帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge

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Title

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

Author(s) Bolige, Aoen, Goto, Ken

CitationJournal of photochemistry and photobiology. B,

Biology, 86: 97-108

Issue Date 2007

URL http://ir.obihiro.ac.jp/dspace/handle/10322/720

Rights

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: kgotoken@obihiro.ac.jp

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.

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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

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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

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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

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(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.

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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

Figure 1

Wavelength (nm)

200 250 300 350 400 450 500 550 600 650 700 750 800 8500.00

0.05

0.10

0.15

0.20

0.25

390 nm (40 µmol m-2 s-1) 480 nm (50 µmol m-2 s-1)595 nm (50 µmol m-2

s-1)610 nm (50 µmol m-2 s-1)644 nm (50 µmol m-2 s-1) 653 nm (40 µmol m-2 s-1)673 nm (50 µmol m-2 s-1)751 nm (25 µmol m-2 s-1)

200 250 300 350 400 450 500 550 600 650 700 750 800 850

Pho

ton

fluen

ce ra

te ( µ

mol

m-2

s-1

)

0.00

0.05

0.10

0.15

0.20

0.25

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)

Pho

ton

fluen

ce ra

te ( µ

mol

m-2

s-1

)

Wavelength (nm)

A

B

FRR

25

Figure 2

Time(h) in DD

-36 -24 -12 0 12 24 36 48 60

5000

10000

15000

AB

CD

testing lightphotindcution of cell division

26 %

Pho

toin

duct

ion

0

2

4

6

8

10

W-L

LFR FR

FR/R

FR/R

/FR

FR/R

/FR

FR/RFR

W-L

L

LED595 (540 mmol m-2)610-HIR

FR/R

FR/R

/FR

+FR

-LL

+FR

-LL

+FR

-LL

W-L

LR

R/F

RRRR

/FR

R/F

RR

/FR

/R

R/F

R/R

R/F

R/R

A LED644 (540 mmol m-2)640-HIRB C LED751 (270 mmol m-2)

740-HIR

FR = LED751 (14 mmol m-2

); R = LED653 (29 mmol m-2

)

Figure 3

27

Figure 4 Wavelength (nm)

300 350 400 450 500 550 600 650 700 750 800 850

300 350 400 450 500 550 600 650 700 750 800 850%

Pho

toin

duct

ion

by L

ED75

1 (2

5 µm

ol m

-2 s

-1 fo

r 3 h

)

0

1

2

3

4

5

6

7

8

9

MCL (20') after LED751 (12')MCL (24') after LED751 (12') MCL (12')EOLW-FR controlW-LL control

20 µmol m-2 s-1

●

28

Figure 5

Dec

reas

e fr

om F

R-c

ontr

ol (%

)

380480610640670740

Fluence (mmol m-2)

1 10 100 10000

1

2

3

4

0

1

2

3

4

Incr

ease

from

con

trol

(%) 380

480610640670740

A

B

29

Figure 6 Wavelength (nm)

300 350 400 450 500 550 600 650 700 750 800 850

[(A) x

(B) x

(C)]-1

/2 (%

mm

ol -1

m-2

)

0.0

0.1

0.2

Satu

ratio

n le

vel (

%)

0

1

2

3

Slop

e (%

mm

ol-1

m-2

)

0.00

0.01

0.02

0.03

0.04

300 350 400 450 500 550 600 650 700 750 800 850(C

ritic

al fl

uenc

e)-1

(1 m

mol

-1 m

-2)

0.0

0.2

0.4

0.6

0.8

W-LL-antagonizingFR-antagonizing

A

D

C

B

30

Figure 7

% P

hoto

indu

ctio

n by

LED

751

(25

µmol

m-2 s

-1 fo

r 3 h

)

0

2

4

6

8

480

(50

mm

ol m

-2)

751

(29

mm

ol m

-2)

751/

480

751/

480/

751

610

(65

mm

ol m

-2)

644

(58

mm

ol m

-2)

610/

644

610/

644/

610

610

(58

mm

ol m

-2)

751

(36

mm

ol m

-2)

751/

610

751/

610/

751

B

Fluence (mmol m-2)10

0

2

4

6

8

740640740/640740/640/740

A

5 30

673

(72

mm

ol m

-2)

644

(72

mm

ol m

-2)

644/

673

644/

673/

644

673

(50

mm

ol m

-2)

751

(50

mm

ol m

-2)

751/

673

751/

673/

751

390

(86

mm

ol m

-2)

644

(58

mm

ol m

-2)

644/

390

644/

390/

644