-
Ubiquity and quantitative significance ofdetoxification
catabolism of chlorophyllassociated with protistan
herbivoryYuichiro Kashiyamaa,1,2, Akiko Yokoyamab,1, Yusuke
Kinoshitaa, Sunao Shojia, Hideaki Miyashiyac, Takashi
Shiratorid,Hisami Sugae, Kanako Ishikawaf, Akira Ishikawag, Isao
Inouyeb, Ken-ichiro Ishidab, Daiki Fujinumah, Keisuke Aokih,Masami
Kobayashih, Shinya Nomotoi, Tadashi Mizoguchia, and Hitoshi
Tamiakia,2
aGraduate School of Life Sciences, Ritsumeikan University,
Kusatsu, Shiga 525-8577, Japan; bFaculty of Life and Environmental
Sciences and dGraduate Schoolof Life and Environmental Sciences,
University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; cGraduate
School of Human and Environmental Studies, KyotoUniversity, Kyoto
606-8501, Japan; eInstitute of Biogeosciences, Japan Agency for
Marine-Earth Science and Technology, Yokosuka, Kanagawa
237-0061,Japan; fSystem Analysis Division, Lake Biwa Environmental
Research Institute, Otsu, Shiga 520-0022, Japan; gGraduate School
of Bioresources, Mie University,Tsu, Mie 514-8507, Japan;
hInstitute of Materials Science, University of Tsukuba, Tsukuba,
Ibaraki 305-8573, Japan; and iDepartment of Chemistry, Universityof
Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
This Feature Article is part of a series identified by the
Editorial Board as reporting findings of exceptional
significance.
Edited by David M. Karl, University of Hawaii, Honolulu, HI, and
approved August 2, 2012 (received for review May 2, 2012)
Chlorophylls are essential components of the photosynthetic
appa-rati that sustain all of the life forms that ultimately depend
on solarenergy. However, a drawback of the extraordinary
photosensitizingefficiency of certain chlorophyll species is their
ability to generateharmful singlet oxygen. Recent studies have
clarified the catabolicprocesses involved in the detoxification of
chlorophylls in landplants, but little is understood about these
strategies in aquaticecosystem. Here, we report that a variety of
heterotrophic protistsaccumulate the chlorophyll a catabolite
132,173-cyclopheophorbidea enol (cPPB-aE) after their ingestion of
algae. This chlorophyll de-rivative is nonfluorescent in solution,
and its inability to generatesinglet oxygen in vitro qualifies it
as a detoxified catabolite of chlo-rophyll a. Using a modified
analytical method, we show that cPPB-aE is ubiquitous in aquatic
environments, and it is often the majorchlorophyll a derivative.
Our findings suggest that cPPB-aE metab-olism is one of the most
important, widely distributed processes inaquatic ecosystems.
Therefore, the herbivorous protists that con-vert chlorophyll a to
cPPB-aE are suggested to play more significantroles in the modern
oceanic carbon flux than was previously recog-nized, critically
linking microscopic primary producers to the mac-roscopic food web
and carbon sequestration in the ocean.
phototoxicity of chlorophylls | microbial herbivory |
phagocytosis |biodiversity of eukaryotes | microbial loop
Chlorophylls are crucial to sustaining most life forms on
Earth,the majority of which ultimately depend on solar
energy.Photoexcitation of chlorophylls initiates various
photosyntheticreactions that convert the energy of photons into
chemical poten-tials, which in turn, drive the full range of
metabolic reactionsthroughout the global ecosystem. Chlorophylls
play a central rolein the photosynthetic apparatus by absorbing
light and trans-ferring the excitation energy to the reaction
centers of photo-systems before photosynthetic electron transport.
However,without measures to contain the excited energy,
chlorophylls canharm organisms because of their high
photosensitizing potential.Photoexcited chlorophylls generate
singlet oxygen, a highly re-active oxygen species that can cause
severe cellular damage (1).Therefore, the phototoxicity of
chlorophylls has been a continu-ous concern for the Earth’s
ecosystem since the global rise inatmospheric oxygen about 2.3
billion y ago (2).Given its potential risks, chlorophyll metabolism
is thought to
be carefully controlled in the cells of phototrophic
organisms.Recent works have revealed that the biodegradation of
chloro-phyll a (Chl-a) (Fig. 1) in land plants (embryophytes) is
regulatedas carefully as its biosynthesis (3). The chlorin moiety
found inChl-a is a highly π-conjugated tetrapyrrolic macrocycle
that acts
as a potentially phototoxic chromophore or fluorophore. It is
con-verted stepwise into an unconjugated and hence, colorless
andnonfluorescent linear tetrapyrrole (SI Text, section 1.1).
However,many algae and cyanobacteria apparently lack the
programmeddetoxifying catabolism of chlorophyll observed in
embryophytes (SIText, section 1.2). The inability of unicellular
phototrophs to detoxifychlorophyll can be rationalized by their
lack of the need to remo-bilize nutrients before death, unlike
multicellular land plants. Re-garding heterotrophs, the digestive
systems of most terrestrialherbivores are dark, whereas the
digestive systems of most aquaticherbivores, such as multicellular
and unicellular zooplankton, aresmall and translucent. This finding
renders the microscopic aquaticherbivores susceptible to damage by
the singlet oxygen generatedwhen ingested chlorophylls are exposed
to light (SI Text, section 1.3).Thus, strategies to protect against
the accumulation of phototoxicchlorophyll derivatives should be
critical for the survival of the mi-croscopic aquatic herbivores
feeding under illumination.We conducted feeding experiments on
several microorganisms
to screen for potentially detoxified chlorophyll catabolites.
