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Mevalonosomes: Specific vacuoles containing


the mevalonate pathway in Plocamium
brasiliense cortical cells (Rhodophyta)

Article in Journal of Phycology April 2015


DOI: 10.1111/jpy.12270

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J. Phycol. 51, 225235 (2015)
2015 Phycological Society of America
DOI: 10.1111/jpy.12270

MEVALONOSOMES: SPECIFIC VACUOLES CONTAINING THE MEVALONATE PATHWAY


IN PLOCAMIUM BRASILIENSE CORTICAL CELLS (RHODOPHYTA)1

Wladimir Costa Paradas


Departamento de Biologia Marinha, Universidade Federal Fluminense, Outeiro S~ao Jo~ao Batista,
s/no., Niter
oi, Rio de Janeiro, Brazil

Thalita Mendes Crespo, Leonardo Tavares Salgado


Diretoria de Pesquisas, Instituto de Pesquisas Jardim Bot^anico do Rio de Janeiro, Rua Pacheco Le~ao, 915, Rio de Janeiro, Brazil

Leonardo Rodrigues de Andrade


Centro de Ci^encias da Sa
ude, Instituto de Ci^
encias Biom
edicas, Departamento de Histologia e Embriologia, Universidade Federal
do Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, 373, bloco: B, sala F2-27, Rio de Janeiro, Brazil

Ang
e lica Ribeiro Soares
N
ucleo de Pesquisas em Ecologia e Desenvolvimento Social de Maca e, Universidade Federal do Rio de Janeiro, Rua Rotary Club,
s/no., S~ao Jos
e do Barreto, Maca
e, Rio de Janeiro, Brazil

Claire Hellio
Universite de Bretagne Occidentale, LEMAR UMR 6539, IUEM - Technopole Brest-Iroise, Rue Dumont dUrville, Plouzan
e,
France

Ricardo Rogers Paranhos


Departamento de Biologia Marinha, Universidade Federal Fluminense, Outeiro S~ao Jo~ao Batista,
s/no., Niter
oi, Rio de Janeiro, Brazil

Lilian Jorge Hill, Geysa Marinho de Souza


Diretoria de Pesquisas, Instituto de Pesquisas Jardim Bot^anico do Rio de Janeiro, Rua Pacheco Le~ao, 915, Rio de Janeiro, Brazil

Alphonse Germaine Albert Charles Kelecom


Departamento de Biologia Geral, Universidade Federal Fluminense, Outeiro S~ao Jo~ao Batista, s/no., Niter
oi, Rio de Janeiro,
Brazil

Bernardo Ant^
o nio Perez Da Gama, Renato Crespo Pereira
Departamento de Biologia Marinha, Universidade Federal Fluminense, Outeiro S~ao Jo~ao Batista,
s/no., Niter
oi, Rio de Janeiro, Brazil

and Gilberto Menezes Amado-Filho2


Diretoria de Pesquisas, Instituto de Pesquisas Jardim Bot^anico do Rio de Janeiro, Rua Pacheco Le~ao, 915, Rio de Janeiro, Brazil

This paper has identified, for the first time in a halogenated monoterpenes. P. brasiliense specimens
member of the Rhodophyta, a vacuolar organelle were submitted to a cytochemical analysis of the
containing enzymes that are involved in the activity of the 3-hydroxy-3-methylglutaryl-CoA
mevalonate pathwayan important step in red algal synthase (HMGS). Using transmission electron
isoprenoid biosynthesis. These organelles were microscopy (TEM), we confirmed the presence of
named mevalonosomes (Mev) and were found in HMGS activity within the Mev. Because HMGS is
the cortical cells (CC) of Plocamium brasiliense, a necessary for the biosynthesis of halogenated
marine macroalgae that synthesizes several monoterpenes, we isolated a hexanic fraction (HF)
rich in halogenated monoterpenes from
1
P. brasiliense that contained a pentachlorinated
Received 20 April 2014. Accepted 21 January 2015.
2
Author for correspondence: e-mail gilbertoamadofilho@
monoterpene as a major metabolite. Because
gmail.com. terpenes are often related to chemical defense, the
Editorial Responsibility: J. Raven (Associate Editor) antifouling (AF) activity of pentachlorinated

225
226 W L A D I M I R CO S T A PA R A D A S E T A L .

