Biochim. Biophys. Acta, Bioenergetics

Excitation energy relaxation in a symbiotic cyanobacterium, Prochloron didemni, occurring in

coral-reef ascidians, and in a free-living cyanobacterium, Prochlorothrix hollandica

Fumiya Hamada1, Makio Yokono2, Euichi Hirose3, Akio Murakami1,4, Seiji Akimoto1,2,5,*

1Graduate School of Science, Kobe University, Kobe 657-8501, Japan,

2Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan,

3Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan,

4Kobe University Research Center for Inland Seas, Awaji 656-2401, Japan,

5JST CREST, Kobe 657-8501, Japan

*Corresponding author:

Dr. Seiji Akimoto

Address: Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan

Tel & Fax: +81-78-803-5705

E-mail: akimoto@hawk.kobe-u.ac.jp

Abbreviations used: PS, photosystem; Chl, chlorophyll; TRFS, Time-resolved fluorescence spectra;

FDAS, fluorescence decay-associated spectra.

Keywords

Prochloron; Prochlorothrix; symbiosis; time-resolved fluorescence; in hospite spectroscopy

Abstract

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in

obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different

host species and cultured Prochlorothrix by means of spectroscopic measurements, including

time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies

between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb,

PS II-PS I connection, and pigment status, especially that of PS I Red Chls, were different between

the four samples. We also discuss relationships between these observed characteristics and the light

conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host

colonies, presence or absence of tunic spicules, and microenvironments within the ascidians'

habitat.

1. Introduction

In most cyanobacteria, chlorophyll (Chl) a and phycobilins are the major photosynthetic

pigments, but certain species contain other Chls and lack phycobilisomes. Prochloron didemni

(hereafter referred to as Prochloron), Prochlorothrix hollandica (hereafter referred to as

Prochlorothrix) and Prochlorococcus marinus were previously classified as Prochlorophyta on

account of their unique pigment compositions, but molecular phylogenetics now places them among

the cyanobacteria [1-4]. These species possess Chl b in addition to Chl a (Prochlorococcus contains

3,8-divinyl (DV) Chls b and a), but do not have phycobilisomes, which serve as light-harvesting

antennae in typical cyanobacteria, although it was found that P. marinus CCMP 1375 have trace

amount of phycoerythrin-like pigment [5]. Their common photosystem antennae instead comprise

three types of Pcb (Prochlorophyte chlorophyll-binding protein), PcbA, PcbB, PcbC, encoded by

pcbA, pcbB, pcbC genes [6]. By contrast, Chl a/b-containing green algae and higher plants possess

eukaryotic-type antennae, LHC (light-harvesting chlorophyll protein complex) I and LHC II,

encoded by the cab gene family [7-9].

Prochlorothrix is a free-living cyanobacterium, whereas Prochloron is anomalous in that

it lives as an obligate symbiont in colonial ascidians inhabiting tropical/subtropical coral reefs;

free-living Prochloron has not been reported to date [10,11]. Moreover, at present, Prochloron

cannot be reliably cultured in vitro. The taxonomy of Prochloron remains controversial. In

molecular phylogeny based on 16S rRNA gene sequencing, no significant differences have been

found between Prochloron cells from different host species and from geographically different sites

[12]. In contrast, three morphological groups of Prochloron have been described in various host

ascidians [13]: Prochloron cells with small 'vacuoles' filling the central region of the cell (Group I),

those containing a large central 'vacuole' (Group II), and those with little 'vacuolation' and many

granular inclusions (Group III). Group-I Prochloron inhabit the colony surface or the tunic of the

host ascidian. Tunic is an integumentary extracellular matrix in which the ascidian zooids are

entirely embedded. On the other hand, Group-II is always distributed among peribranchial–cloacal

cavities of the host colony, and Group-III is exclusively found in Didemnum molle. These different

subcellular microenvironments may underlie the different photosynthetic properties between

Prochloron.

All of the host ascidians that established obligate symbiosis with Prochloron, belong to

four genera of the family Didemnidae: Didemnum, Trididemnum, Lissoclinum, and Diplosoma [11].