Here,we report that 132,173-cyclopheophorbide a enol (cPPB-aE)
(Fig.1) is the dominant product derived from Chl-a that
accumulatesin cells of various aquatic heterotrophic protists
(i.e., unicellulareukaryotes) that feed on algae and that cPPB-aE
is a virtuallynonfluorescent and nonphotosensitizing chlorophyll
derivativeincapable of generating singlet oxygen. These results
stronglysuggest that herbivorous protists generate cPPB-aE as a
detoxi-fied catabolite of Chl-a. We also show that cPPB-aE is
ubiqui-tous in all of the aquatic environments that we tested,
frequentlyas the most abundant Chl-a derivative in the surface
sediments,and that it is actively generated in the water near
illuminatedsurfaces.
Author contributions: Y. Kashiyama, A.Y., H.M., H.S., K.I.,
A.I., I.I., K.-i.I., M.K., S.N., T.M., andH.T. designed research;
Y. Kashiyama, A.Y., Y. Kinoshita, S.S., H.M., T.S., D.F., K.A., and
S.N.performed research;Y. KashiyamaandA.Y. analyzeddata; andY.
Kashiyamawrote thepaper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
See Commentary on page 17311.1Y. Kashiyama and A.Y. contributed
equally to this work.2To whom correspondence may be addressed.
E-mail: [email protected] or [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1207347109/-/DCSupplemental.
17328–17335 | PNAS | October 23, 2012 | vol. 109 | no. 43
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Results and DiscussionFeeding Experiments. We showed that three
genetically distantheterotrophic protists—a stramenopile, a
cercozoan (Filosa),and a heliozoan (Centrohelida)—accumulated
cPPB-aE as theonly major chlorophyll derivative associated with
herbivory. Weestimated that levels of cPPB-aE in extracts from
these protistsafter they were fed on fresh unialgal cells are as
high as 80 mol%of total amounts of chlorins derived from Chl-a
(Fig. 2). Howe-ver, a heterotrophic discicristoidean did not
accumulate any cPPB-aE. No trace of cPPB-aE was detected in control
experiments,where the unialgal cultures fed to the protists were
incubatedunder conditions identical to those conditions used for
thefeeding experiments (Table S1). Organic synthesis of
cPPB-aErequires a strong base to form the C–C bond between 132
and173 carbons (4, 5), suggesting that it is difficult to generate
cPPB-aE in vitro under normal conditions. In fact, the generation
ofcPPB-aE has not been reported in so-called dark
incubationexperiments involving pure unialgal cultures sealed and
pre-served in darkness for months to years (6–8). Therefore,
cPPB-aE detected in the present experiments must be
conclusivelyattributed to metabolic processes in the heterotrophic
protists.Minor products of Chl-a degradation are pyropheophytin
a
(pPhe-a) (Fig. 1) as well as various polar chlorins, including
(132R)-and (132S)-hydroxychlorophyllones a [(R/S)-hCPLs-a] (Fig.
1). Darkincubation experiments of unialgal cultures revealed
substantialaccumulation of pPhe-a only after the incubations had
proceededfor several months (6–8). Given that pPhe-a is absent from
ourcontrol experiments involving unialgal cultures, its
accumulation canalso be attributed to the heterotrophic process.
However, (R/S)-
hCPLs-amay be a product of biotic processing (9, 10) and/or
abioticoxidation of cPPB-aE (11). All samples of heterotrophic
protistsalso contain variable amounts of intact Chl-a, which may
have beenderived from undigested algal materials in phagosomes.Our
experiments, thus, suggest qualitatively that the herbivo-
rous protists studied actively modify Chl-a into cPPB-aE
duringtheir course of phagotrophic digestion (Fig. 3). Given that
het-erotrophic and phototrophic cells grew together in the
culturesused in our experiments, quantitative estimations of the
rate ofcPPB-aE production from Chl-a as well as the stability of
cPPB-aE in vivo were beyond the scope of the present work.
None-theless, it seems reasonable to conclude that cPPB-aE is a
majorChl-a catabolite produced by the herbivorous protists that
westudied. Therefore, cPPB-aE could potentially emerge as a
bio-chemical marker of protistan herbivory in aquatic
environments.
Photochemical Significance. We determined various properties
ofcPPB-aE by analyzing an authentic sample that was
semisynthesizedfrom Chl-a (Materials and Methods). The most
striking propertyof cPPB-aE is that it is essentially
nonfluorescent (4), despite itshighly π-conjugated structure (Fig.
4 A and B, SI Text, section 1.5,Fig. S1, and Table S2). This
finding is consistent with our
N
N N
N
M
OR1
R2O
O
132
173
N
N N
NH
H
O
OH
N
N N
NH
H
O
OOH
*
Chlorophyll a (Chl-a):
M = Mg; R1 = COOMe; R2 = Phytyl
M = 2H; R1 = COOMe; R2 = Phytyl
M = 2H; R1 = H; R2 = Phytyl
M = 2H; R1 = COOMe; R2 = H
M = 2H; R1 = H; R2 = H
M = 2H; R1 = H; R2 = Me
M = 2H; R1 = H; R2 = Cholesteryl
Pheophytin a (Phe-a):
Pyropheophytin a (pPhe-a):
Pheophorbide a (PPB-a):
Pyropheophorbide a (pPPB-a):
Methyl pPPB-a:
Cholesteryl pPPB-a:
Cyclopheophorbide a enol
(cPPB-aE)
(132R/S)-Hydroxychlorophyllones a
((R/S)-hCPLs-a)
Fig. 1. Chemical structures of Chl-a and its derivatives
discussed in thepresent work.