monoterpene was tested. We found that the and 1-deoxy-D-xylulose-5-phosphate synthase, respec-
settlement of the mussel Perna perna was reduced by tively (Hemmerlin et al. 2012, see Lohr et al. 2012
HF treatment (2.25 times less than control; 40% and for a review). Traditionally, the MVA pathway occurs
90% of fouled surface, respectively; P = 0.001; in the cytoplasm and mitochondria, and the MEP
F9,9 = 1.13). The HF (at 10 lg  mL1) also pathway is localized in chloroplasts (C); however,
inhibited three species of fouling microalgae the exact cellular localization of isoprenoid synthesis
(Chlorarachnion reptans, Cylindrotheca cloisterium, and and storage is poorly understood (Lohr et al. 2012).
Exanthemachrysis gayraliae), while at a higher Recent studies suggest that in some species of red
concentration (50 lg  mL1), it inhibited the algae, the MVA pathway has been lost (Lohr et al.
bacteria Halomonas marina, Polaribacter irgensii, 2012), but in Galdieria sulphuraria (Rhodophyta),
Pseudoalteromonas elyakovii, Shewanella putrefaciens, both the MVA and MEP pathways are present
and Vibrio aestuarianus. The AF activity of (Schwender et al. 1997). In Cyanidioschyzon merolae
P. brasiliense halogenated monoterpenes and the (Rhodophyta), a close relative of G. sulphuraria, the
localization of HMGS activity inside Mev suggest only terpenoid synthesis gene identified was related
that this cellular structure found in CC may play a to HMGS, whereas all of the genes encoding
role in thallus protection against biofouling. enzymes for the subsequent reactions in the MVA
pathway are absent (Matsuzaki et al. 2004). How-
Key index words: 3-hydroxy-3-methylglutaryl-CoA syn-
ever, to date, only one study has provided biochemi-
thase; antifouling activity; chemical defense; cortical
cal data supporting the presence of the MVA
cell cytochemistry; halogenated monoterpenes;
pathway in Florideophyceae (Barrow and Temple
mevalonate pathway; mevalonosome; osmiophilic
1985). Similarly, cellular microscopy studies of the
bodies; Plocamium brasiliense
cellular localization of secondary metabolites biosyn-
Abbreviations: AF, antifouling; CC, cortical cells; C, thetic steps have not yet been reported for marine
chloroplast; CW, cell wall; GC-MS, gas chromatogra- macroalgae.
phy mass spectrometry; HF, hexanic fraction; HMGS, The spatial distribution throughout the cell of
3-hydroxy-3-methylglutaryl-CoA synthase; LB, Luria components related to secondary metabolism has
Hinton Broth; MC, medullary cells; Mev, mevalono- been detailed for filamentous fungi and plants
some; MIC, minimum inhibition concentration; (Lendenfeld et al. 1993, Hoppert et al. 2001, Kut-
NMR, nuclear magnetic resonance; OB, osmiophilic chan 2005, Lunn 2007, Hong and Linz 2008, 2009,
bodies; OM, optical microscopy; P, pit connections; Saikia et al. 2008). For example, in Aspergillus para-
SEM, scanning electron microscopy; SER, smooth siticus, aflatoxisomes constitute a group of special-
endoplasmic reticulum; SG, floridean starch grains; ized trafficking vesicles that participate in the
TEM, transmission electron microscopy; V, vacuole biosynthesis and transport of aflatoxin to the cell
exterior (Chanda et al. 2009). In general, sub-cellu-
lar localization studies indicate that the enzymes,
substrates, intermediates, and end products of sec-
Studying the localization, storage, and exudation ondary metabolism often accumulate at different
mechanisms of biologically active secondary subcellular locations (Roze et al. 2011, see Ziegler
metabolites in macroalgae is necessary to enrich our and Facchini 2008 for a review).
understanding of the ecological significance of sur- The intracellular storage sites of secondary metab-
face-active inhibitors (Dworjanyn et al. 1999, Paul olites and the locations of their biosynthetic path-
et al. 2006, Salgado et al. 2008). Numerous studies ways and release mechanisms are highly reflective of
have focused on the activity of macroalgal secondary their ecological roles (Pereira and Da Gama 2008).
metabolites against fouling organisms (Da Gama The production of antifouling (AF) metabolites is
et al. 2008, Nylund et al. 2008, Bazes et al. 2009, Bi- frequently associated with the capability of some
anco et al. 2009, Hellio et al. 2009, Plouguerne species to store some isoprenoids derivatives or sec-
et al. 2010, Fusetani 2011, Silkina et al. 2012). How- ondary metabolites in specialized structures near
ever, little is known about the cellular mechanisms the surface cell layer (Dworjanyn et al. 1999, Paul
involved in secondary metabolite biosynthesis et al. 2006, Salgado et al. 2008). In macroalgae,
(Dworjanyn et al. 1999, Paul et al. 2006, Salgado three types of specialized structures have been
et al. 2008). described so far: gland cells (or vesicle cells), in
More than half of the reported secondary metabo- which a storage vesicle occupies almost the entire
lites from macroalgae are isoprenoid derivatives cellular space (Young and West 1979, Dworjanyn
such as terpenes, steroids, carotenoids, prenylated et al. 1999); the corps en cerise, which are refractive
quinines, and hydroquinones, which are derived cell inclusions found in some species of Laurencia
from either the classical mevalonic acid pathway (Young et al. 1980, Salgado et al. 2008); and the
(MVA) or from the 2C-methyl-D-erythritol 4-phos- physodes found in Ochrophyta (Schoenwaelder
phate (MEP) pathway (Maschek and Baker 2008). 2002, Ank et al. 2014).
The key enzymatic precursors in these pathways are With regard to the exudation mechanisms of sec-
3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) ondary metabolites, in brown algae, for example,
ME V A L O N A T E P A T H W A Y I N P . B R A S I L I E N S E C O R T I C A L C E L L S 227

