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Phytochrome-like responses in Euglena: a low
fluence response that reorganizes the spectral
dependence of the high irradiance response in
long-day photoperiodic induction of cell
division
Bolige, Aoen, Goto, Ken
Journal of photochemistry and photobiology. B,
Biology, 86: 97-108
2007
http://ir.obihiro.ac.jp/dspace/handle/10322/720
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帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
1
Phytochrome-like responses in Euglena: a low fluence response that reorganizes the
spectral dependence of the high irradiance response in long-day photoperiodic
induction of cell division
Aoen Bolige1, 2 and Ken Goto1
1.
Laboratory of Biological Rhythms, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro, Japan 080-8555.
2.
JSPS Research Fellow
Corresponding author: Ken Goto
Laboratory of Biological Rhythms, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro 080-8555, Japan
Tel: +81-155-49-5612
Fax: +81-155-49-5612
E-mail: [email protected]
Keywords: cell division, circadian rhythms, end-of-day, Euglena, photoperiodism,
phytochrome, shade avoidance
Abbreviations:
CRdusk: circadian rhythm shaping photoinductive phase occurring at dusk
CRG2: circadian rhythm regulating the timing of G2 cells progressing into the M phase
DD: continuous darkness
EOD: end of day
EOL: end of continuous light
EOL+FR: end of far-red light-enriched continuous light
EOLW: end of continuous light by cool-white fluorescent light
FR: far-red light (700-800 nm)
+FR-LL: far-red light-enriched continuous light
HIR: high-irradiance response
2
LEDxxx: LED with emission maximum at xxx nm
LFR: low-fluence response
LL: continuous light
MCL: monochromatic light
R: red light (630-700 nm)
W: cool-white fluorescent light
W-LL: LL by W
X-HIR or X-LFR: high-irradiance response or low-fluence response by either X nm light
or the X region of the visible spectrum.
3
Abstract
Irradiance spectra change spatiotemporally, and angiosperms adapt accordingly, mainly
through phytochromes. This study challenges the long-held belief that the flagellated alga
Euglena gracilis lacks phytochromes and is therefore unaffected by spectral changes. We
photoautotrophically cultured the alga under continuous light (LL), then transferred it to
darkness. After about 26 h in darkness, different irradiations for 3 h enabled cell division
in dark-arrested G2 cells evoking a high-irradiance response (HIR). The spectral
characteristics of the irradiation during the LL period (pre-irradiation) defined the
spectral sensitivity in the subsequent dark period. LL with light rich in the red spectrum
led to a HIR to the red spectrum (R-HIR), whereas light rich in the far-red spectrum (FR)
led to a FR-HIR. Finishing the period of pre-irradiation consisting of continuous
cool-white fluorescent light by a FR pulse enhanced the characteristics of the FR-HIR 26
h later. By contrast, a R pulse given at the end of the pre-irradiation rich in FR
potentiated the R-HIR. The effects were completely photoreversible between R and FR
with critical fluences of about 2 mmol m-2, satisfying the classic diagnostic feature of
phytochromes. The action spectrum of the FR effect at the end of pre-irradiation
consisting of continuous cool-white fluorescent light (rich in R) had a main peak at 740
nm and a minor peak at 380 nm, whereas antagonization of the FR effect had a main peak
at 640 nm and a minor peak at 480 nm. Wavelengths of 610 and 670 nm appeared in both
spectra. We also demonstrated the photoreversibility of 380/640, 480/740, and (610 and
670)/(640 and 740) nm. We conclude that Euglena displays phytochrome-like responses
similar to the ‘shade avoidance’ and ‘end-of-day FR’ effects reported in angiosperms.
4
1. Introduction
Phytochromes and homologous proteins are found in various organisms, including
cyanobacteria, non-photosynthetic bacteria, and fungi, although their physiological
function is known only in plants and green algae [1]. Nevertheless, it is thought that the
flagellated alga Euglena lacks phytochromes, based on Holowinsky and Schiff [2], who
demonstrated that potentiation, or enhanced capability by prior illumination, of
light-dependent chlorophyll synthesis does not reveal evidence for the involvement of
phytochromes in this alga, as it does in plants.
Euglena occupies a unique position in the evolutionary history of eukaryotes, and has
attributes of both plants and animals. Eukaryotes are currently divided into eight groups;
Euglena is a member of the discicristates, which have discoid mitochondrial cristae,
unlike both opisthokonts (including animals and fungi) and plants (including plants and
chlorophyte algae, i.e., green algae) [3, 4]. It would therefore perhaps not be surprising if
Euglena lacked phytochromes, and attempts to identify these structures in the alga ceased
following the report of Holowinsky and Schiff [5, 6].
The green alga Chlamydomonas may have played a role as a secondary symbiont [7]
in the evolution of Euglena, giving the latter its plant-like functions. Although a
monoclonal antibody (Pea-25) raised with pea phytochromes [8] showed evidence of
phytochrome-like proteins, functional phytochromes have not yet been reported in
Chlamydomonas [9, 10]. More recently, searches for homologous proteins have failed to
convincingly detect phytochrome-like sequences [11]. These findings may further
suggest that Euglena lacks phytochromes.