0% 20% 40% 60% 80% 100%
fed on Nitzschia sp.
Nitzschia sp.
Heterotrophic stramenopile
Heterotrophic cercozoan fed on Skeletonema sp.
Skeletonema sp.
Heterotrophic heliozoan fed on Pyramimonas sp.
Pyramimonas sp.
Heterotrophic discicristoidean fed on
fed on
fed on green juice powder
a pennate diatom
(Pannate diatom)
Cryptomonas tetrapyrenoidosa
Cryptomonas
tetrapyrenoidosa
Entrobacter aerogenes
Daphnia pulex
Green juice powder (made from young barley leaves)
Chl-a pPhe-a
Steryl chlorin esters
cPPB-aE
Mg-chelated Chl-a derivatives
Free base Chl-a derivatives
Fig. 2. Protistan herbivory and associated chlorophyll
catabolism/degrada-tion. Relative abundances of chlorophyll
derivatives in extracts of culturedheterotrophic protists. Four
experiments involved feeding a stramenopile,a cercozoan, a
heliozoan, and a discicristoidean with the diatoms Nitzschia sp.and
Skeletonema sp., a prasinophyte Pyramimonas sp., and a pannate
diatom,respectively.We observed that, whereas cPPB-aEwas the
dominant componentin the cultures of the stramenopile, the
cercozoan, and the heliozoan, it wasabsent from the culture of the
discicristoidean. Crustacean zooplankton(Daphnia pulex) was fed a
cryptophyte, Cryptomonas tetrapyrenoidosa, fromwhich we did not
identify any cPPB-aE production. When the γ-proteobacte-rium
Entrobacter aerogenes was grown on a media containing green
juicepowder made from young barley leaves, we did not identify any
cPPB-aE pro-duction. None of the unialgal control cultures
contained any cPPB-aE either,suggesting that the Chl-a metabolite
was only produced after phagotrophicfeeding by the three
heterotrophic protists. Additional details are provided inTable S1,
and additional discussions are in SI Text, section 1.4.
Kashiyama et al. PNAS | October 23, 2012 | vol. 109 | no. 43 |
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microscopic observation that the autofluorescence of
chlor-ophylls in the plastids of ingested algae rapidly disappears
duringtheir phagocytosis by herbivorous protists (Fig. 3 and Fig.
S2).This observation suggests very rapid and nonradiative
quenchingof the photoexcited singlet state of cPPB-aE, which is
ascribed tointernal conversion(s) to the ground singlet state (12)
and/or in-tersystem crossing to the excited triplet state.
Considering thebiochemical significance of producing cPPB-aE by
some protists,the latter possibility seems unreasonable owing to
the highly toxicsinglet oxygen that is readily produced from the
triplet state(Fig. S3).Fig. 4C shows that production of singlet
oxygen by photoex-
cited cPPB-aE is negligible relative to the levels generated
fromChl-a and Phe-a. Here, the generation of singlet oxygen was
de-tected using Singlet Oxygen Sensor Green (SOSG), a
commerciallyavailable fluorescent probe. No change in SOSG
fluorescencewas observed over the course of the 180-min experiment
withcPPB-aE, which recapitulated the result of the control
experi-ment. This finding contrasts strikingly with the results for
Chl-a and Phe-a. The increase in the SOSG fluorescence caused
by
rapid generation of singlet oxygen was approximately three
timesmore rapid for Chl-a than Phe-a. The observed difference
be-tween Chl-a and Phe-a should partially reflect differences in
theabsorption intensities of Qy bands (Table S3). In
conclusion,cPPB-aE is likely to be a colored but
nonphotosensitizing Chl-acatabolite, which is comparable with the
colorless nonfluores-cent chlorophyll catabolites (NCCs) of higher
plants (SI Text,section 1.1) insofar as its generation during the
detoxification ofchlorophylls protects against the accumulation of
singlet oxygenin vivo.Previous studies have frequently regarded
cPPB-aE as an an-
tioxidant that accumulates in marine macrofauna (11,
13–16).However, other workers have failed to highlight the
possibilitythat the primary evolutionary advantage of cPPB-aE
productionby single-celled organisms is the detoxification of the
phototoxicchlorophyll derivatives that are inevitably ingested on
feedingchlorophyll-containing diets to protists in an illuminated
andaerated environment. The reported antioxidant function ofcPPB-aE
accumulated in the intestines of metazoans would, thus,be a
secondary adaptation if the derivative, indeed, functions asan
antioxidant. We note that its antioxidant activity, which
shouldprotect against the oxidative damage exerted by reactive
oxygenspecies, has not been shown experimentally. Furthermore,
nodirect evidence has yet been presented that metazoans
producecPPB-aE. Geochemical evidence that is consistent with the
abilityof microscopic plankton to produce cPPB-aE is discussed
below.