In this report, we examine spectral properties of Prochloron harbored in four species of ascidians,

Diplosoma sp., Trididemnum cyclops, Trididemnum miniatum, and Lissoclinum timorense. The

locality of Prochloron cells within their host colonies varies between the host species: Prochloron

cells are harbored in the tunic of T. miniatum and the cloacal cavities of the other three ascidians

[11,14]. More specifically, T. miniatum hosts Group-I Prochloron cells, while the remaining species

host Group-II Prochloron cells. Calcareous spicules are present in the tunics of all host ascidians,

excepting the Diplosoma species. Since the number of spicules depends on the ambient light within

the habitat, the spicules are purported to protect against intense solar radiation in shallow tropical

waters [15]. Although the spicules may also have an anti-predatory function, the cytotoxic

compounds contained in the Prochloron would be enough for the host ascidian to prevent from the

predation [16]. In T. miniatum, the tunic contains both spicules and Prochloron cells, with spicule

density higher in exposed colonies than in shaded colonies. In colonies of T. cyclops, spicules are

dense in the basal part of the colonies but sparse in the upper tunic overlying the cloacal cavities in

which Prochloron cells are distributed. In L. timorense, spicule density in the upper tunic depends

on the habitat of the host colony. Other than the spicules, ascidians have mycosporine-like amino

acids in their colonial tunics; these mycosporine-like amino acids, which are also in Prochloron

itself, protect against UV radiation damage [17].

In this study, Prochloron in hospite in the host ascidians and cultured Prochlorothrix

were analyzed by means of spectroscopic measurements including time-resolved fluorescence

spectroscopy. We discuss the light-harvesting strategy in Prochloron in hospite and how it varies

between four ascidian hosts, with emphasis on the light conditions in ascidian habitats and the

microenvironments offered by ascidians, especially their spicules.

2. Experimental

Didemnid ascidians hosting Prochloron were collected from the coral reef lagoon at

depths of less than one meter. Diplosoma sp. was collected from Yonehara (Ishigakijima Island,

Japan) in February 2010. T. cyclops was collected from Bise (Okinawajima Island, Japan) in

November 2010, while T. miniatum and L. timorense were collected from the same locality in May

2011. In this study, we investigated Prochloron cells dwelling in host ascidians in hospite and in

situ to avoid damage caused by the isolation processes and acidic substances in the host tissues.

Prochlorothrix hollandica (PCC 9006) was cultured autotrophically in BG11 medium at ~ 293 K

(20 °C). Light intensity was ca. 30 µmol photons m-2� s-1.

Steady-state absorption spectra were measured by a spectrometer equipped with an

integrating sphere (JASCO V-650/ISVC-747) at 77 K (–196 °C). Time-resolved fluorescence

spectra (TRFS) were measured with a time-correlated single-photon counting system at 77 K as

described previously [18]. The excitation wavelength was 425 nm, at which all pigments (Chl a,

Chl b and carotenoids) in Prochloron and Prochlorothrix were simultaneously excited. The time

window of measurement was 10 ns with an interval of 2.4 ps/channel, or 100 ns with an interval of

24.4 ps/channel. For each sample, measurements were carried out at least 10 times for the

steady-state absorption and at least 3 times for the time-resolved fluorescence. To construct the

TRFS, fluorescence rise and decay curves were measured in the Chl b to Chl a fluorescence region

(670–760 nm) with an interval of 1 nm/channel. We attempted to observe fluorescence signals at

wavelengths below 670 nm, but we obtained no signals that could be attributed to Chl b, as is also

the case for DV-Chl b in the high-light adapted strain of Prochlorococcus (CCMP1986) [19].

Therefore, our time-resolved measurements are discussed in terms of the signal from Chl a only.

3. Results

3.1 Steady-state absorption spectra of Prochloron in hospite and Prochlorothrix

We first discuss the effects of light conditions on Prochloron dwelling in their host

ascidians. Figure 1 shows low-temperature (77 K) steady-state absorption spectra of Prochloron

harbored in Trididemnum spp. (i.e. the complete ascidian plus Prochloron), together with that of

Prochlorothrix cells cultured as a reference. T. cyclops and T. miniatum both contain calcareous

spicules in their colonies Two peaks around 440 nm and 470 nm correspond to the Soret bands of

