DOI: 10.1002/cbic.
200500343
Chloroplastic Glycolipids Fuel Aldehyde
Biosynthesis in the Marine Diatom Thalassiosira
rotula
Adele Cutignano,[a] Giuliana d’Ippolito,[a] Giovanna Romano,[b] Nadia Lamari,[a]
Guido Cimino,[a] Ferdinando Febbraio,[c] Roberto Nucci,[c] and
Angelo Fontana*[a]
Enzymatic preparations and specialized analytical tools have
shown that chloroplast-derived glycolipids are the main sub-
strates for the biosynthetic pathway that produces antiprolifera-
tive polyunsaturated aldehydes in broken cells of the marine
diatom Thalassiosira rotula. This process, which is associated
with the formation of free fatty acids and lyso compounds from
polar lipids but not triglycerides, is largely dependent on glycoli-
pid hydrolytic activity, rather than phospholipase A2 as previously
Introduction
Production of oxygenated derivatives of lipids is a common
phenomenon in higher organisms. In plants, volatile C6 and C9
aldehydes, together with their corresponding alcohols, contrib-
ute to the characteristic flavors, the so-called “fresh green
odor”, and play an important role in wound healing and pest
resistance.[1] Recently, occurrence of volatile polyunsaturated
aldehydes (PUAs) has been also reported in several marine di-
atoms that, in analogy to plants, seem to employ these mole-
cules for defense.[2] In the absence of genetic data on lipid me-
tabolism, the only information we have on aldehyde synthesis
in this group of microalgae stems from chemical investigations
on the species Thalassiosira rotula and Skeletonema costa-
tum.[3–5] In these organisms, the production of PUAs starts
within seconds after damage, whereas the concentrations of
the aldehydes in unperturbed cells are undetectable. It has
also been shown that this biosynthesis, which involves lipoxy-
genase (LOX) and hydroperoxide lyase (HPL) enzymes, begins
with free fatty acids (FFAs),[3] but when and how these precur-
sors are made available is not well understood. FFAs are not
generally found in large amounts in healthy, intact diatom cells
and, in analogy to plants, their release from membranes has
been thought to be an important step in controlling this pro-
cess. On the basis of the analysis of site-specific transforma-
tions of fluorescent analogues of phospholipids in crude cell
preparations, a wound-activated defense mechanism triggered
by phospholipase A2 (PLA2) activity was proposed to account
for the synthesis of PUAs in T. rotula.[4] However, the presence
of such a wound-inducible PLA2 activity has not been charac-
terized in this or other diatom species. In contrast, PUA synthe-
sis in S. costatum involves a glycolipid-hydrolyzing activity and
450 @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
suggested. Preliminary characterization of lipolytic enzymes has
revealed protein bands of 40–45 kDa. Under native conditions
these proteins seem to be associated with soluble aggregates
that have an apparent molecular weight of approximately
200 kDa. The biochemical process, which is similar to that de-
scribed in the algal-bloom forming diatom Skeletonema costa-
tum, suggests a mechanism based on decompartmentalization
and mixing of preexisting enzymes and substrates.
the process relies above all on chloroplastic fatty acids, such as
6,9,12-hexadecatrienoic acid (HTrA, C16:3 w-4) and 6,9,12,15-
hexadecatetraenoic acid (HTA, C16:4 w-1), in addition to eico-
sapentaenoic acid (EPA, C20:5 w-3).[5]
Since multiple lipolytic enzymes are activated after wound-
ing in plants, discerning the role of different lipolytic reactions
is a central facet of understanding the function and regulation
of PUA synthesis in diatoms. We examined the role of lipid
hydrolysis in damaged T. rotula cells by monitoring the sub-
strate–product relationship in enzymatic fractions and by char-
acterizing the lipolytic activities. The results provide evidence
that hydrolysis of chloroplastic glycolipids plays a crucial role
in the process and multiple lipolytic activities, other than PLA2,
are activated in damaged microalgae. In addition, this study in-
dicates that synthesis of aldehydes and other oxylipins in
marine diatoms can be interpreted by a simple decompart-
[a] Dr. A. Cutignano,+ Dr. G. d’Ippolito,+ N. Lamari, Dr. G. Cimino,
Dr. A. Fontana
CNR, Istituto di Chimica Biomolecolare
Via Campi Flegrei 34, 80078 Pozzuoli, Napoli (Italy)
Fax: (+ 39) 081-867-5096
E-mail: afontana@icb.cnr.it
[b] Dr. G. Romano
Stazione Zoologica “A. Dohrn”
Villa Comunale 1, 80121 Napoli (Italy)
[c] Dr. F. Febbraio, Dr. R. Nucci
CNR, Istituto di Chimica delle Proteine (IBP)
Via Pietro Castellino 111, 80131 Napoli (Italy)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://www.chembiochem.org or from the author.