An important role of phytochromes is to sense spectral changes in the photic
environment, thereby providing spatiotemporal information. For example, the proportion
of blue light and the ratio of R to FR light change throughout the day and become
maximal and minimal, respectively, at midday. One way that plants respond to these
temporal changes is by the “end-of-day FR” (EOD-FR) response [12, 13]. Spatially,
shade changes the balance of light wavelengths, triggering the “shade avoidance”
response, which physiologically and morphologically resembles the EOD-FR response.
The aim of this study was to investigate whether Euglena either does not need to, or is
unable to, respond to spatiotemporal changes, whether it has different types of
5
photoreceptors that are capable of sensing spatiotemporal changes, or whether it indeed
has as yet unidentified phytochromes.
Studies of algal photosensing have centered on blue light syndromes, and as a result, a
novel UV-A/blue photoreceptor has been identified. Named photoactivated adenylyl
cyclase, it generates a second messenger, cyclic adenosine monophosphate, and mediates
a step-up (but not a step-down) photophobic response [14] and possibly also phototaxis
[15]. Photoreceptors in other spectral regions have not yet been identified, although
600-nm light enhances the potentiation of chlorophyll synthesis [16, 17]. It remains
unknown what types of photoreceptor are responsible for photoperiodic induction in
algae.
Photoperiodism is much simpler in algae than in plants and animals [18]. G2 cells of
Euglena grown photoautotrophically do not multiply in the dark unless they are irradiated
before the dark cycle [19]. A circadian rhythm (designated by CRdusk) that persists in
continuous darkness (DD) regulates the response to light, and irradiation at dusk
profoundly induces this capability, whereas irradiation at dawn has no effect. Thus, under
daily light/dark (LD) cycles, light becomes progressively important toward the end of the
photoperiod, and the alga displays photoperiodism with a long-day response of cell
reproduction [18, 20]. Note that CRdusk has only recently been discovered and is different
from another circadian rhythm called CRG2, which regulates the timing of cell division
[21–24]. Both of these phenomena interact to regulate cell-cycle progression during 24-h
LD cycles [24].
One method of studying photoperiodic photoinduction is to expose cells to 3 h of
irradiation at dusk (occurring at the 26th h in DD) to induce G2 cells to divide in the
subsequent dark; the increase in the cell count ≥12 h later represents the extent of
photoinduction [25]. This response is called the high-irradiance response (HIR) because
it depends on exposure time, and the critical fluence is typically well over 10 mmol m-2
[25]. Photoinduction experiments on Euglena using cool-white fluorescent light (W)
suggest that a signal generated by photosynthetic electron transport is involved [18]. In
contrast, studies using monochromatic light (MCL) have indicated otherwise [26], and
that the action maxima include 380, 450 (or 460), 480, 610 , 640, 660, 670 (or 680), and
740 nm [25]. Furthermore, the action spectra change dramatically when the R/FR ratio of
6
LL is manipulated [25]. In the present study, when the R/FR ratio was very high
(hereafter, designated W-LL), the 450 nm peak did not change and the peaks at 480, 640,
and 680 nm prevailed, whereas when the R/FR ratio was lowered using FR-enriched LL
(hereafter, designated +FR-LL), the peaks at 380, 610, 660, and 740 nm prevailed.
Because the major peaks occurred at 640 and 740 nm, photoinduction at these
wavelengths was designated R-HIR and FR-HIR, respectively; in other words, W-LL (or
+FR-LL) potentiated the R-HIR (or FR-HIR). The action spectra and HIRs will be
characterized in another paper [25]. Taken together, these findings suggest that Euglena
senses spatiotemporal changes in light quality and FR, but how it does so remains
unknown.
Phytochromes have been defined physiologically (but not biochemically) as
photoreceptors with photoreversibility between R and FR, which is characterized by a
low-fluence response (LFR) that occurs at 10-7 to 10-3 mol m-2 after relatively short
exposure (seconds to minutes) [27]. We thus investigated whether brief exposure to FR at
the end of W-LL (EOLW) would cancel the effect of W-LL and thus disable the R-HIR
and potentiate the FR-HIR; whether a short exposure to R at the end of +FR-LL (EOL+FR)
would cancel the effect of +FR-LL and thus decrease the FR-HIR and enhance the
R-HIR; and whether the FR (or R)-termination effect would be alleviated by subsequent
exposure to R (or FR). We also characterized the action spectra after inducing and
antagonizing the EOLW-FR effect.
For practical reasons, we used MCL from both LED lamps and the Okazaki Large
Spectrograph. The effect of MCL at EOL was mainly evaluated as the change in the
FR-HIR (photoinduction of cell division) after 3 h of irradiation with LED751 (LED
lamp, or its light, with an emission maximum at 751 nm) at the 26th h (subjective dusk) in
DD.
2. Materials and methods
The alga was photoautotrophically cultured in modified Cramer-Meyer medium
according to Bolige et al. [28] at 25°C for at least 2 days under LL. The light was
generated by an array of cool-white fluorescent lamps (Toshiba, Mellow-white) at 84
µmol m-2 s-1 in the absence (W-LL) or presence (+FR-LL) of a supplemental FR-lamp
7
(Toshiba, FL20S-FR74) covered with a sheet of 5-mm thick Delaglass A (Asahi Kasei
Chemicals, Tokyo) at 24 µmol m-2 s-1. Due to the lighting setup, intensity of W was
slightly (8%) reduced in +FR-LL. The irradiance spectra are shown (Fig. 1A).