Chl a and Chl b, respectively, whereas carotenoids peak around 500 nm. Three peaks around 580

nm, 620 nm, and 680 nm arise from the Qx and Qy bands of Chl a and Chl b. Similar absorption

bands due to photosynthetic pigments are observed in all samples, but large spectral differences

occur between Prochloron in hospite and Prochlorothrix. Especially in the spectrum of Prochloron

in T. cyclops, there appears to be an upward-sloping structure toward the shorter wavelength region,

which are in consistent with the absorption spectra of didemnid ascidians having symbiotic

relationship with Prochloron [17]. It should be noted that Prochloron cells themselves do not

exhibit the upward-sloping structure that exceeds absorption bands of pigments [11]. These suggest

that particles in the host ascidians scatter and/or absorb light in the UV to visible region. On the

other hand, a much reduced upward-sloping structure is seen in the spectrum of Prochlorothrix

(compared to that of Prochloron in hospite). Furthermore, Prochloron in T. cyclops and in T.

miniatum have absorption bands in 690-710 nm region that is not seen in the spectrum of

Prochlorothrix, which suggests Prochloron utilizes light of longer wavelength spectral region

compared to Prochlorothrix. Spectral differences between Prochloron in T. cyclops and Prochloron

in T. miniatum are also apparent; the spectrum of Prochloron in T. cyclops has a shoulder at 710 nm

that is lacking in that of Prochloron in T. miniatum. Therefore, we expect that Prochloron in T.

cyclops possesses more Red Chls (low energy Chl a) emitting in this region than Prochloron in T.

miniatum, whose purpose is to broaden the range of utilizable wavelengths when UV-visible light is

scattered and/or absorbed by calcareous spicules.

3.2 Steady-state fluorescence spectra of Prochloron in hospite and Prochlorothrix

Figure 2 shows steady-state fluorescence spectra of Prochloron harbored in the four

ascidian species and Prochlorothrix. These spectra were constructed by integrating the TRFS. Chl a

fluorescence peaks are marked by vertical dotted lines. Peaks at 685 nm (686 nm for P. hollandica)

and 698 nm arise from Chl a in CP43 and CP47 (the core antenna complexes of photosystem (PS)

II), respectively, while the 715 nm peak is due to Red Chl in PS I [20,21]. Some differences in these

spectra are apparent, especially in the PS I fluorescence region. Prochloron in Diplosoma sp. and L.

timorense display a more intense PS I signal than do Prochloron in T. cyclops and T. miniatum. For

all Prochloron samples, the 715 nm band dominates in the PS I fluorescence region, and shoulders

appear to exist around 720 nm for Prochloron in three of the four ascidians (the exception is T.

miniatum, which harbors Prochloron in the tunic), while, for Prochlorothrix, the PS I signal is

much smaller than that of PS II. These results suggest differences in light-harvesting strategies

between Prochloron in four different host ascidians (and also between Prochloron and

Prochlorothrix), depending on light conditions such as host ascidian habitat and ascidian colony

microenvironment, including calcareous spicules. We discuss these differences in detail using

time-resolved data in the next sections.

3.3 Time-resolved fluorescence spectra (TRFS) of Prochloron in hospite and Prochlorothrix

Figure 3 shows TRFS of Prochloron harbored in the four ascidian species and

Prochlorothrix. Intensities of these spectra are normalized relative to their maximum intensities,

and the scale factors are provided in the individual windows. In the 0–5 ps spectra, signals of Chl a

in CP43 at 685 nm dominate all samples [20]. After 210 ps, a peak due to Chl a in CP47 appears

around 698 nm and increases in relative intensity. This suggests that excitation energy is transferred

from CP43 to CP47 within this time region. Behavior of Chl a signal in the PS I region (longer than

700 nm) is considerably different between the five samples. Fluorescence peaks originating from

PS I Chl a appear at wavelengths longer than 700 nm. For Prochloron in Diplosoma sp. and L.

timorense, fluorescence peaks with relatively large intensities appear immediately after excitation

(0–5 ps), at 710 nm and 715 nm, respectively. However, for Prochloron in T. cyclops and T.

miniatum, significant appearance of these peaks is delayed slightly; Prochloron in T. cyclops

fluoresces at 715 nm at 85–100 ps, whereas Prochloron in T. miniatum, at 715 nm at 570–700 ps.

Apart from Prochloron in T. miniatum (which, as mentioned above, harbors Prochloron in the

tunic), these peaks shift to 720 nm within 3 ns, and exhibit a shoulder around 730 nm.

Prochlorothrix shows much smaller PS I signal than four Prochloron samples in all time stamps.