ChemBioChem 2006, 7, 450 – 456
Aldehyde Biosynthesis in Thalassiosira rotula
mentalization process that, following the disruption of the cell,
leads to mixing of substrates and enzymes.
Results
Lipid Analysis
T. rotula samples were found to contain about 13 % tryglycer-
ides (TG), 21 % phospholipids (PL), and 66 % glycolipids (GL) as
shown by three independent fractionation of lipidic extracts of
the diatom. Fatty acid composition was not dissimilar to that
described in ref. [6] which shows that EPA was the major spe-
cies and made up to 32 % in weight of the whole fatty acid
content. Qualitative composition and distribution of fatty acids
in the extractable lipids of T. rotula are reported in Figure 1.
Except for EPA, which was uniformly distributed in the major
classes, GC–MS profiling revealed strict variation between TG,
PL, and GL, with a major content of saturated and monounsa-
turated species in TGs. However, the most pronounced differ-
ence between the lipid classes was due to the characteristic
presence of C16 and C18 polyunsatutared fatty acids (PUFAs) in
GLs (Figure 1). Within this class of lipids, monogalactosyldiacyl-
glycerols (MGDG; ranging from 56.9 % to 65.7 %) were the
major component. MGDG showed an extremely high level of
polyunsaturated fatty acids, and HTrA (approximately 31 %)
and HTA (approximately 27 %) predominated. The remaining
species included EPA (C20:5 w-3, approximately 19 %) and
C18:4 w-3 (approximately 16 %). Guella’s method[7] for the anal-
ysis of acyl chain distribution at sn-1/sn-2 of MGDG revealed
Figure 1. Fatty acid analysis by using GC–MS. TG = triglycerides; PL = phospholipids; GL = glycolipids.
ChemBioChem 2006, 7, 450 – 456 @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
eight major molecular species: 20:5/16:3 (20.5  2.8), 20:5/16:4
(15.6  2.0), 18:4/16:3 (12.0  2.8), 16:3/16:3 (9.8  2.3), 18:5/
16:4 (9.7  1.3), 18:4/16:4 (8.5  1.2), 18:5/16:3 (6.2  1.3), 16:3/
16:4 (4.8  1.5). These strictly contained HTrA and HTA at sn-2
and C18 PUFAs and EPA at sn-1 (for the complete data, see
Table S1 in the Supporting Information). This positional distri-
bution appears almost entirely chloroplastic in origin, with C16
fatty acids at the sn-2 position as with cyanobacteria.[8] No
data were found in the literature about the composition of the
lipid classes in T. rotula. However, it is interesting to note that a
high level of glycolipids and a low level of PL were reported
for other marine diatoms, and HTrA and HTA were described
as characteristic components of GLs of the marine diatom
S. costatum.[5b, 9]
Lipolytic activity of cell homogenates
Except for the evidence about PLA2 activity,[4] no data are avail-
able in the literature on the lipolytic properties of marine diat-
oms. Because this aspect was crucial in our study, the degree
of hydrolysis of complex lipids in T. rotula was monitored by
quantifying the levels of free and bound fatty acids in lysed
cells. The quantification relied first on the basic hydrolysis of
diatom extracts in 99.9 % deuterated methanol (CD3OD) to
convert only lipid-bound fatty acids into [D3]FAMEs. This was
followed by neutralization of the reaction mixture and esterifi-
cation of the remaining FFA fraction with ethereal diazome-
thane to give the corresponding FAMEs. After extraction, GC–
MS analysis of the resulting material provided the ratio of es-
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 A. Fontana et al.

terified and nonesterified pools by comparison of the areas of
the peaks at M (unlabeled species) and M+3 (labeled species)
in the mass spectra. Table 1 summarizes the results with four
different samples of lysed T. rotula cells (for the complete data,
ing the hypothesis that these compounds were largely present
in a nonesterified form in the cell homogenates. Other compo-
nents, such as palmitic acid (C16:0) and myristic (C14:0) acid,
were almost equally distributed between free and bound fatty
acids (free/bound ratio 0.9  0.1 and 0.8  0.1, respectively).