the Golgi apparatus plays a role in the synthesis and (OM), transmission (TEM), and scanning electron
transport of phlorotannins to the cell wall (CW) via microscopy (SEM); (ii) to localize mevalonic acid
the exocytic pathway (Schoenwaelder 2002). In Lau- enzyme activity inside CC by using a specific cyto-
rencia dendroidea (Rhodophyta), exudation occurs by chemical assay; and (iii) to investigate the AF activity
way of a complex system involving the microfila- of P. brasiliense halogenated monoterpenes using in
ment- and microtubule-mediated vesicular transport vitro bioassays.
of halogenated metabolites from corps en cerise (stor-
age sites) to the cell periphery (Salgado et al. 2008,
MATERIALS AND METHODS
Paradas et al. 2010, Reis et al. 2013). The metabo-
lites are then exocytosed at the CW and reach the Algal sampling. Specimens of P. brasiliense were collected
algal surface, where they act as AF chemicals (Sal- by self-contained underwater breathing apparatus in the sub-
gado et al. 2008, Paradas et al. 2010). tidal zone at Forno beach in 2011 from June to October (Rio
de Janeiro State; Brazil; 22450 42.17 S; 41520 29.25 W).
In this context, the study of the genus Plocamium After collection, living algal samples were stored in filtered
has been restricted mainly to the chemical character- seawater inside a dark isothermic chamber and transported
ization of the products of secondary metabolism and to the laboratory.
their activity on other organisms (Kladi et al. 2004). Optical microscopy. Morphological characterization of living
These algae produce cyclic and acyclic halogenated CC of P. brasiliense was performed using the optical micro-
monoterpenes (Diaz-Marrero et al. 2002), which are scope Olympus BX 50 (Olympus, Tokyo, Japan) equipped
known to decrease the activities of herbivores, foul- with a 1009 N.A. 1.3 objective lens using oblique illumina-
tion and connected to a CoolSnap-Pro Color RS Photometrics
ing organisms, fungi, and competitors (Kladi et al.
camera (Photometrics, Tucson, AZ, USA). Digital images
2004). Monoterpenes isolated from Plocamium costa- were analyzed using ImageJ software (Abramoff et al. 2004)
tum displayed anti-settling activity against cyprid lar- to describe the morphological aspects of the organelles, such
vae of the barnacle Balanus amphitrite at 1 lg  cm2, as area, circularity, and diameter (n = 60).
where the unit lg  cm2 indicates the amount of Transmission electron microscopy. Specimens were fixed in a
material (lg) used to coat one square centimeter solution of 4% formaldehyde and 5% glutaraldehyde (Sigma-
(cm2) of the petri dish internal glass used in B. am- Aldrich Company, Saint Louis, MO, USA) in 0.1 M cacodylate
buffer pH 7.3 diluted in sterile seawater. Post-fixation was
phitrite assays (K
onig et al. 1999a).
performed in OsO4 1% (Sigma-Aldrich Company) for 2 h at
Thus, although many studies have highlighted the room temperature. Algal samples were then dehydrated in a
ecological properties of Plocamium compounds crescent acetone series (to 100%) and embedded in Spurr
(Konig et al. 1999a,b, Pereira et al. 2002, Kladi et al. resin (Sigma-Aldrich Company) at room temperature. The
2004), the cellular structures involved in isoprenoid polymerization process was performed at 70C. Ultrathin sec-
derivative biosynthesis and storage in Plocamium cor- tions (50 nm) were obtained by using an ultra-microtome Le-
tical cells (CC) have not been studied. Moro et al. ica EM UC7 (Leica Microsystems Company, Wetzlar, Hessen,
Germany), collected on copper grids (300 mesh; Electron
(2003) described the ultrastructure of Plocamium Microscopy Sciences Company, Hatfield, PA, USA) and
cartilagineum but did not identify any specific organ- observed on a JEOL 1010 EX (Jeol Company, Tokyo, Japan)
elles related to isoprenoid biosynthesis or storage. TEM microscope operated at 80 kV.
However, published data (Barrow and Temple Localization of mevalonic acid enzyme activity. Specimens of
1985) guide the present work to investigate the loca- P. brasiliense were exposed to enzymatic bioassays in which
tion of the mevalonate pathway and the possible algal fragments were treated with prospected substrates for
existence of cellular structures involved in the syn- the enzymes acetyl-CoA and acetoacetyl-CoA (from Sigma-
Aldrich Company). This treatment is based on prior knowl-
thesis and storage of the substances involved. Radio- edge of a mevalonate-dependent pathway, in which the
active [3H, 14C] mevalonate precursors have been HMGS transforms acetyl-CoA and acetoacetyl-CoA into
successfully incorporated into halogenated monot- 3-hydroxy-3-methylglutaryl-CoA. Another enzymatic product
erpenes in Plocamium cartilagineum (Rhodophyta), of this reaction is free Coenzyme A-SH (CoA-SH). The first
thus, confirming the occurrence of this metabolic step of the cytochemical reaction occurs when potassium fer-
pathway in the Plocamium genus and its role in ricyanide (Sigma-Aldrich Company) is combined with the
incubation solution and reacts with CoA-SH, reducing it to
monoterpene synthesis (Barrow and Temple 1985). ferrocyanide. The ferrocyanide then reacts with added uranyl
The species chosen for the present investigation acetate (Sigma-Aldrich Company) to form uranyl ferrocya-
was Plocamium brasiliense ([Greville] M.A.Howe & nide, which precipitates and appears as a highly particulate
W.R.Taylor [Rhodophyta]). Although studies of the electron-dense material in TEM.
chemical ecology of P. brasiliense are scarce, it was According to Croteau et al. (2000), the enzymology of iso-
demonstrated that P. brasiliense monoterpenes have pentenyl pyrophosphate (IPP) biosynthesisthe precursor of
high anti-herbivore activity (Pereira et al. 2002, all terpenoids compoundsby the acetate/mevalonate path-
way is widely accepted. This cytosolic IPP pathway involves
Pereira and Vasconcelos 2014). In an applied con- the two-step condensation of three acetyl-CoA molecules cata-
text, these molecules showed activity as herbicides lyzed by thiolase (TH) and HGMS (Croteau et al. 2000). The
(Fonseca et al. 2012) and against both herpes virus resulting product, 3-hydroxy-3-methylglutaryl-CoA, is subse-
(Ferreira et al. 2010, Pinto et al. 2014) and snake quently reduced by HMGR in two coupled reactions to form
venom (Claudino et al. 2014). Thus, the aims of mevalonic acid (Croteau et al. 2000). The TH, HMGS, and
the present work were: (i) to describe the HMGR reactions are some of the many chemical reactions in
ultrastructure of P. brasiliense CC using optical which free CoA-SH is released (Croteau et al. 2000). For
228 W L A D I M I R CO S T A PA R A D A S E T A L .