Fluorescence signals remain 31 ns after the excitation pulse (Fig. 3), and the longest

lifetimes extend to 21–26 ns (see Sec. 3.4 and Fig. 4). These lifetimes are much longer than that of

Chl a itself; therefore, they are attributed to the delayed fluorescence originating from charge

recombination occurring in the PS II reaction center [22]. This delayed fluorescence showed two

peaks in the PS II region at 685 nm and at 698 nm for all Prochloron samples, suggesting that

transfer of excitation energy from the reaction center to CP43 and CP47 occurs. In Prochlorothrix,

contribution of the 698 nm band was considerably small, indicating that connection between the

reaction center and CP47 is weaker. With the exception of Prochloron in T. miniatum and

Prochlorothrix, delayed fluorescence also peaks in the PS I region at 715 or 720 nm, a possible

manifestation of excitation energy transfer from PS II to PS I [23].

3.4 Fluorescence decay-associated spectra (FDAS) of Prochloron in hospite and Prochlorothrix

To further investigate excitation energy transfer in Prochloron and Prochlorothrix, we

performed global analysis, and obtained fluorescence decay-associated spectra (FDAS) [19].

Throughout the analysis procedures, we did not assume spectral profiles of FDAS, but analyzed the

fluorescence rise and decay curves at each wavelength by fitting the sum of exponentials with

common time constants as follows:

€

F(λ, t) = An (λ)exp(−tτ n)

n∑ (1)

The FDAS of each time-constant (

€

τ n) is given by the amplitudes (

€

An (λ) ). Positive and negative

amplitudes indicate fluorescence decay and rise, respectively. A pair of positive and negative

amplitudes indicates excitation energy transfer from donor with positive amplitudes to acceptor

with negative amplitudes. In this analysis, the fitted decay curves of the 2.4 ps/channel interval and

the 24.4 ps/channel interval of each wavelength were connected in the 2–5 ns region and treated as

a single decay curve across the total time range. The time-constants (n) were determined as reported

previously [22], and FDAS of all samples consisted of six components (Figure 4).

In the fastest components of FDAS (11-51 ps), positive amplitudes around 680 nm and

negative amplitudes in the longer wavelength region were obtained in all samples (Fig. 4). The peak

around 680 nm was assigned to Chl a in Pcb [20]. Of the three kinds of Pcb, PcbA and/or PcbB,

which emit(s) fluorescence around 680 nm, are the most probable candidate(s); PcbC emits at 688

nm [9,21]. PcbA and PcbB rarely combine strongly with PS I [9,21], therefore, it is conceivable that

these Pcbs function as antenna complexes of PS II, while PcbC is the antenna of PS I. Besides PcbC,

the PS II core antenna complexes, CP43 and CP47, emit in the 685–698 nm region. Profiles of

FDAS around 688 nm depend strongly upon samples; the emission amplitudes of Prochloron in T.

cyclops, L. timorense, and T. miniatum and Prochlorothrix are negative, whereas those of

Prochloron in Diplosoma sp. are positive. Within this time region, CP43 and CP47 could accept

excitation energy from PcbA or PcbB, while PcbC transfers energy to the PS I complex; the former

gives negative amplitudes around 688 nm, whereas the latter, positive ones. Therefore, the decay

and rise kinetics are mixed in this spectral region.

In the second to fourth components of FDAS (the second, 150–260 ps; the third, 440–890

ps; and the fourth, 1.3–1.7 ns), positive amplitudes are obtained in the fluorescence regions from

685 to 698 nm and from 710 to 730 nm (Fig. 4). At wavelengths shorter than 700 nm, positive

amplitudes of CP43 (685 nm) dominate the second FDAS, while those of CP47 (698 nm) appear

after the third FDAS, indicating that CP43 transfers excitation energy to CP47 in the 150–260 ps

timeframe. At wavelengths longer than 700 nm, a distinct positive peak at 710 nm appears in the

second FDAS, while peaks at 715 and 720 nm occur in the third and the fourth FDAS. For

Prochloron in T. cyclops, Diplosoma sp., and L. timorense, a shoulder around 730 nm is also

observed in the fourth FDAS. In the fifth FDAS (3.1–4.9 ns), only the signal from PS II core

antenna is observed. The sixth FDAS (21–26 ns) exhibits delayed fluorescence, in which two

distinct peaks at 685 and 698 nm are recognized for all Prochloron samples and only the 686 nm

band is observed for Prochlorothrix. An additional peak is recognized at 715 nm for Prochloron in