Table 1. Composition of fatty acids occurring as free and bound forms in

damaged cells of T. rotula.[a] Metabolism of complex lipids by crude enzymatic
preparations
1
Fatty acid mg mg pellet Free/bound ratio[b]
To determine the ability of crude enzymatic preparations to
C14:0 2.93  0.57 0.9  0.1
C16:4 0.49  0.18 3.0  0.1
metabolize the different classes of complex lipids, diatom ex-
C16:3 1.83  0.27 3.1 1.3 tracts were processed by using a size-exclusion membrane
C16:2 0.47  0.16 1.3  0.6 with a cut-off of 10 kDa. The excluded fractions (retentates)
C16:1 5.16  0.58 1.6  0.3 containing high molecular weight molecules were incubated in
C16:0 2.18  0.17 0.8  0.1
C18:5 trace n.c.
triplicate with TG (2 mg), PL (6 mg), and GL (8 mg) purified
C18:4 0.72  0.36 3.2  0.4 from T. rotula lipids. As shown in Figure 2, the incubation of GL
C18:4 1.53  0.23 3.8  0.6 with the diatom retentates induced synthesis of 2E,4Z-octadi-
C18:2 1.29  0.41 1.6  0.4 enal (4.10  0.18 mg per 107 cells) and 2E,4Z,7Z-decatrienal
C18:1 trace n.c.
C20:5 4.82  0.37 2.6  0.5
(2.63  0.12 mg per 107 cells). Similar results were obtained in
C22:6 0.12  0.07 1.1  0.4 experiments with MGDG (1 mg) which is the major glycolipid
class of microalgae (5.06  0.14 mg per 107 cells for 2E,4Z-octa-
[a] Results are expressed as mean  standard deviation of four independ-
ent analyses. [b] Relative amount was established as described in the text dienal and 1.87  0.33 mg per 107 cells for 2E,4Z,7Z-decatrienal).
by measuring the M/(M+3) ratio in the MS spectrum of each component. Under the same conditions, PL metabolism led only to the for-
n.d. = not detectable; n.c. = not calculable. mation of decatrienal (2.72  0.21 mg per 107 cells). On the
other hand, TGs were apparently not metabolized and most of
this lipidic fraction was recovered unaltered after the experi-
see Table S2 in the Supporting Information). Using pentadeca- ment, without any detectable formation of aldehydes. In incu-
noic acid (C15:0) as internal standard, recovery and quantifica- bation experiments of MGDG with retentates, synthesis of
tion of fatty acids was performed by measuring the area of PUAs was also accompanied with release of HTrA, HTA, and
each GC peak. On the contrary, the ratio of free/bound species EPA as detected by GC–MS with crude incubation mixtures
was determined by comparing the area of M or M+3 peaks (data not shown). Since the major molecular species (sn-1/sn-2)
that were obtained by using selected-ion monitoring mode. of MGDG were 20:5n-3/16:3n-4 and 20:5n-3/16:4n-1, the con-
About 75 % of the content of HTrA, HTA, EPA, and C18:4 were comitant presence of these fatty acids indicates hydrolysis
found to be FFAs (free/bound ratio of about 3:1) thus support- from both sn-positions of MGDG. Accordingly, soon after the

Figure 2. Conversion of complex lipids by YM-10 Amicon retentates from T. rotula. GC profiles of aldehydes after incubation with: GL = glycolipids, PL= phos-
pholipids, TG = triglycerides. 4-trans-Decenal (30 mg) was used as internal standard (std) for quantification and [D]6-HTrA as internal control for the enzymatic
conversion. Aldehydes were analyzed as the corresponding carbethoxyethylidene (CET) derivatives.

452 www.chembiochem.org @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2006, 7, 450 – 456
Aldehyde Biosynthesis in Thalassiosira rotula
addition of MGDG to the retentates, SiO2-TLC revealed two
spots (Rf of 0.2–0.3 in chloroform/MeOH, 90:10, v/v) that were
due to the formation of monogalactosylmonoacylglycerols
(Figure 3 A), which rapidly decreased after a few minutes of in-
cubation (data not shown). Boiled retentates were not able to
promote either the synthesis of aldehydes or release of fatty
acids from complex lipids (Figure 3 B).