example, carnitine acetyltransferase (ACT) also releases CoA- ethanol/water solutions (30%, 50%, 70%, and 100%). Algal
SH. The discussed reactions are described below: samples were dried using the critical point dryer LEICA CPD
(Leica Microsystems Company), and the dried pieces were
TH: Acetyl-CoA + Acetyl-CoA ? Acetoacetyl-CoA (+ CoA- mounted on aluminum sample holders for SEM using dou-
SH) ble-sided carbon tape (SPI Supplies Company, West Chester,
HMGS: Acetyl-CoA + Acetoacetyl-CoA ? PA, USA). The thalli of P. brasiliense were fractured, and dou-
3-hydroxy-3-methylglutaryl-CoA (+ CoA-SH) ble-sided adhesive was used to remove tissue fragments (in
HMGR: 3-hydroxy-3-methylglutaryl-CoA ? Mevalonic acid the following order: stub/carbon tape/alga/adhesive tape).
(+ CoA-SH) The tissue fracture resulted in cellular fracture, thus exposing
ACT: Carnitine + Acetyl-CoA ? Acetylcarnitine (+ CoA- the intracellular contents of the fractured cells. The samples
SH) were then coated with a thin gold layer (~20 nm) with a
In this way, the localization of acetyl-CoA, acetyl-CoA C- Sputter coater BalTec SCD 050 (Baltec Company, Manches-
transferase (TH), and 3-hydroxy-3-methylglutaryl-CoA reduc- ter, NH, USA).
tase (HGMR) are indistinguishable from that of HGMS Extraction, fractionation, and chemical analyses. Crude extract
because both reactions produce CoA-SH, which precipitates from P. brasiliense (0.6% dry weight, DW) was obtained by
as uranyl ferrocyanide (Curry 1987). The HGMS reaction nec- extraction in dichloromethane (Merck & Co. Inc., Reading-
essarily labels HGMR. The highest level of precipitation is ton Township, NJ, USA) for 15 d (the solvent was exchanged
expected to be associated with HGMS and not TH because a three times) following previously described procedures for
high quantity of enzymatic substrate was presented to HGMS natural product extraction (Da Gama et al. 2002). To obtain
(Curry 1987). a chemical profile of the compounds in the extract, it was ini-
In these experiments, specimens of P. brasiliense were fixed tially analyzed with 1H nuclear magnetic resonance (NMR;
for 30 min in 4% formaldehyde and 1% glutaraldehyde in 300 MHz, CDCl3) and gas chromatography mass spectrometry
0.05 M sodium cacodylate buffer (pH 7.0) diluted in sterile (GC-MS). After that, the crude extract (97.3 g) was subjected
seawater, followed by a buffer rinse (0.05 M cacodylate, pH to a preparative chromatography fractionation to isolate the
7.0). Subsequently, the algal tissue was pre-incubated at ambi- most abundant terpenes. Then, 10 g of silica gel (Merck
ent temperature for 20 min in 3 mM potassium ferricyanide 60 F254 0.5 mm; Merck & Co. Inc.) was placed inside a glass
in 0.05 M cacodylate buffer (pH 7.0) followed by a buffer filter holder (Merck & Co. Inc.) attached to a Kitasato glass
rinse (Curry 1987). Then, algae were incubated for 45 min at (1,000 mL). Afterward, the crude extract was carefully
ambient temperature to allow enzyme catalysis and precipita- inserted into the silica gel and then filtered under a vacuum
tion to occur (Curry 1987). Complete media including aceto- with 25 mL of hexane (Merck & Co. Inc.) four times. The fil-
acetyl-CoA or carnitine were used along with several controls tered and hexanic fractions (HF) were collected separately
(Table 1). for solvent elimination under reduced pressure in a rotary
evaporator at room temperature. The HF was analyzed by 1D
Specimens of P. brasiliense were then post-fixed for 1 h at and 2D NMR techniques (at 300 MHz to 1H and 75 MHz to
ambient temperature in 2% (w/v) osmium tetroxide in 13
C in CDCl3) and by GC-MS. All the GC-MS analyses were
0.05 M sodium cacodylate buffer (pH 7.0) followed by a buf- performed using a QP2010 plus series instrument (Shimadzu
fer rinse. Then, the algal tissue was dehydrated in a crescent Corporation, Kyoto, Japan). The GC-MS was equipped with a
acetone series (to 100%) and embedded in Spurr resin at polysiloxane capillary column (RTX-1MS; 30 m 9 0.25 mm
room temperature. The polymerization process was per- i.d. 9 film thickness 0.25 lm; Restek Corporation, Bellefonte,
formed at 70C. Thin sections (200 nm) were obtained on an PA, USA). The temperature was programmed to hold at
ultra-microtome Leica EM UC7 and collected on single slot 100C for 2 min and then increase from 8C  min1 to
copper grids (Electron Microscopy Sciences Company) 310C, where it was maintained for 1 min. Helium was used
coated with a Formvar film (~20 nm). The grids were as a carrier gas at a flow rate of 1.2 mL  min1. The injector
observed in a FEI TECNAI G20 (FEI Company, Hillsboro, and interface temperatures were 260C and 320C, respec-
OR, USA) TEM with an accelerated voltage of 200 kV. tively. Electron impact spectra were recorded at 70 eV with
Scanning electron microscopy. To provide three-dimensional scan time of 1 s. The compounds were identified by compari-
information on organelle distribution, SEM was performed son of their mass spectra with those provided in a mass spec-
using a Zeiss EVO 40 (Zeiss Company, Oberkochen, BW, Ger- tral database (NIST 2008) and in the literature (Mynderse
many) with an accelerated voltage of 15 kV. Fragments of and Faulkner 1975, Afolayan et al. 2009). The GC-MS solu-
P. brasiliense were fixed in 4% glutaraldehyde and 5% formal- tion version 2.53 software (Shimadzu Corporation) was used
dehyde in 0.1 M pH 7.4 sodium cacodylate buffer diluted in for data processing.
seawater. Thereafter, samples were washed with buffered ster- Antifouling assays. Mussel assay: The HF from P. brasiliense
ile seawater (0.05 M sodium cacodylate, pH 7.4) and post- was used in assays of the response of the fouling mussel Perna
fixed in 1% OsO4 for 1 h at 20C. After washing in buffered perna. AF activity was measured by following the method
sterile seawater, the samples were dehydrated in a series of described by Da Gama et al. (2003) with some modification.

TABLE 1. Enzymatic substrate mediums used to treat to Plocamium brasiliense tissue. (A) Complete acetoacetyl-CoA, (B) com-
plete carnitine, (C) substrate control for acetoacetyl-CoA, (D) substrate control for carnitine, (E) primary reaction product
control, and (F) final reaction product control (Curry 1987).

