and exhibited maximal sensitivity to blue light (approx. 450 nm) with a minor peak in the near ultra-violet spectral region. Recently, substantial progress has been made toward characterizing the sequence of events between photon absorption and chloroplast aggregation in Vaucheria. Blatt and Briggs (1980) found that aggregation was preceded by and dependent upon formation of a network or reticulum of cytoplasmic fibers, composed of large actin bundles (Blatt et al. 1980). Also associated with aggregation was an outward-directed current, possibly carried by protons, which appeared simultaneously with fiber reticulation (Blatt et al. 1981). In contrast to the Fischer-Arnold's results, however, measurements of the wavelength sensitivity for reticulation (Blatt and Briggs 1980), and early results at two wavelengths for chloroplast aggregation and for the current (Blatt et al. 1981) indicated that all three events were more sensitive to light at 470 nm that at 450 nm. Indeed, the fluence-rate-response data were consistent with a very sharp action maximum near the longer wavelength. Thus, a reinvestigation of the aggregation action spectrum was in order. The action spectrum was of interest, too, because of a possible association of aggregation with three growth-related responses to blue light in Vaucheria, namely phototropic bending, branching and apical expansion (Kataoka 1975a, b, 1981). The present paper deals with this reinvestigation. Additional evidence supports a mechanistic relationship between chloroplast aggregation and the three growth-affiliated photoresponses. Moreover, the kinetic results are fitted to a model involving a photostationary level of excited photoreceptor in the steady state. Material and methods Algal culture and experimental protocol. Axenic cultures of Vaucheria sessilis D.C. were maintained on 0.75 mB1/BBT (a modified Bold's medium with added biotin, vitamin B12 and thiamine; see Blatt and Briggs 1980) agar plates and transferred to liquid 0.75 roB1 medium for experimentation at weekly intervals. Unbranched filaments 2-4 cm in length were mounted in microphotometer chambers (Blatt and Briggs 1977, 1983) in 0.75 mB1 and the chambers placed in the dark 12 h before the beginning of each experiment. All subsequent operations
M.R. Blatt: Action spectrum for Vaucheria chloroplast movements were performed under dim green safelight (see Video analysis below, also Blatt and Briggs 1983). Photometric measurements were carried out as described below. Multiple recordings from single cells were made with the fiber optics repositioned, and test sites were chosen at l-mm intervals along the algal filament. Measurements at the individual sites were repeated, when necessary, only after an intervening l-h dark period. An ambient temperature of 20+ 1~ C was maintained throughout these experiments.
Microphotometry. Chloroplast aggregation was measured with a fiber-optic microphotometer as described previously (Blatt and Briggs 1977, 1983). Individual cells were irradiated locally with light from a single, 55-gin-diameter optical fiber positioned within 10 gm of the cell. Transmitted light was collected end-on at the opposing cell surface with a second optical fiber and conducted to a phototransistor, the output of which was amplified and recorded on a strip-chart recorder. Wavelengths of light at 675 nm (AL675 interference filter, Schott and Gen., Mainz, F R G ; and CS3-66 cut-off filter, Corning Glass, Coming, N.Y., USA) and 743 nm (AL743 interference filter, Schott and Gen. ; and CS2-60 cut-off filter, Corning) were used to measure transmission changes resulting from chlorophyll absorption and to correct for changes in light scattering during chloroplast aggregation, respectively. The measuring beams were chopped alternately, combined with an actinic light beam, and focused on one end of the optical fiber used to irradiate the cells. Transmitted actinic light was removed with two small filter discs (Roscolene 823 and 809, Edmund Scientific, Barrington, N.J., USA) placed in front of the phototransistor. For the present studies, actinic light was provided by a high-pressure mercury arc lamp (PEK 401, PEK Industries, Sunnyvale, Calif., USA), selected interference filters (see Table 1) and an appropriate combination of neutral density filters (Schott and Gen.). Measuring and actinic beams passed through a heat filter (KG-1, 4 ram; Schott and Gen.) to remove wavelengths of light beyond 800 nm. The microphotometer was calibrated and light intensities at the algal cell surface calculated as before (Blatt and Briggs 1980; Blatt et al. 1981; Blatt and Briggs 1983). Measuring-beam energy fluence rates (time averaged) of 100 mW m -z at 675 nm and 10 mW m z at 743 nm were sufficient to follow the transmittance changes during chloroplast
Table 1. Wavelength maxima and halfband widths for Schott (Schott and Gen., Mainz, FRG) DAL and AL, and Balzers (Balzers, Liechtenstein) B-40 series interference filters
Wavelength maximum (nm)
Halfband width (nm)
Filter series
385
11 9 17 18 20 15 9 9 23 20 14 18 22
B-40 DAL DAL DAL DAL DAL B-40 B-40 AL DAL B-40 AL AL
411 431 454 473 491 512 528 554 579 598 675 743
M.R. Blatt: Action spectrum for Vaucheria chloroplast movements
Video analysis and photomicrography. For direct microscopic observation a Leitz Ortholux microscope (Leitz, Wetzlar, FRG) was employed. Non-actinic background lighting (579 nm, 1-3 W m 2) was provided by the microscope illuminator and a 6-mm heat filter (Leitz) plus a DAL579 interference filter (Schott and Gen.). Chloroplast densities (Pc) and scalar streaming velocities (re) were obtained from real time video recordings (Mikroscop Video, Grundig, F/irth, FRG) taken immediately preceding each photometric measurement. A calibrated grid was placed on the video monitor screen and chloroplasts counted in triplicate at 30-s intervals (recording time) over an area corresponding to at least 200 ~tm2. Chloroplast scalar streaming velocities were measured by following the movements of 10-15 organelles during three consecutive 30-s intervals. At first, mean velocities for each interval were recorded separately as a precaution against any photokinetic effects of the background lighting required for video recording. This practice was dropped when no consistent differences in streaming velocities were observed within any set of three intervals. Photomicrographs were taken on a Zeiss Universal microscope equipped with Nomarski Differential Interference Contrast optics (see Blatt and Briggs 1980), and recorded on 25 mm Tri-X Pan film "Eastman Kodak, Rochester, N.Y., USA).