When the cell titer reached 1.0–2.5 × 104 cells/mL in LL, the alga was transferred to
DD; all cell-cycle transitions ceased within 12 h in DD [19]. At the LL-to-DD transition
(EOL), the alga was either transferred to DD immediately or briefly exposed (typically 12
min, but this varied from 3 to 117 min) to MCL from the Okazaki Large Spectrograph, or
to LED390, -480, -610, -653, -673, or -751, before transfer. The irradiance spectra of the
LEDs are shown (Fig. 1B).
For 3 h around subjective dusk (26th h of DD after W-LL or 28th h of DD after
+FR-LL), photoinduction was performed using LED595 (50 µmol m-2 s-1), LED644 (50
µmol m-2 s-1), or LED751 (25 µmol m-2 s-1) unless otherwise stated (Fig. 1B). At ≥12 h
after treatment, the alga was fixed in 1.4% formalin containing 1.1% NaCl, and cells were
counted using a Coulter Electronic Particle Counter.
Action spectra were derived for the LFR at EOLW using fluence rates of 20–100 µmol
m-2 s-1 and 3–117 min of exposure; the determinant of the response was the fluence,
according to the reciprocity law. To derive the action spectrum for the EOLW-FR effect,
samples were irradiated immediately with MCL at EOLW; to derive the action spectrum
for the EOLW-FR antagonizing effect, samples were irradiated with MCL after 12 min of
exposure to LED751 (20 µmol m-2 s-1). These effects were evaluated by the change in
photoinduction following 3 h of exposure to LED751 at around the 26th h. See Fig. 2 for
irradiation schema in this study.
3. Results
3.1. R/FR photoreversibility phenomenon
The alga was photoautotrophically cultured in W-LL or +FR-LL. All of the irradiance
spectra derived in this study are shown in Fig. 1. When transferred to DD, cell cycle
progression ceases within 12 h, and about 22% of the cells arrest in the G2 phase [19].
Light treatment enables dark-arrested G2 cells to progress through cell division in
subsequent DD, and this photoinduction follows a circadian rhythm, peaking at
8
subjective dusk and reaching a minimum, with no occurrence, at subjective dawn [18,
25].
Photoinduction also depends on the irradiance spectra of both the LL and the
inductive light. When the alga cultured under W-LL were immediately transferred to DD
and treated with 3 h of irradiation at the 26th h, exposure to LED644 (Fig. 3B) was much
more effective than exposure to either LED595 (Fig. 3A) or LED751 (Fig. 3C). The
reverted pattern was observed for the alga cultured under +FR-LL (with 3 h of irradiation
at the 28th h). Thus, the R/FR ratio in the LL prior to DD determined the photoinductive
pathways of cell division. Interestingly, a higher R/FR ratio made the R-HIR by LED644
dominant, whereas a lower R/FR ratio made the FR-HIR by LED751 dominant (see also
Introduction).
Terminating the prior W-LL with a short exposure (12 min) to FR by LED751 at a low
fluence rate (20 µmol m-2 s-1) before transfer to DD reverted the photoinduction pattern,
and the resultant dependence of photoinduction on LED wavelengths was exactly as that
obtained for the +FR-LL control (Fig. 3A–C), namely, lower photoinduction by LED644
and higher photoinduction by LED595 and LED751. We designated this phenomenon the
EOLW-antagonizing or EOLW-FR effect. It was completely reversed by a short exposure
(12 min) to R from LED653 at a low fluence rate (40 µmol m-2 s-1), which we designated
the EOLW-FR antagonizing effect. This effect, in turn, was completely counteracted by
exposure to FR from LED751 for 12 min at 20 µmol m-2 s-1.
A mirror image of the results was obtained for the alga cultured under +FR-LL (Fig.
3A–C). Using the same protocol as above, we found that EOL+FR-R negated the
photoinduction pattern of the +FR-LL culture and resulted in the same photoinduction
pattern as the W-LL control (EOL+FR-antagonizing or EOL+FR-R effect). Moreover,
subsequent exposure to FR counteracted this effect (EOL+FR-R antagonizing effect). This,
in turn, was counteracted by exposure to R from LED653.
In conclusion, irradiation with R and FR at the EOL photoreversibly antagonized the
effect of the R/FR ratio in the prior LL to reorganize the photoinduction patterns so as to
lower the FR-HIR and R-HIR, respectively, and potentiate the R-HIR and FR-HIR,
respectively. Because these were LFRs, as detailed in the next section, it can be said that
the R-LFR and FR-LFR (or EOL+FR-R and EOLw-FR, respectively) photoreversibly
9
potentiated the R-HIR and FR-HIR, respectively.
3.2. Action spectra for the effects of R and FR
Equal-quantum action spectra were derived for both the EOLW-FR effect and the
EOLW-FR antagonizing effect of irradiation in the alga cultured under W-LL with MCL
(20 µmol m-2 s-1 for 12, 20, or 24 min) using the Okazaki Large Spectrograph (Fig. 4).