Chemical substrates and reagents A B C D E F

2.0 mg  mL1 potassium ferricyanide X X X X X X


1.0 mg  mL1 uranyl acetate X X X X X X
0.8 mg  mL1 acetyl-CoA sodium salt X X X
1.6 mg  mL1 acetoacetyl-CoA sodium salt X X
1.6 mg  mL1 DL carnitine X X
0.05 M sodium cacodylate buffer (pH 7.0) X X X X X X
ME V A L O N A T E P A T H W A Y I N P . B R A S I L I E N S E C O R T I C A L C E L L S 229

Juvenile specimens of P. perna collected during low tide from


the rocky coastal area of Itaipu beach (Niter oi City, Rio de
Janeiro State, Brazil; 23000 34 S; 44260 10 W) were accli-
mated in a 2,301 recirculating laboratory aquarium
(equipped with biological filtering, protein skimming and
activated carbon) at a constant temperature (20C), salinity
(35) and aeration for 12 h. Treatment filters were soaked in
the P. brasiliense HF fraction at physiological concentration
(0.24%, DW). Next, all filter paper circles were allowed to air
dry. Filter circles were then placed in the bottom of sterile
polystyrene petri dishes (9 cm diameter 9 1.0 cm height) FIG. 1. Image of a longitudinal section of the living thallus
and completely filled with sterile seawater (~80 mL), and from Plocamium brasiliense obtained by optical microscopy (a, b).
three mussel specimens (1.52.5 cm length) were added to The large spherical organelles (arrowheads) were observed in all
each petri dish (n = 10). cortical cells; bar = 10 lm.
Antibacterial assay: The P. brasiliense HF was tested for
inhibitory activity against growth of five strains of biofilm-
forming marine bacteria (Bressy et al. 2014) obtained from TABLE 2. Morphometry of the cortical and medullary cells
the collection of the University of Portsmouth (School of and spherical organelles of Plocamium brasiliense. Mean
Biological Sciences): Halomonas marina (ATCC 25374), Polarib- and SD in lm (n = 60).
acter irgensii (ATCC 700398), Pseudoalteromonas elyakovii (ATCC
700519), Shewanella putrefaciens (ATCC 8071), and Vibrio aestu- Cell diameter Spherical organelles
arianus (ATCC 35048). Each treatment condition and control
(culture media) was replicated six times. The bacteria Cortical Medullary Area Diameter
(lm2)
(2 9 108 cells  mL1) were incubated in 96 well-plates with (lm) (lm) (lm) Circularity
the HFs at concentrations of 0.1, 10, 50, 125, 250, and Mean 22.03 99.25 24.12 10.00 0.92
500 lg  mL1 in Luria Hinton Broth (LB) medium (Sigma- SD 4.7 14.25 1.30 2.00 0.02
Aldrich Company) supplemented with NaCl (35 g  L1) at
30C for 72 h (Mar echal et al. 2004). Minimum inhibitory
concentrations (MICs) compared with the control were deter- these refractive organelles were circular (Fig. 1, a
mined using the microtiter broth dilution method (Amster-
dam 1996, Plouguern e et al. 2008, 2010). and b; Table 2).
Antimicroalgal assay: The P. brasiliense HF was tested for Transmission electron microscopy. Images obtained
inhibitory activity against five strains of marine microalgae by TEM show the presence of a thin biofilm mainly
obtained from Algobank-Caen (Universit e de Caen Basse-Nor- composed of bacteria and microalgae on the P. bra-
mandie, France): Chlorarachnion reptans (AC132), Cylindrotheca siliense surface (Fig. 2a). In P. brasiliense CCs, the fol-
cloisterium (AC170), Exanthemachrysis gayraliae (AC15), Navicula lowing cellular structures were observed: the large
jeffreyi (AC181), and Chlorarachnion globosum (ATCC 8071). All spherical organelles (Mev), C with electron-dense
microalgal cultures and assays were performed under con-
trolled conditions (23C  2C under 54 lmol pho-
inclusions (350 nm  50, n = 60), inclusions of
tons  m2  s1 from a cool-white fluorescent lamp). F/2 osmiophilic bodies (OB), pit connections (P), flori-
medium (Guillard and Ryther 1962) was used for cultivation. dean starch grains (SG), smooth endoplasmic retic-
Microalgae for AF assays were cultivated as outlined by ulum (SER), and small vacuoles (V; Fig. 2b).
Tsoukatou et al. (2002). All experiments were carried out in Osmiophilic vesicles were observed in association
six replicates. Briefly, 100 lL of a culture at 0.4 lg  mL1 of with Mev (Fig. 2c), and a large number of OB were
chl a was introduced into 96-well plates containing the frac-
tions at concentrations of 0.1, 1, 10, 50, and 100 lg  mL1
observed forming clusters in many regions of the
(Plouguerne et al. 2010). After 48 h, MICs were determined cytoplasm and near the CW (Fig. 2, b and e).
by comparison of the cell growth between treatments and Localization of mevalonic acid enzyme activity. The
controls (Tsoukatou et al. 2002). TEM images of P. brasiliense CC treated with com-
Statistical analyses. A t-test for independent samples was plete acetoacetyl-CoA medium (A) indicated the
performed to compare mussel byssal thread attachment data presence of granular, electron-dense material within
between filters in HF treatment and control conditions using Mev (Fig. 3a), CCs in control mediums (C and E)
the Statistica 8.0 software (Statsoft Inc., Tulsa, OK, USA).
When normality or variance homogeneity assumptions were
did not present granular electron-dense material
not met, data were transformed using the square root of within Mev (Fig. 3b). In algae submitted to the com-
X + 1 prior to t-test. Differences were considered significant plete carnitine medium (B), the TEM images
whenever P < 0.05 (a = 5%). showed the presence of electron-dense vesicles sur-
rounding the Mev (Fig. 3, c and d) and electron-
dense clusters near C (Fig. 3e). In carnitine substrate
RESULTS
control medium (D), no electron-dense material was
Optical microscopy. A large spherical and refractive observed (Fig. 3f). No electron-dense material was
organelle not previously described, here termed the observed in TEM images of algae treated with the
mevalonosome (Mev), was observed in the CC (1 control mediums C and F (data not showed).
per CC, n = 60) of P. brasiliense (Fig. 1, a and b). Scanning electron microscopy. The SEM images of
These organelles had a diameter, circularity, and P. brasiliense cells revealed the thallus surface and
area of 10  2, 0.92  0.027, and 24.12  1.30 lm, the intracellular constituents of fractured CC and
respectively. Several contour measures revealed that medullar cells (MC; Fig. 4a). The Mev was observed
230 W L A D I M I R CO S T A PA R A D A S E T A L .