Ubiquity and Geochemical Significance of cPPB-aE in Aquatic
Environ-ments. To determine the distribution of cPPB-aE
metabolismamong various aquatic settings, we used a geochemical
approachthat involved detecting cPPB-aE directly from particulate
organicmatter (POM) in water columns as well as surface
sedimentarysamples just beneath the columns. We analyzed extracts
of glassmicrofiber filters (GF/F grade;Whatman) that presumably
collectedany particulate matters larger than 0.7 μm in diameter
from thewater samples examined, and therefore, they should have
includedany eukaryotic cells and most prokaryotic cells present as
well asfecal materials and any organic aggregates.The results
showed that cPPB-aE is ubiquitous and abundant in
all of the aquatic environments tested, confirming the
ecologicaland biogeochemical significance of cPPB-aE metabolism
(Fig. 5).In summary, we detected cPPB-aE in all POMsamples
fromdiverseenvironments, although its abundance relative to the
other Chl-aderivatives ranged from5mol% (samples collected from Ise
Bay) to42 mol% (samples collected from a dammed creek). These
valuescorrespond to ranges in the cPPB-aE/Chl-a ratios from 0.07 to
1.53.However, the relative concentrations of cPPB-aE in the
surfacesediments were generally very high (51–81%), representing
vir-tually the most abundant Chl-a derivatives. In fact, the
presenceof cPPB-aE in sediments has been occasionally reported in
pre-vious works (11, 17–19), although it has been probably
completelymissed in other reports because of analytical artifacts
(20).The abundance of cPPB-aE relative to Chl-a in water
columns
implies that a considerable proportion of photosynthetic
primaryproduction should initially be processed by herbivorous
protiststhat produce cPPB-aE. Given that Chl-a is generally
regarded asstanding biomass of phototrophic plankton, we similarly
expectthat the cPPB-aE content in POM should represent the mass
ofherbivorous protists as well as their excreta in water.
Depthprofile patterns of cPPB-aE and the other chlorophyll
derivatives(Fig. 6) are perhaps best explained by (i) in situ
feeding activitiesof herbivorous protists in the upper parts of
water columns and/or (ii) selective preservation into deeper water
and sediments,which would explain the elevated relative
concentration of cPPB-aE in the surface sediments.Evaluations of
those data in terms of either the rate of protistan
herbivory or the rate of cPPB-aE production require
additionalinvestigation, including investigation of the rate of
cPPB-aE pro-
NP
EP
LP
NP
EP
LP
Skeletonema sp.never ingested
(unfocused)
Skeletonema sp.never ingested
(unfocused)
A
B
Fig. 3. A typical differential interference image (A) and a
correspondingfluorescent image (B; excitation light = 400–440 nm)
of cercozoans in dif-ferent stages of phagocytosis. The cercozoan
cells are indicated by the dashedlines on the fluorescent image.
EP, a cell in an earlier stage of phagocytosiscontaining two chains
of Skeletonema sp. in different stages of digestion;LP, a cell in a
later stage of phagocytosis containing a chain of Sketetonemasp. in
a later stage of digestion displaying no autofluorescence; NP, a
cellexhibiting no phagosome formation. (Scale bars: 50 μm.) In EP
cell, the longerchain of diatoms on the left is partially digested,
and therefore, the plastids ofseveral diatom cells still display
the autofluorescence of chlorophyll (arrows),whereas the shorter
chain on the right is in a later stage of digestion andcontains
remnant plastids (dark brown grains) displaying no
autofluores-cence. Additional information is given in Fig. S2.
17330 | www.pnas.org/cgi/doi/10.1073/pnas.1207347109 Kashiyama
et al.
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duction by various herbivorous protists, its lifetime in their
cells andthe environment (e.g., in the excreta), and the sinking
rate of POMin each environment. In particular, a better
understanding of thechemical properties of cPPB-aE in vivo is
required to explain itsapparently high preservation potential in
environmental samples,despite its instability in organic solutions.
In fact, we do not exclude
the possibility that protistan catabolism responsible for
producingcPPB-aE would occur even in darkness (e.g., in deep water
columnsand sediments), despite our hypothesis that the process
evolvedprimarily to avoid the phototoxicity of chlorophyll
derivatives.