The experimental methods were the same as those described in the left half of Fig. 3C,
except that samples were irradiated immediately with MCL at the EOLW to examine the
EOLW-FR effect, and irradiated with MCL after exposure to LED751 (12 min of 20 µmol
m-2 s-1; 14.4 mmol m-2) at the EOLW to evaluate the EOLW-FR antagonizing effect. Both
effects were measured as the extent of the FR-HIR by LED751 (25 µmol m-2 s-1 for 3 h;
270 mmol m-2). The action spectrum for the EOLW-FR effect showed a single main peak
at 740 nm and small peaks at 380 and 610 nm, whereas that for the EOLW-FR
antagonizing effect showed a major peak at 640 nm and minor peaks around 480 nm (Fig.
4).
Fluence responses were measured for all of the wavelengths examined (Fig. 4) by
varying both the fluence rate (20–100 µmol m-2 s-1) and exposure time (3–117 min). The
patterns followed the Bunsen-Roscoe law, and fluence determined response. The fluence
responses of the peak wavelengths are shown as representative examples (Fig. 5). Each
had a region of log–linear dependence, enabling the identification (via linear regressions)
of fluences that were critical to the response. The inverses of the critical fluences were
plotted against the wavelengths, according to the traditional method of plotting action
spectra (Fig. 6A).
Not all of the linear regression slopes and saturation levels were the same (Fig. 5; also
see [29] for an example of different slopes). Because the slopes and saturation levels both
represent the effectiveness of each wavelength, both were plotted (Fig. 6B and C,
respectively). To express all three measures of effectiveness at once, we plotted the
square-roots of their multiples against wavelengths to express them in a meaningful unit
(% photoinduction per mmol m-2; Fig. 6D). The action maxima occurred at 380, 480, 610,
640, 670, and 740 nm, and did not depend on the measures of effectiveness; however,
their relative strengths did.
The EOLW-FR effect was most pronounced at 740 nm (Fig. 6), which had a critical
10
fluence of about 2 mmol m-2; the maximum response occurred at 20–30 mmol m-2 (Fig.
5A). The EOLW-FR antagonizing effect was most pronounced at 640 nm (Fig. 6), which
had the same critical fluence (about 2 mmol m-2); the maximum response occurred at a
fluence 1.3–2.0 times higher, and at about 40 mmol m-2 (Fig. 5B). MCL at 380 and 480
nm behaved like 740 and 640 nm light, respectively, in that their effects were restricted to
the EOLW-FR effect and the EOLW-FR antagonizing effect, respectively, although the
former two wavelengths were much weaker. In contrast, MCL at both 610 and 670 nm
produced a stronger EOLW-FR antagonizing effect than the EOLW-FR (or W-LL
antagonizing) effect.
3.3. Photoreversibility between the maxima
The action spectra were derived based on photoreversible responses, strongly
suggesting photoreversibility between 740 nm and 480, 610, 640, and 670 nm and
between 640 nm and 380, 610, 670, and 740 nm. Indeed, there was photoreversibility
between 640 and 740 nm, using both LED653 and LED751 (Fig. 3) and MCL generated
by the Okazaki Large Spectrograph (Fig. 7A). The FR-HIR (photoinduction) by LED751
was photoreversibly potentiated at EOLW by brief exposure to 740 nm, and counteracted
by 640 nm. We also confirmed photoreversibility between 380 and 640 nm, 480 and 740
nm, and (610 and 670 nm) and (640 and 740 nm) (Fig. 7B), using LED390, -480, -610,
-644, -673, and -751 (Fig. 1).
In summary, in addition to R (640)/ FR (740) photoreversibility, these LFRs displayed
380/640, 480/740, 610/640, 610/740, 640/670, and 670/740 photoreversibility. It is
reasonable to also expect photoreversibility between the minor peaks at 380/480,
380/610, 380/670, 480/610, and 610/670.
4. Discussion
We demonstrated phytochrome-like responses, categorized into LFRs, for the first
time in Euglena, or in any discicristate for that matter, and derived their action spectra.
Although an R/FR photoreversible LFR has long been considered a diagnostic feature of
phytochromes in plants [27], several of the phytochromes that have been molecularly
identified do not express phytochrome-like physiological responses [1, 30]. It is possible
that the reverse may also hold true: the phytochrome-like responses in Euglena may not
11
represent phytochromes.
Molecular identity aside, it is clear that Euglena senses and responds to
spatiotemporal changes in environmental irradiance spectra. The alteration of the
prevalent isoforms of the phytochrome-like photoreceptors by manipulating the R/FR
ratio of the prior LL (see below) is reminiscent of the shade-avoidance response, and the
photoreversible LFR at EOLW and EOL+FR is similar to the EOD-FR (or R) effect [12,
13]. Although both phenomena have so far been reported only in angiosperms [13], they
may be more widespread than previously thought, and more ancient in origin.