Figure 3. A) TLC analysis (chloroform/MeOH, 90:10) of MGDG with YM-10
supernatants of T. rotula. B) Inactivated preparations from T. rotula as control
sample. Lane 1: fatty acids (reference); lane 2: fatty acids (reference) and in-
cubation mixture; lane 3: incubation mixture; lane 4: MGDG (reference) and
incubation mixture; lane 5: MGDG (reference).
Fractionation and preliminary characterization of the
lipolytic activity
When the diatom extracts were processed by centrifugation at
102 000 g, lipolytic activity towards 4-methylumbelliferyl buty-
rate (MUF-butyrate) and MGDG derived from diatoms was
found to be associated with both the supernatant and pellet
(data not shown). Analysis of SDS-PAGE gels showed the pres-
ence of more than one protein band t approximately 40–
45 kDa when MUF-butyrate was used as substrate in both frac-
tions, but not with MUF-oleate (data not shown). Repetitive
cycles of suspension and ultracentrifugation allowed the con-
centration of the fraction above 45 kDa into a pellet and left
the prominent lipolytic activities (approximately 41–43 kDa) in
the supernatant (Figure 4). This material displayed significant
hydrolytic activity with MGDG (0.5 mg), sulfoquinovosyldiacyl-
glycerol (SQDG; 0.5 mg), and L-a-phosphatidyl-d,l-glycerol-di-
oleyl, but not with tristearine and natural digalactosyldiacylgly-
cerol (DGDG). Native molecular mass determination of the
lipid-hydrolyzing activity in the supernatant was carried out by
using a size-exclusion column calibrated with standard pro-
teins. Assuming a globular structure, after treatment with
MUF-butyrate (Figure 4 C) and incubation with MGDG (0.5 mg)
in phosphate buffer, the lipolytic activity appeared to have a
molecular mass of around 200 kDa. In particular, the 200 kDa
band (1 mg mL 1 of protein content) showed a hydrolytic activ-
ity that was about 60-times higher than supernatants obtained
ChemBioChem 2006, 7, 450 – 456 @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 4. SDS-PAGE of T. rotula fractions stained with A) Comassie brilliant
blue and B) MUF-butyrate for lipase activity; each lane holds 15 mg of sam-
ple. Lane 1: extract after sonication; lane 2: supernatant from sample centri-
fuged at 10 600 g; lane 3: pellet from sample centrifuged at 102 000 g;
lane 4: supernatant from sample centrifuged at 102 000 g. Protein markers
consisted of phosphorylase b (97 kDa), bovine serum albumin (66 kDa), oval-
bumin (45 kDa), and carbonic anhydrase (30 kDa). C) Native molecular mass
of lipolytic activities (*) determined by Superdex 200 size exclusion gel
chromatography, by using bovine serum albumin (1; 67 kDa), aldolase (2;
158 kDa), and catalase, (3; 232 kDa; *).
1
after centrifugation at 102 000 g (0.8 mg mL of protein con-
tent).
Discussion
Our findings show that the formation of volatile aldehydes in
T. rotula involves, above all, glycolipid PUFAs as the major sub-
strates.[10] This is reflected by the GC–MS distribution of fatty
acids in this class of lipids (Figure 1) and, in particular, by the
specific presence of HTrA and HTA in MGDG, the dominant
chloroplast component. Accordingly, experiments with enzy-
matic preparations clearly demonstrated the strict substrate–
product relationship between glycolipids and aldehydes
(Figure 2), which suggests that PUA biosynthesis is dependent
on glycolipid-hydrolyzing activity. This process was associated
with the disappearance of complex lipids and formation of
FFAs and lyso compounds, which was rigorously shown after
incubation of MGDG with crude enzymatic preparations
(Figure 3). Synthesis of PUAs also involves PLs but not TGs, as
previously observed.[4] It is, however, evident that phospholi-
www.chembiochem.org 453

pids do not represent a limiting factor in the biosynthetic pro-
cess, as this class of lipids accounts only for the formation of
part of decatrienal (Figure 2). Since galactolipids are restricted
to diatom chloroplast, our data strongly suggest that these or-
ganelles have a direct role in the production of antiprolifera-
tive aldehydes in diatoms. This is consistent with recent results
on the molecular basis of chemical defense in land plants.[1]
Incubation experiments with pure substrates localized the
lipolytic activity on MGDG, SQDG, and PL in soluble fractions
of T. rotula. In particular, under native conditions, analysis of
this material revealed the presence of 200 kDa soluble aggre-
gates capable of efficiently hydrolyzing MGDG. The biochemi-
cal mechanism involves removal of fatty acids from both sn-
positions of glycolipids—as shown by release of HTrA and HTA
from sn-2—and EPA and C18 PUFAs from sn-1 of MGDG purified
from T. rotula. It remains, however, to be elucidated if the en-
zyme(s) can directly act at both glycerol sites or if isomeriza-
tion of fatty acid is involved as supposed for some lipases. Pu-
rification and characterization of the putative enzymes are cur-
rently in progress. Preliminary analysis of soluble fractions de-
rived from T. rotula samples centrifuged at 102 000 g revealed
the presence of more lipolytic bands at approximately 42 kDa,
as suggested by the SDS-PAGE staining pattern when MUF-bu-
tyrate was used as substrate.