Evolutionary Significance of Protistan Herbivory. We postulate
thatthe ingestion of Chl-a by small, transparent, or translucent
microbesin illuminated and oxygenated environments places an
evolutionaryconstraint on microbial herbivory by necessitating
appropriatebiochemical strategies to contain levels of singlet
oxygen-gener-ating molecules after feeding on microalgae,
cyanobacteria, orphototrophic bacteria. The evolution of aquatic
microbial her-bivory should have been accompanied by the
establishment ofsome enzymatic pathways that catabolize
chlorophylls into non-fluorescent substances. Unlike the NCCs of
higher plants, cPPB-aE is a colored catabolite, despite its
nonphotosensititizing prop-erties. Given that conversion of Chl-a
to cPPB-aE apparentlyrequires fewer enzymatic steps than the
generation of NCCs, ithas emerged as a simple and efficient
strategy for rapid detoxifi-cation of chlorophyll among aquatic
protists.Our evidences regarding the synthesis of cPPB-aE by
protistan
herbivores are comparable with the results of similar feeding
ex-periments in the work by Goericke et al. (17) using
heterotrophicprotists. The work by Goericke et al. (17) also
identified cPPB-aEas a major Chl-a derivative in the fecal material
of three protists,the ciliate Strombidinopsis acuminatum and the
heterotrophicdinoflagellates Amphidinium sp. and Noctiluca
scintillans. Fig. 7illustrates that cPPB-aE producers, thus,
distribute widely amongthe protistan lineage, at least in two
supergroups. These are theStramenopile-Alveolate-Rhizaria (SAR)
clade and the Cryptophyte-Centrohelid-Telonemid-Haptophyte (CCTH)
clade (21, 22). Incontrast, one species belonging to the opistkonta
clade lacks theability to synthesize cPPB-aE. Significantly, over
70% of hetero-trophic protists in the marine environment are known
to belong
0
20
40
60
80
100
120
300 350 400 450 500 550 600 650 700 750 800
500
1000
1500
2000
ce
nc
e in
te
ns
ity
cre
me
nt
-500
0
500
Flu
oresc
in
Irradiation time (min)
C
BA
Wavelength (nm)
Ex
tin
ctio
n c
oe
ffic
ie
nt
(m
M-1c
m-1)
cPPB-aE
Chl-a
Phe-a
Chl-a Phe-a cPPB-aE
N
N N
NH
H
O
OH
132,173-Cyclopheophorbide a enol(cPPB-aE)
0 30 60 90 120 150 180
Fig. 4. Photochemical properties of cPPB-aE. (A) Chl-a, Phe-a,
and cPPB-aE in anisole (50 μM in a 4-mL quartz cuvettete) under
white (Upper) and UV (Lower)light, showing the absence of red
fluorescence from cPPB-aE, which is typical of chlorophyll
derivatives. (B) Absorption spectra of Chl-a, Phe-a, and cPPB-aE
inanisole. (C) Experiments detecting production of singlet oxygen
in vitro using SOSG. When Chl-a is codissolved with SOSG in
methanol-anisole (1:1, vol/vol; ),relatively rapid generation of
singlet oxygen was indicated on illumination of red light (>630
nm). Phe-a (■) also acted as a photosensitizer for the gen-eration
of singlet oxygen. By contrast, no generation of singlet oxygen was
evident from cPPB-aE ( ) or the control experiment (only SOSG; ).
In eachexperiment, the concentrations of pigments and SOSG used
were 10 and 1 μM, respectively.
Kumano-Nada in the Pacific OceanWater (50 m)
Water (8 m)
Sediment
Water (5 m)
Sediment
Water (10 m)
Sediment
Dammed creek water (BKC)
Paddle water in a garden rock
Lake Biwa
Tokyo Bay
Ise Bay
0% 20% 40% 60% 80% 100%
Chl-a pPhe-a
Steryl chlorin esters
cPPB-aE
Mg-chelated Chl-a derivatives
Free base Chl-a derivatives
Fig. 5. Relative abundances of chlorophyll derivatives in
extracts of POMfrom various aquatic samples and surface sediments.
The samples analyzedare summarized in Table S4.
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to either the SAR or CCTH clade (23), which is consistent
withour geochemical data.The SAR and CCTH clades include various
phototrophic and
mixotrotrophic protists other than the obligate
heterotrophsanalyzed here. Many of these phototrophic and
mixotrophicprotists possess plastids that had been derived from
ancestral redalgae through secondary symbiosis (Fig. 7). They
include majormarine algae, such as diatoms and dinoflagellates, as
well as di-verse picophytoplankton species (24). The rest of the
SAR andCCTH protists are colorless and lack plastids, and thus,
they are
presumably heterotrophic. Nonetheless, it is still not
certainwhether these protists lost plastids during the course of
evolution(i.e., secondary evolution of heterotrophy) or had never
possessedplastids (i.e., multiple origins of secondary
phototrophy). Our evi-dence implies that the generation of cPPB-aE,
a key metabolicstep in the heterotrophic lifestyle, is likely to
have evolved atthe root of the two clades, if not at an earlier
evolutionarystage. The ability to synthesize cPPB-aE must, thus, be
retainedin heterotrophic protists of distant lineages, regardless
of thereason for the absence of plastids.
A
D
(pmol/L)W
ater
dep
th (m
)
0
100
200
300
400
500
600
0.1 1 10 100 1000
0% 20% 40% 60% 80% 100%
0 m10 m20 m30 m40 m50 m60 m70 m80 m90 m
100 m150 m200 m300 m500 m
B
E
(pmol/L)
Wat
er d
epth
(m)
0% 20% 40% 60% 80% 100%
C
F
(pmol/L)
Wat
er d
epth
(m)
0% 20% 40% 60% 80% 100%
0 m
8 m
20 m
32 m
Sediment
0
5
10
15
20
25
30
35
0 500 1000 1500 2000 2500
0 m
5 m
10 m
15 m
20 m
80 m
Sediment
0102030405060708090
0 200 400 600 800 1000 1200
Chl-a
pPhe-a
Steryl chlorin esters
cPPB-aE
Mg-chelated Chl-a derivativesFree base Chl-a derivatives
Chl-a
pPhe-a
Steryl chlorin esters
cPPB-aE
Mg-chelated Chl-a derivativesFree base Chl-a derivative
0102030405060708090
100
0 200 400(pmol/L)
Fig. 6. Depth profiles of absolute and relative abundances of
chlorophyll derivatives in water columns. (A–C) Absolute
abundances. (D–F) Relative abun-dances. POM in the pelagic Pacific
Ocean (Kumano-Nada; A and D), POM and surface sediments from Ise
Bay (B and E), and POM and surface sediments fromfreshwater Lake
Biwa (C and F) are shown.