4. 1. Action spectra
4.1.1. Overview
Because this alga contains large amounts of pigment, such as chlorophyll and
carotenoids, the fluence rate of MCL reaching the phytochrome-like photoreceptors
changes considerably depending on the light wavelength (except for the peak at 740 nm,
to which the alga is highly transparent), and thus, the action maxima do not directly
reflect the absorption maxima of the photoreceptors. This screening effect is responsible
for the difference in phytochrome action spectra between etiolated and de-etiolated plant
tissues, and for the shift to shorter wavelengths, i.e., 610–650 nm, in the latter [31]. Thus,
the major action maximum at 640 nm in the alga is within the range of de-etiolated plant
tissues. The action maxima of plant phytochromes in the FR spectral region range from
720–740 nm [29, 32, 33]. Thus, the maximum at 740 nm in Euglena is not significantly
different from plant systems.
Although it is unclear whether the phytochrome-like photoreceptors are true
phytochromes, the two major peaks at 640 and 740 nm very likely represent
photoreceptors, either homologous or analogous to plant phytochromes, because the
action maxima of the two systems resemble each other and display a diagnostic feature of
phytochromes. Thus, we tentatively call them Euglena-phytochrome and classify them as
640-nm and 740-nm absorbing forms, or Euglena-PR and Euglena-PFR, respectively.
Other action maxima, i.e., at 380 and 610 nm, closely resemble in vitro absorption
maxima from plant phytochromes [34]. In contrast, the maxima at 480 and 670 nm do not
appear in the absorption spectra of plants.
12
On the other hand, all of the alga maxima appear in the action spectra derived for
photoinduction of hook-opening in seedlings of Phaseolus vulgaris, which show minor
peaks near 400 and 500 nm, a shoulder at 620 nm, and a major peak at 660 nm [29]. In our
action spectra, the minor peaks at 480 and 610 nm and the major peak at 640 nm for the
EOLW-FR antagonizing effect correspond to the 500, 620, and 660 nm peaks reported for
P. vulgaris; the minor peak at 380 nm for the EOLW-FR effect (or FR-related
photoresponse) corresponds to the 400 nm peak of P. vulgaris. Thus, the peaks shown by
the alga were consistently at wavelengths 10–20 nm shorter than those of P. vulgaris,
suggesting that the two organisms have the same photoreceptors. However, it is unknown
whether the peaks in P. vulgaris are all ascribable to phytochromes [29].
A tentative conclusion is that the major maxima at 640 and 740 nm may excite
Euglena-PR and Euglena-PFR, respectively, whereas the minor peaks at 380, 480, 610, and
670 nm do not completely conform to plant phytochromes.
A novel feature of the photoresponse reported here is that nearly all of the action
maxima were photoreversible as follows: R (640)/FR (740), 380/640, 480/740, 610/640,
610/740, 610/670, and 670/740. The action maxima may be classified into three types: R,
FR, and bimodal. 480 and 640 nm were of the R type, exerting both EOL+FR
antagonization and EOLW-FR antagonization to potentiate the R-HIR; 380 and 740 nm
were of the FR type, exerting both EOLW antagonization and EOL+FR-R antagonization to
potentiate the FR-HIR; and 610 and 670 nm behaved like both, but more closely
resembled the R type. Therefore, it is reasonable to expect that photoreversibility may
also hold true for 380/480, 380/610, 380/670, 480/610, and 610/670, although there are
currently no supporting data. All of the wavelengths may represent
Euglena-phytochrome; 380 and 480 nm may exclusively excite Euglena-PFR and
Euglena-PR, respectively, whereas 610 and 670 nm may excite both.
An alternative explanation may be that the action maxima other than 640 and 740 nm
represent photoreceptors that are distinct from Euglena-phytochrome because they have
features never before documented in plant phytochromes. Thus, the photoreversibility
reported here may result from both Euglena-phytochromes and other as-yet-unidentified
photoreceptors antagonistically regulating a common signaling pathway. This pathway
may serve as a secondary switch for reorganization of the photoreceptors for HIR (i.e.,
13
photoinduction of cell division), which may be photoreversible. Differences in the slopes
of the fluence responses (Fig. 5) may reflect those in phototransduction pathways or their
coupling pathways, favoring this view. Whereas 640 and 740 nm may excite
Euglena-phytochrome, other maxima may excite other photoreceptors. Indeed, the
photoreversibility of 480 and 600 nm has recently been found in an in vitro preparation of
Anabaena sensory rhodopsin [35]. Purification of Euglena-phytochrome will resolve this
issue.
4.1.2. Relations to other action spectra in the alga
The action maxima at 380 and 480 nm have also been reported for photophobic
responses in the alga and may be involved in UV-A/blue photoreceptors [36]. Whereas
the action maxima of the UV-A/blue photoreceptors behaved similarly, those reported
here for the alga behaved antagonistically (Fig. 6), suggesting that they are not related to
the action maxima of the UV-A/blue photoreceptors.
The action maximum at 610 nm reported here is very similar to that (600 nm) of
dark-adapted heterotrophically cultured Euglena [16, 17]. However, they differ markedly
in other aspects: the former involves a LFR, whereas the latter involves a HIR, and the
latter study did not examine photoreversibility. However, both may represent an identical
photoreceptor because our HIR, the action maxima of which were at 610 nm, as well as
380, 480, 640, 670, and 740 nm, resulted from repetition of LFRs (at the corresponding
wavelengths), which were mutually photoreversible [25].