In conclusion, physical damage resulted in the production of
aldehydes and other oxylipins in T. rotula.[9, 10] This response oc-
curred only in lysed organisms where cellular decompartmen-
talization led to mixing of complex lipids and lipolytic, lipoxy-
genase and hydroperoxide lyase activities (Scheme 1). Above
all, as the formation of the antiproliferative aldehydes is imme-
diate after cell disruption, the process seems to have a simple
regulation based on a combination of pre-existing glycolipid-
hydrolizing enzymes and substrates, mostly chloroplastic lycoli-
Scheme 1. Biosynthetic proposal for the origin of antiproliferative aldehydes in lysed
T. rotula cells.
454 www.chembiochem.org @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A. Fontana et al.
pids. To date there is no evidence of activation of signaling
pathways and wound-induced response within planktonic
communities, so the definition of wound-activated defense, al-
though formally correct, can be misleading in this context.
The release of fatty acids in T. rotula extracts can be ex-
plained by the co-occurrence of proteins with glycolipase and
phospholipase activity. However, the enzymatic preparations of
T. rotula showed (Figure 2) the typical substrate specificity of
lipolytic acyl hydrolases (LAHs)—a large class of plant and bac-
terial proteins that selectively catalyze deacylation of galacto-
and phospholipids but not triacylglycerols. No genetic data are
available on T. rotula, but genes coding for patatins, a group of
glycoproteins with lipid acyl hydrolase activity,[11] have been re-
ported in Thalassiosira pseudonana—a cogeneric diatom the
genome of which was recently sequenced.[12] We do not ex-
clude the presence of PLA2 activity in T. rotula, as previously
observed.[4] Nevertheless, the data discussed above show that
glycolipid hydrolyzing activity, rather than a PLA2, plays the
major role in regulating PUA synthesis in the diatom—the hy-
drolysis of glycolipids being the only way to feed the down-
stream LOX pathway(s) with the whole set of fatty acids
(Scheme 1). The biochemical mechanism discussed above re-
captures the role of glycolipids and the enzymatic strategies
described in the algal-bloom forming diatom S. costatum.[5b]
Analogies from taxonomically unrelated species seem to indi-
cate a general model for producing high local concentrations
of ecologically relevant substances by the exploitation of cellu-
lar resources as abundant as chloroplastic glycolipids, with
little or no additional energy costs during normal growth of
healthy individuals. PUA synthesis in T. rotula and S. costatum
points out the role of chloroplasts in providing signaling mole-
cules that mediate eco–physiological processes of marine diat-
oms. Elucidating compartmentalization and substrate–enzyme
specificity in different diatom species and during the
cell cycle of microalgae is likely to reveal the regula-
tory connections between the biosynthetic pathways
silent in intact cells and the eco–physiological role of
PUAs or other oxylipins in marine diatoms.
Experimental Section
General: Solvents were purchased from Carlo Erba
(Milan, Italy) and distilled prior to use. HTrA and
[6,7,9,10,12,13-2H6]-6,9,12-hexadecatrienoic acid ([D]6-
HTrA) were prepared as described by d’Ippolito et al.[5a]
MUF-butyrate, p-nitrophenyl-acetate, and all other chem-
icals were obtained from Sigma–Aldrich. SO2-TLC and
silica-gel columns were performed by using precoated
Merck SiO2 60 F254 plates and Merck Kieselgel 60
powder. Rotatory TLC was carried out by using Chroma-
totronQ (Harrison Research Inc., Palo alto, USA) on pre-
coated silica plates. GC–MS data were obtained by using
a Hewlett & Packard 5989B mass spectrometer equipped
with a 5890 Series II plus gas chromatograph. Routine
NMR analysis of products was carried out with a Bruker
AMX 500 and Bruker Avance DPX 300 spectrometers.