T. cyclops, Diplosoma sp., and 720 nm for Prochloron in L. timorense. This indicates a transfer of

excitation energy to PS I in these time regions [23]. For Prochloron in T. miniatum, relative

intensity of delayed fluorescence in the PS I region to that in the PS II region is negligibly small,

suggesting that PS II and PS I are not strongly combined in this sample. For Prochlorothrix, relative

intensities in the PS I region to those in the PS II region are smaller than those for Prochloron after

the third FDAS.

4. Discussion

The TRFS and FDAS of Prochloron in four different ascidians indicate that pigment

composition and efficiency of excitation energy transfer between antenna proteins in Prochloron

change with host species. First, in FDAS, the time constants of excitation energy transfer from Pcb

to PS II and PS I can be classified into two groups. For Prochloron in T. cyclops and Diplosoma sp.,

the excitation energy transfer occurs rapidly (11–18 ps), while it is slower (50–51 ps) for

Prochloron in T. miniatum and L. timorense (Fig. 4). Differences between these samples might be

due to varying light conditions in the host ascidians’ habitat. In the present study, the collected

colonies of T. miniatum and L. timorense inhabited sites strongly exposed to light, while the

colonies of T. cyclops and Diplosoma sp. inhabited shaded sites with low light exposure. It is

therefore suggested that Prochloron changes the excitation energy transfer efficiency among its

antenna proteins depending upon light conditions, and that Prochloron in shaded ascidians possess

a more effective light harvesting system. These are supported by the fact that the first FDAS of

Prochlorothrix (50 ps), which is not shaded by ascidian, shows almost the same behavior as

Prochloron in T. miniatum. Furthermore, in the fifth component of FDAS, the emission amplitudes

are smaller for Prochloron in T. miniatum and L. timorense than in the other two samples. This

suggests that excitation energy within antenna Pcb is quenched in the presence of excess excitation

energy under sunny conditions. In this sense, smaller amplitude in the fifth FDAS in Prochlorothrix

than those of Prochloron in T. miniatum and L. timorense suggested that Prochlorothrix is in

relatively high-light adapted condition.

Differences between the four samples regarding the PS I Red Chls were also apparent. All

samples show fluorescence of the PS I Chls at 710 nm, 715 nm, and 720nm (Figs. 3 and 4). In

addition, Prochloron in T. cyclops, Diplosoma sp., and L. timorense fluoresce around 730 nm. As

discussed above, T. cyclops and Diplosoma sp. inhabit shaded areas; therefore Prochloron in these

two ascidians require an efficient light harvesting system. The likely presence of Red Chls in these

ascidians would broaden their utilizable wavelength region. By contrast, L. timorense (and T.

miniatum) reside in sunny habitats, in which the efficiency of the light-harvesting system is less

stringent. The presence of Red Chls in Prochloron in L. timorense is difficult to explain based on

the hosts' habitat, but the ascidian tunic and calcareous spicules both absorb light of shorter

wavelengths. In T. miniatum, Prochloron cells and spicules reside together in the tunic, which

might partially enable the Prochloron cells to absorb sunlight directly without being shaded by

spicules. On the other hand, in L. timorense, Prochloron cells are in the cloacal cavity, and

therefore receive light through calcareous spicules. Therefore, it is likely that by possessing the Red

Chls, Prochloron in L. timorense can utilize light energy of longer wavelengths under appropriate

conditions. Moreover, strong self-shading would be expected, because the Prochloron cells are at a

very high density in the cloacal cavities. The difference of subcellular structure in Prochloron may

also cause the presence or absence of Red Chls; Prochloron cells in T. miniatum are belonged to

Group-I, while those in T. cyclops, Diplosoma sp., and L. timorense are Group-II [11,14]. The PS I

Chl signals of Prochlorothrix, are much smaller than those of Prochloron. Therefore, it seems that

Prochlorothrix used in this study utilizes shorter wavelength region and has relatively

high-light-adapted nature than Prochloron.