The action maximum at 670 nm is very similar to that of photosynthetic electron
transport in plants, suggesting that the photoreceptor involved may be chlorophyll a
(Chla). Photosynthetic electron transport converts light energy to metabolic energy as
ATP and NADPH, but it also generates redox signals, which help regulate photosystems
[37–39]. The HIR reported here also involves signaling via photosynthetic electron
transport, at least when photoinduction is performed with cool-white fluorescent light
(see Fig. 1A for the irradiance spectrum) [18]. Thus, photosynthetic electron transport
may be involved in the 670 nm maximum, although it is also possible that it is related to
Euglena-PR, as discussed above.
Interestingly, all the action maxima for the LFRs at EOL reported here also appeared
14
in those for the algal HIRs (the present study addressed only 610-, 640-, and 740-HIRs);
an exception was the maximum at 450–460 nm, which was responsible for only the HIR.
Using 3-min cycles of intermittent irradiation lasting 3 h, we found that the HIRs by 480,
610, 640, 670, and 740 nm were photoreversibly cancelled out by 640 and/or 740 nm; 380
nm has not yet been examined [25]. Thus, whereas these maxima regulated different
photoresponses (LFRs at EOL or HIRs for photoinduction), they most likely involved the
same photoreceptors, excluding 460-HIR.
The widespread and long-held belief that Euglena lacks phytochromes was sparked
by studies on the potentiation of chlorophyll synthesis [2, 16, 40]. We think that a
reexamination of this issue is warranted. In previous studies, FR did slightly affect
potentiation, and it is uncertain whether appropriate fluences for R and FR were chosen.
Moreover, as shown here, phototransduction pathways are inevitably reorganized
according to the prior illumination conditions.
4.2. Shade avoidance, end-of-day FR, complementary chromatic adaptation, and
more
In sun-adapted plants, a lower R/FR ratio in the daily photoperiod induces a
shade-avoidance response, and end-of-day FR (EOD-FR) invokes the equivalent
response [12, 13]. Our simple experimental protocols simulated the daily cycles and may
provide a clear experimental advantage over plant systems where repeated cycles of FR
irradiation at EOD are required to induce the EOD-FR effect (e.g., stimulation of
hypocotyl elongation), although the final photomorphogenic responses were different.
The culture condition of +FR-LL can be considered equivalent to FR-rich shade
environments, and the FR-termination at EOLW equivalent to EOD-FR. Reciprocally, the
R-termination at EOL+FR produced a response similar to W-LL. Although shade
avoidance or EOD-FR has not been documented in green algae or gymnosperms, such
responses may not be limited to angiosperms.
How might shade avoidance serve Euglena? As previously mentioned, prior W-LL
(or EOL+FR-R) and +FR-LL (or EOLW-FR) potentiated the HIR involving Euglena-PR
and Euglena-PFR, respectively, in the subsequent dark, indicating that R- or FR-enriched
environments potentiate the R- or FR-HIR responsible for photoinduction of cell
reproduction. The reorganization of photoinduction signaling may help the alga
15
reproduce as quickly as possible, regardless of environmental changes in light quality.
Such reorganization may seem unnecessary for Euglena because it is motile and can
move to favorable environments; for example, if shaded by crowding, it could swim
toward a brighter location. It may indeed do so under controlled laboratory conditions,
but blooms of various organisms, sometimes Euglena itself [41], frequently occur in
nature. When they do, virtually no free spaces are available. The self-potentiation of
phototransduction would be advantageous under such conditions.
The self-potentiation is also reminiscent of complementary chromatic adaptation [42]
and autosensitization (or self-potentiation) by phytochromes, in which prior irradiation
by FR enhances the response to FR in plants [43]. Relevantly, prior irradiation by
UV-A/blue light is also known to enhance the response to FR [44].
There are five families of phytochromes, i.e., phyA–E, in angiosperms. Of these,
phyA and phyB are the most abundant, and are thought to have diverged when seed plants
evolved. Both a very low fluence response (VLFR) and FR-HIR are ascribed to phyA,
and LFR and R-HIR are ascribed to phyB. Thus, Euglena-phytochromes resemble the
behavior of both plant phyA and phyB. Future studies should elucidate whether
Euglena-phytochromes are ascendants of plant phytochromes or represent novel
photoreceptors analogous, but not homologous, to plant phytochromes.
Finally, photoperiodic induction of cell division also involves Euglena-phytochrome
[25]. Euglena-phytochrome may be responsible for phase-shifting the circadian
UV-resistance rhythm [24]; enhancing UV resistance, particularly at subjective midnight
(the most sensitive phase) [24]; and inducing Chla synthesis in DD, when it has been
assumed synthesis does not occur in the alga. Thus, Euglena-phytochrome plays a major
role and seems to be a representative photoreceptor in the alga.
16
Acknowledgments
The action spectra were obtained under the NIBB Cooperative Research Program for
Okazaki Large Spectrograph (5-513). We are very grateful to the OLS staff for their
assistance. We also appreciate the experimental advice of Mr. S. Higashi (OLS) and Prof.