Glauco
phytes
Chlo
roph
ytes
Rhod
ophyt
es
Stre
ptop
hyte
s
Centroh
elids
(heliozo
ans)
Telone
mids
Cyani
diophy
tes
Haptophy
tes
Cryptomonads
Kathablepharids
Picobiliphytes
Sloomycetes
StramenochomesPlacidids
Bicosoecids
Labyrinthurids
Slopalinids
Chrom
erids
Dinoflagellates
Apicomplexa
Colpodellids
PerkinsidsE
llobiopsids
Cilia
tes
Endo
myx
aFi
losa
Fora
min
ifera
Radio
zoa
Heterolob
oseaEugle
nozoaJakobids
Parabasalids
Diplomonads + Enteromonads
RetortamonadsCarpediemonads
Trimastix
Oxymonads
Malawimonads
MetazoaChoanoflagellates
Capsaspora
owczarz
aki
Ichthyosporids
Corallochytrium
Ascomycetes
RozellidsBlastocladiomycetes
MicrosporidsZygomycetesNephridiophagids
BasidiomycetesGlome
romycetes
Chytridio
mycetes
Discic
ristoid
ea
Entamoebae
Thecamoebae
Archamoebae
Cochliopodids
Pelobionts
Flabellinida/Discosea
Dic
tyos
telid
s
Tubline
aAcan
thamo
ebae
Proto
stelid
s
Myx
ogas
trids
Breviat
esAp
usozoa
SAR
Archaeplastida
CCTH
Excavata
Opistokonta
Amebozoa
stram
enop
iles
cercozoans
Fig. 7. Unrooted tree of eukaryotes comprising six distinctive
supergroups. Yellow stars beside taxonomic groups denote that they
contain protists thatproduce cPPB-aE, which was reported in this
study or a previous report (17). Examined protists belonging to
discicristoidea did not produce cPPB-aE (red x).The taxonomic
groups with green circles contain phototrophs with true
chloroplasts. Note that the stramenopiles include not only the
cPPB-aE–producingheterotrophs but also phototrophic diatoms used as
diets that did not produce any trace of cPPB-aE (Fig. 2). This
finding suggests that cPPB-aE metabolismevolved primarily for
heterotrophy. Modified from Walker et al. (22).
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Ecological Significance of Protistan Herbivory. We postulate
thatherbivory, particularly of the picophytoplankton, by
cPPB-aE–producing protists is one of the most important processes
in themodern aquatic food web, but it has been poorly
understooduntil now (Fig. 8). Importance of protistan herbivory as
a part ofthe microbial loop in aquatic environments has been
recognizedfor nearly three decades (25). During this time, the
quantitativesignificance of picophytoplankton (ϕ ≤ 3 μm) has also
beenrevealed (23, 24). Picophytoplankton includes coccoidal
cyano-bacteria and eukaryotic picophytoplankton. The former
belongto the two major genera Prochlorococcus and
Synechococcus,which account for up to 50% of pelagic marine
oxygenic pho-tosynthesis (26–28). Eukaryotic picophytoplankton are
believedto account for 20–50% of aquatic photosynthesis (29–32).
Theuse of the PCR to analyze environmental samples has alsorevealed
the amazingly wide diversity and activity of
eukaryoticpicophytoplankton species (33, 34).However, we are still
largely ignorant of abundance as well as
ecological and biogeochemical roles of colorless protists in
theoceans, in large part because the majority of these protists
has
never been studied because of an inability to maintain them
inculture. Indeed, only recent advances showed the
tremendousdiversity of protists in aquatic environments (32, 35,
36), thankslargely to molecular biological approaches, such as PCR
surveysof the diversity of 18S rRNA sequences in environmental
samples.Therefore, the ecological functions of aquatic colorless
protistsremain largely unexplored (37, 38).Our evidence suggests
that picophytoplankton-based protistan
herbivory is a quantitatively important process in aquatic
envi-ronments. In analyses of POM samples from the water column
ofLake Biwa, cPPB-aE was substantially enriched in the fine
POMfractions, which contained POM with diameters of 5–0.7 μm(Fig.
S4), which indicates that cPPB-aE was either accumulatedin
picozooplanktonic protists (ϕ ≤ 3 μm) and/or concentrated invery
fine particulate excreta most likely derived from variouslysized
zooplanktonic protists. Therefore, picophytoplankton areprobably
consumed by these protistan herbivores (Fig. 8). In fact,the small
size of picophytoplankton precludes their consumptionby metazoan
zooplankton such as copepods, suggesting that theyare instead
likely to be preyed on by protists (23, 39).
Protistanbacteriovory
Dissolvedorganicmatter
Dissolvedinorganic
matter
Nutrients(N, P, S, etc.)
CO2
Phototrophy(solar energy)
Phytoplankton(>3 μm)
Pico-phytoplankton(
-
ConclusionsThe singlet oxygen generated when oxygen is
photosensitized bychlorophylls is probably a major concern among
the organismsliving on the oxygenated and illuminated surface of
the Earth.However, all organisms have evolved to somehow protect
them-selves against the potential phototoxicity of the chlorophylls
intheir respective environments. However, the significance of
thesedetoxification processes is only readily apparent in such
strikingmanifestations as autumnal tints. In the present work, we
haveshown a cryptic process of chlorophyll detoxification that is
widelydistributed among the SAR and CCTH protists, where colored
butnonfluorescent cPPB-aE is the catabolite of Chl-a. The
ubiquitousoccurrence of cPPB-aE in all aquatic environments
indicates thatcPPB-aE catabolism is one of the major chlorophyll
detoxificationmechanisms in the modern global ecosystem.If we
consider cPPB-aE a biomarker of protistan activity, our
results suggest the quantitative importance of herbivory by
theSAR and CCTH protists in aquatic environments.