Amicon YM-10 was purchased from Millipore. Protein
content was determined by using the Lowry protein
ChemBioChem 2006, 7, 450 – 456
Aldehyde Biosynthesis in Thalassiosira rotula
assay kit (Bio-Rad) by following the manufacturer’s instructions to-
gether with bovine serum albumin as standard. SDS-PAGE was per-
formed by using a continuous buffer system[13] in a miniprotein
vertical slab gel apparatus (Bio-Rad). The acrylamide stock con-
tained acrylamide (29.8 %, w/v) and N,N’-methylenebisacrylamide
(0.2 %, w/v). Low molecular weight standards for SDS-PAGE (Amer-
sham Biosciences) were used as calibration proteins: phosphoryl-
ase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin
(45 kDa), carbonic anhydrase (30 kDa). Fluorescence-based lipase
assays were adapted from the method of Diaz et al.[14] by using
either MUF-butyrate or MUF-oleate.
Cell culture: Axenic cultures of T. rotula were prepared as de-
scribed by Miralto et al.[2a] Briefly, diatoms were grown in Guillard’s
(F/2) Marine Enrichment Basal Salt Mixture Powder (Sigma–Aldrich)
medium, on a 12 light:12 dark cycle, at a light intensity of
175 mmol m 2 s 1. Cells were kept in 10 L tanks for one week and
then harvested by centrifugation at 1200 g in a swing-out rotor.
The procedure for preparing and maintaining axenic cultures are
reported elsewhere.[15]
Lipid extraction: Diatoms were harvested by gentle centrifugation
at 1200 g for 10 min at 16 8C. The resulting pellet was suspended in
F/2 medium (about 3 mL) and put in boiling MeOH (12 mL).[16]
Then the suspension was extracted by modified Folch method
with CHCl3/MeOH (2:1).[17] The lipid classes were fractionated by
silica gel column. TGs were eluted with petroleum ether/diethyl
ether (90:10 and 40:60) whereas GLs and PLs were obtained with
acetone/MeOH (9:1) and CHCl3/MeOH/H20 (65:25:4), respectively.[18]
MGDGs were separated from DGDGs and SQDGs by rotatory SiO2-
TLC with CHCl3/MeOH/H2O (65:25:2). Products were identified by
using NMR spectroscopy in CDCl3/CD3OD (1:1). Quantitative analy-
sis of lipid classes was expressed as mean of three independent
fractionations.
Lipid analysis: Fatty acids were analyzed as methyl esters (FAME)
by using GC–MS on an OV-1 column (injector, 260 8C; detector,
260 8C; N2 flow, 1.4 mL min 1; temperature gradient, 135 8C up to
260 8C, 3.5 8C min 1). FFAs were converted into the corresponding
methyl esters by treatment with a saturated solution of diazome-
thane in diethyl ether. For the GC–MS analysis, FAME mixtures
were dissolved in CH2Cl2 to a final concentration of 0.5 mg mL 1.
Quantification was performed by using pentadecanoic acid (C15:0)
as internal standard. Composition of MGDG was carried out on
silica-gel purified fractions as described by Guella and co-workers.[7]
Quantification of lipid hydrolysis in lysed diatoms: The cell pellet
(about 5 R 107 cells) was suspended in F/2 medium (2 mL) and soni-
cated for 30 s. The suspension was transferred to a conical flask
and erucic acid (13Z-docosenoic acid, 0.4 mg) was added as an in-
ternal standard prior to homogenization with CHCl3/MeOH (2:1, v/
v; 3.6 mL). The sample was then filtered on paper and the residue
washed on filter with CHCl3/MeOH (2:1, v/v; 2 mL). The filtrate was
transferred to a separatory funnel in order to obtain phase separa-
tion. The lower layer was recovered and the aqueous residue was
re-extracted with chloroform (2 mL). Organic phases were com-
bined and washed with H2O/MeOH (1:1). The organic solvent was
then removed under reduced pressure and the resulting residue
was dissolved in CD3OD (2.5 mL) and saponified under argon with
dry Na2CO3 (12 mg). After being stirred at 42 8C for 4 h, the reaction
mixture was neutralized (pH 6) by H2SO4 (1 N) and extracted
against diethyl ether. The organic solution was dried on Na2SO4, fil-
tered on paper and methylated with an ethereal solution of diazo-
methane. After removing the excess diazomethane under a N2
stream, the solution was evaporated at reduced pressure and dis-
ChemBioChem 2006, 7, 450 – 456 @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
solved in acetonitrile at a final concentration of 1.5 mg/mL. Analysis
was carried out by using GC–MS as reported above.