In the latest time region of TRFS and the sixth component of FDAS, all Prochloron

samples display definitive signals at 685 nm and 698 nm (Figs. 3 and 4), indicating that charge

recombination in the PS II reaction center occurs in all samples, while Prochlorothrix shows the

definitive signal only at 685 nm with negligible one at 698 nm. In addition, unambiguous delayed

fluorescence in the PS I region occurs in the sixth FDAS of Prochloron in T. cyclops and

Diplosoma sp. (peaking at 715 nm), and in L. timorense (peaking at 720 nm). These results suggest

that (1) excitation energy caused by charge recombination is efficiently transferred to the PS I

complexes through the PS II core antenna (indicating that PS II and PS I are energetically

connected), and (2) the final energy trap following PS II-to-PS I excitation energy transfer depends

on environment.

In summary, Prochloron in T. cyclops and Diplosoma sp. possess efficient

light-harvesting systems, mediated by high efficiency of excitation energy transfer from PS II to PS

I, which are tightly associated. The light harvesting systems of Prochloron in the other two

ascidians, T. miniatum and L. timorense, are less efficient. Prochloron occupying shaded habitats

might be expected to develop effective light-harvesting systems via association between two

photosystems, in order to enhance excitation energy transfer. However, Prochloron in L. timorense

also exhibited PS II-to-PS I excitation energy transfer, although this ascidian inhabits sunny areas.

From the tendencies observed in the other samples, we recognize that Prochloron in hospite adjust

their photosynthetic systems according to the light conditions of their habitats. In L. timorense, we

anticipate “shade” effects from spicules that scatter and/or absorb light of shorter wavelengths.

Prochloron in L. timorense might employ adaptive strategies to utilize longer-wavelength light by

producing more Red Chls and by maximizing the efficiency of the PS II-to-PS I excitation energy

transfer, since the host ascidian has armored itself with spicules as protection against intense

sunlight. While Prochlorothrix shows some behaviors similar to Prochloron, especially that in T.

miniatum, Prochlorothrix has even less PS I Chl and does not utilize lights in longer wavelength

region of photosynthetic active radiation, compared to Prochloron. Therefore, two Chl b-containing

cyanobacteria, symbiotic Prochloron and free-living Prochlorothrix, must have adapted to light

environment in their respective habitat through different adaptive strategy for light-harvesting

system.

Acknowledgements

This work was supported by a Grant in Aid for JSPS Fellows (No. 21·2944) to MY, a Grant in

Aid for Scientific Research from JSPS (23370013) and Ocean Exposition Commemorative Park

Management Foundation (Okinawa) to AM, EH, and SA, a Grant in Aid for Scientific Research

from JSPS to SA (23370017).

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supercomplex isolated from Prochloron didemni retaining its chlorophyll a/b light-harvesting

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Figure captions

Figure 1. Absorption spectra of Prochloron in hospite and Prochlorothrix observed at 77 K: (A)

Prochloron in Trididemnum cyclops, (B) Prochloron in Trididemnum miniatum, and (C)

Prochlorothrix.

Figure 2. Steady-state fluorescence spectra of Prochloron in hospite and Prochlorothrix: (A)

Prochloron in Trididemnum cyclops, (B) Prochloron in Trididemnum miniatum, (C) Prochloron in

Diplosoma sp., (D) Prochloron in Lissoclinum timorense, and (E) Prochlorothrix observed at 77 K.

Excitation wavelength is 425 nm. Spectra are normalized relative to their maximum intensities.

Vertical dotted lines indicate 685 nm (PS II), 698 nm (PS II), and 715 nm (PS I).

Figure 3. Time-resolved fluorescence spectra (TRFS) of Prochloron in hospite and Prochlorothrix:

(A) Prochloron in Trididemnum cyclops, (B) Prochloron in Trididemnum miniatum, (C)

Prochloron in Diplosoma sp., (D) Prochloron in Lissoclinum timorense, and (E) Prochlorothrix at

77 K. Excitation wavelength is 425 nm. Spectra are normalized relative to their maximum

intensities.

Figure 4. Fluorescence decay-associated spectra (FDAS) of Prochloron in hospite and

Prochlorothrix: (A) Prochloron in Trididemnum cyclops, (B) Prochloron in Trididemnum miniatum,

(C) Prochloron in Diplosoma sp., (D) Prochloron in Lissoclinum timorense, and (E) Prochlorothrix

observed at 77 K. These spectra are normalized relative to the intensity at 710 nm in the second

components (150–260 ps).

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