Picophyto-planktons are particularly likely to be an important
prey. Althoughthe microbial loop tends to be pictured as a material
flow startingfrom bacteria and archaea that are fed on POM or
dissolved or-ganic matter (i.e., protistan bacterivory), nano- and
microplanktonicprotists should also play primary roles in the
picophytoplankton-based microbial loop (37, 40, 41) that, hence,
makes substantialcontributions to the flow of carbon and energy in
aquatic envi-ronments (42). Additional studies of cPPB-aE,
including its suit-ability as a proxy for protistan herbivory, may
provide approachesfor quantifying the contribution of
picophytoplankton to flux inthe aquatic geochemical cycle.A better
understanding of cPPB-aE metabolism within and
outside the SAR and CCTH clades of protists may provide
valu-able insights into the evolutionary origin of the modern
aquaticecosystem, founded largely on protistan herbivory. Feeding
onphotosynthetic organisms in situ in the presence of
molecularoxygen and light, would have enabled much more efficient
bio-geochemical recycling and bioenergetic redistribution within
envi-ronments near the water surface. Interestingly, chemical
structuresof fossil biomarkers that could be derived from cPPB-aE
have beenreported from various aquatic sedimentary rocks as old as
the EarlyJurrasic (ca. 183 Ma) (Fig. S5) (10, 43–50).
Interestingly, fossil-based evidence suggests that this timing
correlates roughly withthe emergence of secondary algae containing
red algae-derivedplastids. These plastids include phototrophic
protists within theSAR and CCTH clades, including dinoflagellates,
coccolitho-phores, and diatoms (51). An understanding of aquatic
microbialherbivory gleaned from combining biochemical,
molecularbiological, and geochemical evidences would provide
criticalinsights into the evolutionary dynamics of the aquatic
protistanlineages as well as the global geochemical cycles.
Materials and MethodsPreparation of Authentic Samples. Schemes
for preparation of authenticsamples of Chl-a, Phe-a, pPhe-a,
pPPB-a, cholesteryl pPPB-a, cPPB-aE, and (R/S)-hCPLs-a are
summarized in Fig. S6. Additional details are described in SI
Text,section 2.1.
Development of HPLC Methods. We developed improved analytical
methodsthat enable quantitative identification of unstable cPPB-aE
from microbio-logical and environmental samples with high
sensitivity. Analytical difficul-ties arose as a consequence of the
instability of cPPB-aE during handlingduring extraction and
analysis involving HPLC (11, 13, 17). Given that cPPB-aE is rather
unstable in most organic solvents in the presence of
molecularoxygen, it was rapidly degraded, especially when present
at low concen-trations. Our results, thus, depend largely on the
development of improvedanalytical methods that required the
availability of the semisynthesizedauthentic standard. In short,
the analysis required (i) careful removal ofmolecular oxygen from
the solvents for extraction and the mobile phases ofHPLC, (ii) use
of an end-capped reverse-phase HPLC column as well as
itsdeactivation by preconditioning using mobile phase with 0.5%
(wt/vol)
trifluoroacetic acid, and (iii ) use of stabilizing agents. In
particular, theaddition of imidazole in the mobile phase
dramatically improved the quanti-tative analysis of cPPB-aE using
HPLC. In addition, cPPB-aE was found to beparticularly stabilized
in anisole solution. Therefore, the use of the standardsolution in
anisole permitted accurate calibration on the HPLC analysis
(SIText, section 2.2). Consequently, the current methods
significantly improvedthe quantitative analysis of cPPB-aE (Fig.
S7, Fig. S8, and SI Text, section 2.3).Detection limit of cPPB-aE
was ∼30 fmol per injection on the currentHPLC method.
Analytical HPLC (Fig. S9) was performed using a Shimadzu
Prominence liquidchromatograph system, which comprised a CBM-20A
communications busmodule, a DGU-20A3 degasser, two LC-20AD pumps
constituting a binarypumping system, an SIL-20AAC auto sampler, a
CTO-20AC column oven, and anSPD-M20Avp diode array detector. The
system was coupled to a personalcomputer configured to run Shimadzu
LC Solution software. All solvents usedfor the analytical HPLC were
of HPLC-grade quality, and they were purchasedfromNacalai Tesque.
The standard solutions of various Chl-a derivatives used inthe
following analytical HPLCswereprepared using anisole (SI Text,
section 2.2).Analytical HPLC involved the use of a reverse-phase
monomeric columnZORBAX Eclipse Plus C18 (4.6 × 30 mm, 1.8-μm silica
particle size; Rapid Reso-lution HT). The solvent gradient program
used is summarized in Table S5, in-cluding the conditions used to
precondition the column. Because only a binaryautomated programming
techniquewith two pumps is available in our system,solvents B and C
are introduced to the same second pump using a switchingvalve in
the line. Therefore, switching of the solvents and purging of the
pumpsystem by the solvent were performed promptly within 1 min
after pre-conditioning of the column. All three solvents were
degassed in vacuo withultrasonication and sealed under argon. The
solvent reservoir bottles werespecially customized for the
convenience of degassing or sealing with argon aswell as preventing
the solvent from contacting the air during analysis. The flowrate
of the mobile phase was 1.00 mLmin−1. The column ovenwas set to 25
°C.The auto sampler tray was set to 15 °C. Given that standard
solutions andsamples were prepared in anisole, which is not a
component of the eluent, theinjection volume was generally 1.00 μL
or smaller.