M. Watanabe (OLS and Graduate University for Advanced Studies, Hayama, Japan).
17
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21
FIGURE LEGENDS
Fig. 1. Irradiance spectra of lamps
(A) Prior LL was either W-LL (light rich in R) or +FR-LL (light rich in FR). (B)
Irradiation at EOL was performed using LED390, LED480, LED610, LED644, LED653,
LED673, and LED751. Irradiation at the 26th h of DD (photoinduction of cell division)
was conducted using LED595, LED644, or LED751.
Fig. 2 Irradiation schema
The alga was grown log-linearly in W-LL (protocol “A”), and then transferred to DD
either uninterruptedly (protocol “A”) or after a brief (the order of minutes) exposure of
MCL (protocol “B”); in either case, the cell cycle arrested within 10 h in DD. The effect
of the brief exposure (i.e. ‘testing light’) was evaluated by examining changes in
spectral dependence of HIR (i.e. ‘photoinduction of cell division’) that was performed
around 26 h in DD with a 3-h exposure of MCL. The HIR enabled the dark-arrested G2
cells to reproduce in the subsequent darkness; see the cell number increase after the 3-h
light pulse. The same protocol was taken for the alga cultured under +FR-LL (protocols
“C” and “D”) except that the prior LL was now +FR-LL (enriched in FR). This study
deals with spectral dependence of ‘testing light’, whereas the accompanying paper that
of ‘photoinduction of cell division’.
Fig. 3. R/FR reversibility at EOLW or EOL+FR in changing photoinductive light quality
The alga cultured under W-LL was either uninterruptedly transferred to DD or transferred
after a brief exposure to FR (LED751; 20 µmol m-2 s-1), FR/R (LED653; 40 µmol m-2 s-1),
or FR/R/FR. Similarly, the alga cultured under +FR-LL was transferred to DD either
uninterruptedly or after a brief exposure to R, R/FR, or R/FR/R. R or FR each lasted for
12 min. At the 26th or 28th h of DD, the alga was irradiated for 3 h with LED595 (50 µmol
m-2 s-1), LED644 (50 µmol m-2 s-1), or LED751 (25 µmol m-2 s-1), and the cells were
counted after ≥12 h; the increase in cells compared to the DD control represents the
percent photoinduction. EOL irradiation did not change the number of cells in the DD
22
control. Data represent the mean ± SE of six samples. The difference was statistically
significant (p < 0.0001), shown as solid and hatched bars within each block. LED595,
LED653, and LED751 were used for the 610-, 640- and 740-HIR, respectively.
Fig. 4. Equal-quantum action spectra for EOLW irradiation in potentiating LED751
induction and its antagonizing effect
Experimental protocols were essentially the same as in Fig. 3, except that W-LL was
terminated with either MCL (12 min) or LED751 (12 min) followed by MCL (20 or 24
min), using a fluence of 20 µmol m-2 s-1. The former conditions tested the EOLW-FR
effect on potentiating the FR-HIR (photoinduction of cell division by 3 h of irradiation
with LED751 at 25 µmol m-2 s-1 at the 26th h of DD), and the latter conditions tested the
EOLW-FR antagonizing effect. Horizontal lines, solid or dotted, represent the control
levels of photoinduction in the alga transferred to DD from W-LL, uninterruptedly or
following a 12-min pulse of LED751, respectively.
Fig. 5. Fluence responses to representative wavelengths of potentiating LED751
induction and its antagonizing effect at EOLW
Experimental protocols were essentially the same as in Fig. 4, except that we varied both
the fluence rate (20–100 µmol m-2 s-1) and exposure time (3–117 min) of the MCL of the
Okazaki Large Spectrograph; LED751 at EOLW was the same (20 µmol m-2 s-1, 12 min).
The abscissa represents the fluence in logarithmic scale. (A) EOLW-FR effect; MCL was
uninterruptedly applied at EOLW and its effect was evaluated as potentiation of
photoinduction by LED751 (25 µmol m-2 s-1; 3 h), i.e., the increase in photoinduction of
the unperturbed W-LL control. (B) EOLW-FR antagonizing effect; MCL was irradiated
after 12 min of exposure to FR (LED751) at EOLW and its effect was estimated as the
decrease in photoinduction of the control, EOLW-FR without a subsequent exposure to
MCL. Straight lines were plotted using linear regression.
23
Fig. 6. Action spectra for potentiating LED751 induction and its antagonizing effect at
EOLW
All of the fluence responses shown in Fig. 5 were used to plot the action spectra. (A) The
inverses of critical fluence, obtained using linear regression in Fig. 5, minimally required
for the photoresponse, i.e., either an increase from the photoinduction of the W-LL
control or a decrease from that of the EOLW-FR control. (B) Slopes of the linear
regressions in Fig. 5; inverse of incremental fluence, evaluated from 5 mmol m-2, required
for 1% photoresponse. (C) Saturated levels of the photoresponse. (D) Square-roots of the
multiples of (A), (B), and (C). The incremental fluence was calculated as follows: the
critical fluence (x mmol m-2) and fluence (y mmol m-2) required for 1% photoresponse
were read, and the incremental fluence was calculated as 5 × (y/x – 1). Whereas the
coefficient of 5 mmol m-2 was somewhat arbitrary, we chose this value because most of
the critical fluences at the representative wavelengths varied around it.