tern for halogenated compounds observed at mass


spectra of major component compounds corrobo-
rated the presence of halogenated terpenes in
P. brasiliense crude extract. In light of the GC-MS
data, the crude extract was fractionated and the
chemical structure of the major compound com-
prising HF was suggested by NMR (1H, APT experi-
ments, HSQC and HMBC) and MS analysis. Based
on the comparison of our data with the literature
(Mynderse and Faulkner 1975, Antunes et al. 2011)
the compound was identified as pentachlorinated
monoterpene (Fig. 5), previously reported in differ-
ent species of the Plocamium genus (Mynderse and
Faulkner 1975, Afolayan et al. 2009, Antunes et al.
2011).
Characterization of the pentachlorinated monoter-
pene: 1H NMR (400 MHz, CDCl3) d 6.97 (3H, s, H-
9), 6.38 (1H, dd, 12.6, 3.6, H-5), 6.33 (1H, d, 12.6,
H-6), 6.29 (1H, s, H-8), 6.08 (1H, dd, 12.0, 9.0, H-
2), 5.45 (1H, d, 12.0, H-1a), 5.30 (1H, d, 9.0, H-1b),
4.55 (1H, d, 3.0, H-4), 1.77 (3H, s, H-10). 13C
(100 MHz, CDCl3) d 139.4(CH, C-2), 137.9 (C, C-7),
129.3 (CH, C-6), 126.4 (CH, C-5), 118.6 (CH, C-8),
115.5 (CH2, C-1), 70.8 (C, C-3), 67.9 (CH, C-4),
64.6 (CH, C-9), 24.1 (CH3, C-10). EIMS (70 eV) m/
z (rel. int.): 306/308/310/312/314M+ (<1/<1/<1/
<1/<1), 271/273/275 [M Cl]+ (<1/<1/<1), 235/
237 [M Cl2]+, 217/219/221/223 [M C4H6Cl]+,
89/91 [M C6H5Cl4]+ base peak.
Antifouling assays. In the P. perna assay, greater
byssus attachment was found to control filters than
to P. brasiliense HF treatment filters (F9,9 = 1.13,
FIG. 2. Transmission electron microscopy (TEM) images of P = 0.001; Fig. 6). P. brasiliense HF inhibited three
Plocamium brasiliense cortical cells. (a) Bacteria and microalgae
present in a microfouling community on the alga surface. Aster-
species of fouling microalgae (C. reptans, Cylindrot-
isks: bacteria; Arrowheads: microalgae; CW: cell walls; bar = 1 lm. heca closterium and E. gayraliae) with MIC values of
(b) Common cellular structures found in cortical cells: chlorop- 10 lg  mL1 (Table 2), while MIC values of
lasts (C), large spherical organelle (black asterisk), starch grains 50 lg  mL1 were recorded toward five species of
(GA), osmiophilic bodies (OB), pit connection (P), cell wall fouling bacteria (Halomonas marina, Polaribacter irg-
(CW), smooth endoplasmic reticulum (SER), and vacuoles (V).
White asterisks show electron-dense inclusions; bar = 1 lm. (c) ensii, Pseudoalteromonas elyakovii, Shewanella putrefac-
Osmiophilic bodies are transported from spherical organelles to iens, and Vibrio aestuarianus) and two microalgae
the cytoplasm, where they form clusters among the organelles. (Navicula jeffreyi and Chlorarachnion globosum;
Arrowheads indicate vesicle transport from large spherical organ- Table 3).
elles to the cytoplasm; bar = 1 lm. (d) SER and associated vesi-
cles being released to the cytoplasm. Arrowheads are pointing to
vesicles coming from the SER; bar = 50 nm. (e) Osmiophilic DISCUSSION
bodies accumulating in distinct regions of the cytoplasm, includ-
ing near the cell wall. Arrowheads are pointing to OB clusters in This work identified novel large spherical organ-
the cytoplasm; bar = 1 lm. elles in the CC of P. brasiliense by OM, TEM and
SEM techniques. The organelle is rounded, similar
inside CC (Fig. 4b) and was always surrounded by to other organelles described in the CC of chemi-
vesicles (Fig. 4b). More cellular contents were cally defended macroalgae, such the corps en cerise
observed in CCs than in MCs (Fig. 4,b and c). and glandular vesicles from the Rhodophyta Lauren-
Chemical analyses. The analysis of the 1H NMR cia dendroidea and Asparagopsis armata, respectively
spectra from P. brasiliense crude extract shows (Young et al. 1980, Dworjanyn et al. 1999, Scho-
groups of signals characteristic of halogenated terp- enwaelder 2002, Paul et al. 2006, Salgado et al.
enes between d 7.005.20 ppm, d 4.604.50 ppm, 2008). Morphologically, the organelles possess simi-
and d 1.901.70 (ranges related to the olefin lar luminal content (refractive in OM and electron-
region, to methine groups bonded to halogen dense in TEM), shape (spherical), and size (much
atoms and to methyl groups bonded to quaternary bigger than typical cell vesicles). Due to their sug-
carbons, respectively). The crude extracts were then gested role in secondary metabolite synthesis and/
analyzed by GC-MS. The clear fragmentation pat- or storage, these novel organelles can be considered
ME V A L O N A T E P A T H W A Y I N P . B R A S I L I E N S E C O R T I C A L C E L L S 231