Feeding Experiments. Four combinations of heterotrophic protists
with algae,a combination of a crustacean zooplanktonwith an alga,
and a combination ofabacteriumwithplantmaterialswereexamined.
Experiments, thus, comprise sixfeeding experiments as well as six
control experiments without heterotrophs/crustacean
zooplankton/bacteria. All protistan strains as well as the
copepodDaphnia magna were maintained by feeding unialgal clones
(Table S1). Aftercertain incubation periods, these strains were
collected on sterile glass micro-fiber filters (GF/F grade; 47 mm
ϕ; Whatman), which were then extracted andanalyzed according to the
analytical procedures used for natural samples de-scribed below.
Information including specific names of organisms examined,their
phylogenetic position, strain identities, and experimental
conditions aresummarized in Table S1.
Optical Spectrometry and Singlet Oxygen Detection Experiments.
Electronicabsorption spectraweremeasured using aHitachi U-3500
spectrophotometer.Fluorescence spectraweremeasuredusing aHitachi
F-4500 spectrophotometer,and fluorescence quantum yields were
obtained using a photoluminescencemethod with an absolute
photoluminescence quantum yield measurementsystem model C9920-02
comprising an excitation xenon light source, amonochromator, an
integral sphere, and a multichannel CCD spectrometer(Hamamatsu
Photonics). OD was about 1.0/10 mm at the Soret absorptionmaximum
in both anisole and tetrahydrofuran (THF) for electronic
absorptionmeasurements. THF used for spectroscopy was distilled
from a regent pur-chased from Nacalai Tesque. Anisole (RegentPlus
grade) and tert-butylmethyl ether (ACS regent grade) were purchased
from Sigma-Aldrich. All ofthe other solvents used herein were
spectrometry-grade regents purchasedfrom Nacalai Tesque, and they
were used without additional purification. Inoptical spectroscopy,
absorption and emission properties of chlorophyllderivatives in THF
and anisole are listed in Table S2. In experiments involvingcPPB-aE
dissolved in THF, we, therefore, prepared the solution under
argonin freshly distilled THF and measured all optical properties
immediately. Insinglet oxygen detection experiments, generation of
singlet oxygen was de-tected using SOSG (Invitrogen), a
commercially available fluorescent probe(52). A chlorophyll
derivative (10 μM) and SOSG (1 μM) were dissolved inanisole and
methanol (1:1, vol/vol), placed in a quartz cell, irradiated
withred light that was provided by a 250 W metal halide lamp
(LS-250–7500;Sumita Optical Glass), and passed through a colored
glass filter that failed totransmit light with a wavelength shorter
than ∼630 nm (AGC Techno Glass).Therefore, the irradiated light
selectively excited chlorophyll derivatives butnot SOSG. The
aerated solutions were continuously stirred during irradiation
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experiments using a magnetic stirrer. The increase in
fluorescence intensity(i.e., initial fluorescence ascribable to the
purchased SOSG was subtracted)was plotted against the irradiation
time to show and compare the relativeabilities of singlet oxygen
generation between different Chl-a derivatives aswell as a control
SOSG solution without the addition of any photosensitizers(Fig.
4C).
Analytical Procedures Used for Natural Samples. Water samples
were filteredusing a glass microfiber filter (GF/F grade; 47 mm ϕ;
Whatman). The filterswere then stored below −20 °C before analysis.
Sediment samples werestored at 4 °C before analysis. A wet filter
sample (containing POM) or a wetsediment sample was extracted in
acetone and ultrasonicated for 5 min at0 °C in the dark, with the
extraction and sonication procedures repeateda total of three
times. The combined extracts were then dried undera stream of argon
in the dark. The residue was then dissolved in anisole. Allof the
above operations were performed in an argon atmosphere using
a glove bag. Acetone and anisole were carefully degassed and
purged withargon gas before use. The sample dissolved in anisole
was then transferredto the vial for the autosampler of the HPLC
system.
ACKNOWLEDGMENTS. We thank the crew and scientific party of
theTraining/Research Vessel Seisui-maru (cruise number 1127) and
ResearchVessel Hakken. We also thank Mr. Hokuto Tsutamoto and Dr.
NobutakaImamura for kindly supplying the clone of D. pulex and Dr.
Hiroshi Endohfor favorably providing the clone of E. aerogenes; Mr.
Ryuzou Narita, Dr.Satoshi Ohkubo, Ms. Hiroko Usui, Mr. Koichi
Fujita, and Dr. ShinnosukeMachida for technical assistance; and Dr.
Jun Yokoyama and Dr. JonathanJ. Tyler for critical comments. This
study was supported in part by Grants-in-Aid for Scientific
Research 23870028 (to Y. Kashiyama), 20570081 (to A.Y.),24657060
(to A.Y.), 21247005 (to H.M.), and 21247010 (to I.I.), a
ResearchFellowship for Young Scientists (to Y. Kashiyama), and a
grant from theRitsumeikan Global Innovation Research Organization
(to Y. Kashiyamaand H.T.).
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