Fig. 7. Photoreversibility at EOLW
Experimental protocols were essentially the same as in Fig. 3. MCL from the Okazaki
Large Spectrograph or LED lamps were used to successively irradiate samples at EOLW,
each for 12 min, and their effect was evaluated as the extent of the FR-HIR
(photoinduction of cell division) by irradiating with LED751 for 3 h at the 26th h of DD.
Horizontal lines represent percent photoinduction of the unperturbed W-LL control,
which was not significantly altered by LED480, LED644, or MCL640 at EOLW, of 2.36 ±
0.16 (n = 9) and 2.29 ± 0.12 (n = 19) for (A) and (B), respectively. (A) Photoreversibility
between 640 and 740 nm of the Okazaki Large Spectrograph light at equal fluences (5–42
mmol m-2). (B) Photoreversibility between LED390 and LED644, between LED480 and
LED751, and between (LED610 and LED673) and (LED644 and LED751). The
difference was statistically significant (p < 0.01), shown as solid and hatched bars within
each block.
24
200
Photon fluence rate (µmol m-2 s-1)
0.25
300
350
400
450
500
550
600
650
700
750
800
850
A
W-LL (W at 84 µmol m -2 s-1)
+FR-LL (W at 77 µmol m -2 s-1 + FR at 24 µmol m -2 s-1)
0.20
Wavelength (nm)
0.15
0.10
R
FR
0.05
0.00
0.25
Photon fluence rate (µmol m-2 s-1)
250
B
390 nm (40 µmol m s )
0.20
-2
-1
-2
-1
480 nm (50 µmol m s )
-2 -1
595 nm (50 µmol m s )
-2
-1
-2
-1
-2
-1
610 nm (50 µmol m s )
644 nm (50 µmol m s )
0.15
653 nm (40 µmol m s )
673 nm (50 µmol m-2 s-1)
-2 -1
751 nm (25 µmol m s )
0.10
0.05
0.00
200 250 300 350 400 450 500 550 600 650 700 750 800 850
Wavelength (nm)
Figure 1
25
Time(h) in DD
-36
-24
-12
0
12
24
36
A
B
C
D
testing light
15000
10000
5000
Figure 2
photindcution of cell division
48
60
26
A
10
-2
LED595 (540 mmol m )
610-HIR
B
-2
LED644 (540 mmol m )
640-HIR
-2
C
-2
LED751 (270 mmol m )
740-HIR
-2
FR = LED751 (14 mmol m ); R = LED653 (29 mmol m )
% Photoinduction
8
6
4
Figure 3
+FR-LL
R
R/FR
R/FR/R
W-LL
FR
FR/R
FR/R/FR
+FR-LL
R
R/FR
R/FR/R
W-LL
FR
FR/R
FR/R/FR
+FR-LL
R
R/FR
R/FR/R
0
W-LL
FR
FR/R
FR/R/FR
2
27
300
9
350
400
450
500
550
600
650
700
750
800
20 µmol m s
-2
MCL (20') after LED751 (12')
MCL (24') after LED751 (12')
MCL (12')
●
W
EOL -FR control
W-LL control
% Photoinduction by LED751 (25 µmol m-2 s-1 for 3 h)
8
7
850
-1
6
5
4
3
2
1
0
300
350
400
450
500
550
600
650
Wavelength (nm)
Figure 4
700
750
800
850
28
A
Increase from control (%)
4
380
480
610
640
670
740
3
2
1
Decrease from FR-control (%)
0
B
4
380
480
610
640
670
740
3
2
1
0
1
10
100
Fluence (mmol m-2)
Figure 5
1000
Slope (% mmol-1 m-2)
(Critical fluence)-1 (1 mmol-1 m-2)
29
300
0.8
350
400
450
500
550
600
650
700
750
800
850
550
600
650
700
750
800
850
A
W-LL-antagonizing
FR-antagonizing
0.6
0.4
0.2
0.0
0.04
B
0.03
0.02
0.01
0.00
[(A) x (B) x (C)]-1/2 (% mmol -1 m-2)
Saturation level (%)
C
3
2
1
0
D
0.2
0.1
0.0
300
Figure 6
350
400
450
500
Wavelength (nm)
-2
0
Figure 7
-2
8
10
751 (50 mmol m )
-2
673 (50 mmol m )
751/673
751/673/751
A
-2
644 (72 mmol m )
-2
673 (72 mmol m )
644/673
644/673/644
-2
751 (36 mmol m )
-2
610 (58 mmol m )
751/610
751/610/751
-2
610 (65 mmol m )
-2
644 (58 mmol m )
610/644
610/644/610
5
-2
751 (29 mmol m )
-2
480 (50 mmol m )
751/480
751/480/751
8
644 (58 mmol m )
-2
390 (86 mmol m )
644/390
644/390/644
% Photoinduction by LED751 (25 µmol m-2 s-1 for 3 h)
30
740
640
740/640
740/640/740
6
4
2
0
-2
30
Fluence (mmol m )
B
6
4
2