aggregation. Control experiments (data not shown) were performed in which the measuring-beam pulses (nominally 0.75 s each, frequency 0.2 s - j ) were reduced to a frequency of 0.03 s- 1. The 579-nm interference filter in front of the microscope illuminator (see below) was replaced by a second AL675/ CS3-66 or AL743/CS2-60 filter combination, or by a red cut-off filter (CS2-60; Corning), but no measurable effect of light at these wavelengths on aggregation was observed, even at fluence rates of 1 W m - 2. During chloroplast aggregation, relative cell transmittance to 675- and 743-nm light decreased exponentially. Time points at 15-s intervals following the onset of aggregation and initial decline in 675 nm transmittance (time t = 0 ) were selected and the scatter-corrected relative transmittance to 675 nm light, Toorr, t, calculated as ln(T~ ..... ) =ln(T675.t)--~,ln(T74.3. t)
(1)
where T675, ~ and Tv43, t are the relative transmittances to 675and 743-nm light at time t normalized to their zero-time values, and ~'r is the scattering-coefficient ratio for the two wavelengths and equals 1.32 (Blatt and Briggs 1983). Values of Toom t during the first 3 rain of aggregation were subject to log-linear leastsquares regression analysis (regression coefficients > 0.990) and the slope, -Aln(Too,r ' t)/At taken as a measure of the chloroplast aggregation rate. The rate of aggregation, rather than its extent at any given time, provided a satisfactory index of the photoresponse for two reasons. First, data scatter was minimized, since rate measurement effectively averaged transmittance changes over a 3-min period. Second, the photoresponse itself was independent of the organelles entering the light beam (see Fig. 2 and Results, also Fischer-Arnold 1963; Blatt and Briggs 1980, 1983) and, thus, could be viewed as a light-mediated "trapping potential" proportional to the photometric aggregation rate.
I [--
I.oo
r
r
I
/
A
7
~"
0.80
"~
o
o~
3
269
Results
Before examining the wavelength and fluence-rate dependence of chloroplast aggregation, it was necessary to eliminate, or at least account for, other factors which might influence the photoresponse as measured photometrically. Fischer-Arnold
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o
).40
I
/
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o
o
3.60
g "~ 0.992) at all eight wavelengths (see Table 3), suggesting that a single photoreceptor mediates the response. The action spectrum for aggregation at the 0.63 unit (50%) response level is shown in Fig. 4. Chloroplast aggregation showed a sharp action maximum at 473 rim, a trough between 410 and 450 nm and action in the near-UV. Light at 598 nm resulted in only small changes in corrected transmittance over 10-15 min, even at the highest fluence
272
M.R. Btatt: Action spectrum for Vaucheria chloroplast movements
Table 3. Summary of regression analyses for data in Fig. 3. Data points for each wavelength were subjected to log-linear least-squares fitting. Response thresholds were estimated by extrapolation of the resultant regression lines to the zero response level
which might be attributed also to a change in chloroplast shape (Fischer-Arnold 1963; Blatt and Briggs 1983).
Wavelength (nm)
Slope
Discussion
385 411 431 454 473 491 512 528
0.200 0.217 0.196 0.220 0.217 0.200 0.192 0.191
1.0
_
Response threshold (pmol c m - z s-
1)
32 160 250 110 19 32 270 33,000
Regression coefficient
0.998 0.992 0.998 0.997 0.996 0.992 0.993 0.998
l
0.8
-~
~)
:
--
6
i
~:
0.6
400
-
i
450
I
500
W a v e L e n g t h , nm
E
Z o
0,4
0.2
l__L '400
"~
I 450
500
Wavelength, nm Fig. 4. Action spectrum for chloroplast aggregation in Vaucheria at the 50% response level. Data from Fig. 3. Quantum flux densities giving half-maximal response were determined according to Eq. (4) and Fig. 2 (legend). Inset:Action spectrum plotted logarithmically
rates attainable (