Substrate specificity of lipolytic activity: All procedures were car-
ried out at 4 8C unless otherwise indicated. Typically, a frozen
diatom pellet (6 R 108 cells) was sonicated in F/2 medium (30 mL)
for 1 min. The homogenate (about 27 mL) was centrifuged for
30 min at 9600 g at 6 8C and the clarified supernatant was ultrafil-
tered on Amicon YM-10 membranes (cut-off of 10 kDa) under a N2
flow. The high molecular weight fraction retained by the filter
membrane (retentate) was extensively washed with F/2 medium
and diluted in the same medium (10 mL). This solution was divided
in different aliquots and incubated with GL (8 mg in 80 mL MeOH),
PL (6 mg in 80 mL MeOH), TG (2 mg in 30 mL acetone), and MGDG
(1 mg in 50 mL MeOH), which had been previously purified from
other T. rotula extracts. The incubation experiments were per-
formed in triplicate and [D]6-HTrA (0.2 mg) was added as internal
control. Each incubation was kept at 22 8C for 30 min and vigorous-
ly stirred. The reaction was terminated by adding an equal volume
of acetone (about 1 mL). After addition of the internal reference
(30 mg dec-4-enal) half of each reaction mixture (about 400 mL) was
extracted with dichlomethane, derivatized with carbethoxyethyli-
dene–triphenylphosphorane (CET–TPP) and analyzed by using GC–
MS in acetonitrile (100 mL).[2c] The remaining parts of the incubation
mixtures (about 600 mL) were extracted with CHCl3/MeOH (2:1) and
filtered. The filtrates were combined, quantitatively transferred to a
glass tube, and allowed to stand for 10 min at 4 8C for phase sepa-
ration. The lower chloroform phase was collected, and the upper
alcohol phase was re-extracted with chloroform (30 mL). The com-
bined chloroform extracts were washed twice with NaCl solution
(0.9 %, w/v). The extract was concentrated by rotary evaporation
under reduced pressure at 30 8C and kept under vacuum until a
constant weight was obtained. This material was then dissolved in
chloroform/methanol and analyzed on TLC (silica gel 60 F-254) by
using one of the following solvent systems: chloroform/methanol
(90:10, v/v), chloroform/methanol/water (65:25:4, v/v/v), chloro-
form/methanol/ammonia (65:30:4, v/v/v), petroleum ether/diethyl
ether/acetic acid, (70:30:1, v/v/v). Boiled retentates (supplemented
with lipids) and retentate controls were included in each experi-
ment.
Fractionation of the lipolytic activity: About 20 g of wet T. rotula
were suspended in sodium phosphate buffer (50 mM, pH 7.0). The
suspension was sonicated in three cycles, each 30 s long and the
cell extract was centrifuged at 9600 g (6 8C) for 30 min. The pellet
was resuspended in the same buffer and centrifuged twice. Super-
natants were combined (60 mL) and centrifuged at 102 000 g for
4 h at 6 8C. Lipolytic activity was qualitatively monitored on the re-
sulting supernatant and pellet by assaying small aliquots of sample
with MUF-butyrate (25 mM ; 5 mL), and spotted onto filter paper.[10]
After that, lipid hydrolyzing hydrolysis was monitored on the same
fractions (100 mL) by incubation (in 800 mL of 50 mM sodium phos-
phate buffer, pH 7.0) with MGDG (0.5 mg in 50 mL MeOH), SQDG
(0.5 mg in 100 mL H2O), and DGDG (0.5 mg in 100 mL H2O), which
were purified from natural sources, in addition to commercially
available L-a-phosphatidyl-d,l-glyceroldioleyl (0.5 mg in 100 mL H2O)
and L-a-phosphatidyl-d,l-glyceroldioleyl/tristearine (2:1, 1.5 mg in
100 mL H2O). The reaction course was monitored with SiO2-TLC by
using chloroform/methanol/water (65:25:2, v/v) as eluent and fatty
acid release was analyzed by using GC–MS with erucic acid (10 mg)
as internal standard.