FIG. 3. TEM images of Plocamium brasiliense cortical cells submitted to cytochemical mevalonic acid enzymes assay. (a) The complete
acetoacetyl-CoA medium; images showing typical cellular structures such as nuclei (Nu), starch grains (SG), cell wall (CW), chloroplasts
(C), and the Mev. Arrowheads are pointing to electron-dense granular material produced by HGMS/HGMR activity inside the Mev; bar:
2 lm. (b) The primary reaction product control medium (e); image showing the lack of precipitation inside Mev. (V) vacuole;
bar = 2 lm. (ce) The complete carnitine medium (b); images of electron-dense material in vesicles associated with Mev and in outer
region of chloroplast membranes; arrowheads are pointing to electron-dense vesicles; arrows are pointing to outer region chloroplast elec-
tron-dense deposits; bars: (c) 2 lm; (d) 450 nm; (e) 1 lm. The substrate control medium for carnitine (d); image showing the lack of
electron-dense precipitation; bar: 2 lm.

vacuoles that are specific for secondary metabolite within the Mev. This labeling pattern was not
storage (Wink 1993). observed in control enzymatic assays (C, E, and F).
In this study, conventional TEM images of P. bra- From these data, we suggest that the granular elec-
siliense presented the first description of secondary tron-dense deposits inside the large spherical orga-
metabolite distribution in CC. The TEM images nelle are the product of active enzymes acting in
showed that electron-dense material was found in the mevalonate pathway, the HMGS and/or HGMR.
large spherical organelles, in vesicles associated with We therefore propose to name this organelle the
Mev or with the CWs, and in inclusions among Mev, as the subcellular center of production of
organelles. Previous work has demonstrated that mevalonic acida fundamental isoprenoid precur-
OsO4 also binds to unsaturated sesquiterpenes in sor for monoterpene biosynthesis in P. brasiliense
the leaves of Sauromatum guttatum and other plant CC.
species (Skubatz and Kunkel 1999, Grotewold There is evidence that the mevalonate pathway,
2004). Otherwise, the OsO4 used to fix the material or some of its component steps, may be compart-
during preparation for TEM reacts with unsaturated mentalized in vesicles or vacuoles (Nagegowda
fatty acids, thereby producing an osmiolabelled 2010). Maurey et al. (1986) determined that HGMR
material (Korn 1967, Bozzola and Russel 1998). activity exists in the microssomal fraction of the alga
However, because the TEM fixation reaction Ochromonas malhamensis (Chrysophyta). In addition,
(OsO4) is non-specific and affects both monoterp- vesicles enriched with HMGR were found in Arabid-
enes and unsaturated fatty acids, it could not deter- opsis cells, possibly representing the basis of isopren-
mine the exact intracellular localization of oid biosynthesis in this species (Enjuto et al. 1994).
P. brasiliense monoterpenes. For this reason, cyto- Studies using microscopy and cell fractionation
chemical detection of terpene precursors was car- techniques have revealed that HMGR1S is localized
ried out. to endoplasmic reticulum, inside spherical struc-
We demonstrated a positive HGMS cytochemical tures and within vacuoles (Leivar et al. 2005). In
reaction, which, in TEM images of P. brasiliense CC, addition, in vitro localization studies in Brassica jun-
appeared as a granular electron-dense precipitation cea using GFP-fusion constructs have indicated that
232 W L A D I M I R CO S T A PA R A D A S E T A L .

FIG. 6. Mean number of byssal threads (%) attached to sub-


strata in different treatments: control (CTRL) and HF. Asterisks
indicates significant results (t-test for independent samples)
(F9,9 = 1.13, P = 0.001 t-test for independent samples); error bars
indicate SD.

1987). Similarly, our data suggest that the MVA


FIG. 4. SEM images of Plocamium brasiliense cortical and medul-
lar cells. (a) Image of the outer portion of the cell wall (CW), pathway is constantly supplied with acetyl-CoA from
the cortical cells (CC) and the medullar cells (MC); bar = 40 lm. cytosol regions in P. brasiliense CC.
(b) CCs possess a large quantity of cytoplasmic content; note the The presence of a carnitine ACT reaction product
large number of vesicles associated with the Mev. Arrowheads in vesicles associated with Mev suggests that acetyl-
are pointing to vesicles; bar = 10 lm. (c) MCs exhibited few cel-
lular contents; arrowheads are pointing to cellular struc-
CoA substrates are being transported to sites near
tures = 10 lm. Mev, where they are most likely being internalized
by Mev. In general, acetyl-CoA could be used as a
carbon supply for the synthesis of fatty acids, amino
acids, sterols, and other isoprenoid derivatives (e.g.,
monoterpenes). According to Somerville et al.
(2000), acetyl-CoA may be the most central interme-
diate in cellular metabolism, providing a link
between many disparate pathways. The major path-
ways involved in its production are glycolysis and
fatty acid oxidation (Somerville et al. 2000). During
respiration, acetyl-CoA is the source of carbon input
to the citric acid cycle in mitochondria (Somerville
et al. 2000).
In P. cartilagineum, feeding experiments using 3H
FIG. 5. Chemical structure of the pentachlorinated monoter-
pene in Plocamium brasiliense HF as determined by NMR and GC-
geranylgeranyl (an intermediate precursor of the
MS analyzes. MVA) showed successful incorporation into haloge-
nated monoterpenes (Barrow and Temple 1985). In
HMGS (BjHMGS1) is located in the cytosol (Nage- our work, spectroscopic data indicated the large
gowda et al. 2005), where its biosynthesis could be presence of a pentachlorinated monoterpene in the
induced by wounding or other stress responses HF isolated from P. brasiliense crude extract that was
(Alex et al. 2000). similar to the monoterpenes found in P. cartilagieum.
In this work, P. brasiliense CC that were submitted Thus, based on the results from these two Plocamium
to the complete carnitine ACT medium presented species, we suggest that the Mev is an organelle spe-
precipitates in vesicles associated with Mev and near cialized for terpene synthesis in these algae. This
C. Curry (1987) showed that carnitine ACT and novel characteristic may be essential for the
mevalonic acid enzymatic products were labeled improvement of macroalgal fitness because the stor-
together during anthesis in the floral scent glands age of secondary metabolites within vacuoles in CC
of Stenhopea anfracta (Angiosperms) when the mon- is known to increase the AF capacity of basibionts
oterpenes myrcene (37%) and ipsdienol (40.3%) for defense against epibionts (Da Gama et al. 2014,
are released to attract pollinators. The authors have see review).
suggested that the biochemical pathways involving In higher plants, many lines of evidence suggest
acetate and mevalonic acid could be related (Curry that the storage of secondary metabolites inside
ME V A L O N A T E P A T H W A Y I N P . B R A S I L I E N S E C O R T I C A L C E L L S 233

TABLE 3. Effect of Plocamium brasiliense HF, tested at the MIC, on sensitive strains of marine fouling bacteria and microalgae.
MICs are expressed in lg  mL1.