Native gel filtration of 102 000 g supernatants: Lipolytic activity
was evaluated by gel filtration under native conditions. To this aim,
supernatants from 102 000 g centrifugations were concentrated on
www.chembiochem.org 455

Centricon 10Q as described above and dialyzed at 6 8C against NaCl
(150 mM) in sodium phosphate buffer (50 mM, pH 7.0). The sample
was loaded onto a Superdex 200 HR 26/60 (Amersham Lifescience)
FPLC column, equilibrated in sodium phosphate buffer (50 mM,
pH 7.0) containing NaCl (0.15 M). Gel-filtration chromatography was
performed with the same eluent at a flow rate of 1.0 mL min 1 and
adsorbance was monitored at 280 nm. A high-molecular weight
gel-filtration calibration kit (Amersham Biosciences) which con-
tained the proteins catalase (232 kDa), aldolase (158 kDa), and
bovine serum albumin (67 kDa) was used. Apparent molecular
weights of the protein aggregates were determined by comparing
their elution peaks (calculated as the ratio of elution volume [Ve] to
void volume [V0]) against a calibration curve generated by the elu-
tion peaks of the protein standards. V0 was determined by gel fil-
tration of blue dextran. Fractions (5.0 mL) were collected and ana-
lyzed on paper filter for protein content and lipolytic activity with
MUF-butyrate as substrate. The active fractions were concentrated
on Centricon 10 and assayed in sodium phosphate buffer (50 mM ;
1 mL; pH 7.0) for the galactolipid-hydrolyzing activity by using
MGDG (0.5 mg) purified from T. rotula extracts. The reactions were
kept at 30 8C for 2 h and then stopped by extracting the mixtures
with ethyl acetate. After the addition of pentadecanoic acid
(C15:0) as internal standard, the organic solutions were methylated
by ethereal diazomethane, dried under nitrogen and analyzed by
using GC–MS as described above.
Preliminary characterization of lipolytic activity and zymograms:
Supernatant and pellet were obtained by centrifugation at
102 000 g as described above. These fractions were analyzed on
SDS-PAGE containing acrylamide (10 % or 15 %) in Tris/HCl (0.375 M,
pH 8.8), and SDS (0.1 %). Samples were dissolved in Tris/HCl (0.01 M,
pH 6.8), SDS (1 %), b-mercaptoethanol (0.7 M), glycerol (1.36 M), and
bromphenol blue (0.005 %) as tracking dye, without heating. Elec-
trophoresis was performed at 25 8C at 24 mA for 1 h. The pellet
was repetitively washed with sodium phosphate buffer (50 mM,
pH 7.0) and centrifuged at 102 000 g. The combined supernatants
were concentrated on Centriplus YM-10 (Millipore) and dialyzed
against diethanolamine (20 mM, pH 8.4; 2 L). Aliquots of these frac-
tions were analyzed by SDS-PAGE as described previously.[14] Re-
solved gels were soaked in Triton X-100 (2.5 %) at room tempera-
ture for 30 min, washed for 5 min in sodium phosphate buffer
(50 mM, pH 7.0), and incubated in MUF-butyrate (100 mM, 10 mL), or
MUF-oleate (200 mM) in sodium phosphate (50 mM, pH 7.0). After in-
cubation for 1 min with MUF-butyrate or 15 min with the MUF-
oleate, lipase activity was visualized by using the Chemidoc
System (Bio-Rad). Following zymogram analysis, the same gel was
stained with 0.1 % Coomassie brilliant blue R-250 in methanol
(25 %, v/v). The proteins were monitored spectrophotometrically at
280 nm or were assayed with Bio-Rad protein dye assay reagent.
Acknowledgements
We thank Mr. M. Di Pinto and Dr. F. Esposito for technical support
in diatom culturing and Prof. Graziano Guella of the University of
Trento for the analysis of MGDG. The project was partially sup-
ported by “Programma Nazionale di Ricerche in Antartide” and
“Legge Regionale Campania n. 5/2002”. A.C. is grateful to Prof. P.
456 www.chembiochem.org @ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A. Fontana et al.
Diaz of the University of Barcelona for his support with the lipo-
lytic assays.
Keywords: aldehydes · biosynthesis · chemical ecology ·
glycolipids · lipases
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Received: August 15, 2005
Published online on February 7, 2006
ChemBioChem 2006, 7, 450 – 456