Antibacterial activity (MIC)


Halomonas marina Polaribacter irgensii Pseudoalteromonas elyakovii Shewanella putrefaciens Vibrio aestuarianus
>50 >50 >50 >50 >50
Antimicroalgal activity (MIC)
Chlorarachnion reptans Cylindrotheca closterium Exanthemachrysis gayraliae Navicula jeffreyi Chlorarachnion globosum
10 10 10 >50 >50
HF, hexanic fraction; MIC, minimum inhibitory concentration.

vacuoles increases the metabolite concentration inside the Mev, we suggest that the Mev could be
(Wink 1993, Baldwin 2010). For example, in the latex involved in the biosynthesis of halogenated monot-
vacuoles of Chelidonium majus, the concentrations of erpenes (e.g., pentachlorinated monoterpene) in
sanguinarine, berberine and chelidonine rose to P. brasiliense and also in the protection of the thallus
5001,000 mM, whereas the latex vacuoles of Papaver against macro- and microfouling.
somniferum can sequester up to 500 mM morphine When comparisons were made among MIC values
(Hauser and Wink 1993, Wink 1993). Because many from the present work and those from others stud-
of these compounds are toxic and dangerous to the ies (using the same strains of bacteria and microal-
plants producing them these vacuoles have been gae), the present data had lower MIC values than
referred to as toxic compartments (Matile 1984). those found for different populations of Sargassum
Currently, it is assumed that plants evolved an vulgare (20500 lg  mL1; Plouguerne et al. 2010).
immune defense system, where the production of According to Hellio et al. (2001), a high AF activity
secondary metabolites constitutes one of many was observed in fractions from Rhodophyceae such
weapons (e.g., physical barriers) commonly used as Chondrus crispus, Polysiphonia lanosa, and Laurencia
against natural enemies (Wink 1993, Baldwin 2010). pinnatifida, with MIC inhibition values ranging from
In foliose macroalgae, as well as in higher plants, 24 to 48 lg  mL1. In this way, the present data
much evidence shows that the ability to store toxic corroborate recent studies that have shown that
metabolites near the thallus surface in storage vacu- macroalgae species containing vacuoles to store sec-
oles increases the capacity of some species to defend ondary metabolites presented high AF activity
against fouling organisms, herbivores and competi- (Dworjanyn et al. 1999, Paul et al. 2006, Salgado
tors (Da Gama et al. 2014). et al. 2008, Sudatti et al. 2008, Paradas et al. 2010).
For example, the red macroalgae L. dendroidea In conclusion, this paper describes a novel P. bra-
stores halogenated metabolites inside storage vacu- siliense CC organelle containing enzymes from MVA
oles named corps en cerise (CC) in CC (Salgado et al. pathway. We suggest this organelle as a possible stor-
2008). Recently, we showed that the transportation age vacuole and we named it the Mev. In addition,
of halogenated vesicles from CCs to the thallus sur- the high AF activity of P. brasiliense halogenated
face could decrease the density of the bacterial bio- monoterpenes indicates how this seaweed system is
film (Paradas et al. 2010). In addition, when able to defend itself against a wide array of marine
bromine was depleted from the culture media, CCs fouling organisms.
presented an altered morphology and the bacterial
density at the thallus surface was higher, indicating The authors are grateful to Coordenac~ao de Aperfeicoamen-
that decreased production of halogenated metabo- to de Pessoal de Nvel Superior (CAPES) and Conselho Nac-
lites negatively affected the algal defense system ional de Desenvolvimento Cientfico e Tecnol ogico (CNPq)
for financial support. R.C.P., G.M.A.F., L.T.S., B.A.P.G., and
against fouling bacteria (Paradas et al. 2010). A.R.S. thank CNPq for research productivity fellowships. This
In the present work, settlement of the mussel study is part of the PhD thesis of W.C.P. The authors thank
P. perna was reduced by HF treatment (2.25 times Dr. Marcos Farina (ICB, UFRJ), Dr. Wanderley de Souza
less than the control; 40% and 90% fouled HF-trea- (IBCCF, UFRJ), and Dr. Bechara Kachar (NIDCD, NIH,
ted and control surfaces, respectively), and the HF Bethesda, MD) for the use of the microscopes. The authors
samples exhibited MIC values of 10 lg  mL1 also thank Msc. Thiago Luiz de Barros Moreira (IBCCF,
UFRJ) for obtaining TEM images, Dra Luzineide Tinoco
toward the inhibition of N. jeffreyi and C. globosum, (LAMAR-NPPN/UFRJ) for NMR analyses and Dr. Benoit
while MIC values of 50 lg  mL1 facilitated the Veron (Algobank, Caen, France) for providing the strains of
inhibition of other microalgae (C. reptans, C. cloisteri- microalgae.
um, and E. gayraliae) and bacterial strains
(H. marina, P. irgensii, P. elyakovii, S. putrefaciens, and Abramoff, M. D., Magalh~aes, P. J. & Ram, S. J. 2004. Image pro-
V. aestuarianus). Thus, because it is already know cessing with imageJ. Biophot. Int. 11:3642.
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J. J. & Beukes, D. R. 2009. Antiplasmodial halogenated mon-
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siliense the mevalonate enzymes were observed tochemistry 70:597600.
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