Z. Marina - Interreg IV B Sudoe

Allelopathic effects of Zostera spp on the growth and photosynthetic
activity of the toxic dinoflagellate Alexandrium catenella
M. Laabira, M. Grignon-Duboisb, P. Cecchia, B. Rezzonicob, M. Rouquettea and E. Massereta
a.UMR 5119, Ecosystème Lagunaires, Place Eugène Bataillon, F 34095 Montpellier Cedex 05
b.UMR Environnements et Paléoenvironnements Océaniques (EPOC), Univ. Bordeaux 1, PhyValBio, F 33405 Talence Cedex
Allelopathy is a biological phenomenon by which an organism produces one or more biochemicals that influence the growth,
survival, and reproduction of other organisms. These substances are known as allelochemicals and can have beneficial (positive allelopathy)
or detrimental (negative allelopathy) effects on the target organisms. Aquatic macrophytes have long been suspected of suppressing
phytoplankton growth through the production and excretion of chemical substances.
Alexandrium catenella is a widespread PSP toxin-producing dinoflagellate species. Since 1998, recurrent A. catenella blooms have
been observed in the Thau lagoon (French Mediterranean coast), leading to the closing of shellfish farms (Collos et al., 2007), but never in
Arcachon lagoon (French Atllantic coast). Analyses of REPHY and REBENT monitoring network database showed a low occurrence of
Alexandrium blooms in the vicinity of extensive Zostera beds. This led us to investigate the non-nutrient relationship between Zostera species
and A. catenella.
Red tide in Thau lagoon
• Zostera marina (eelgrass) is found on both coasts of North America, as well as in Europe.
• Zostera noltii (dwarf eelgrass) occurs along the Atlantic European and northen African coasts and the Mediterranean
sea. It s distinguished from Z. marina by its smaller size, and by the colour and shape of the leaves.
• The two species produce bioactive phenolics among which zosteric acid, rosmarinic acid and flavonoids
(Achamlale et al., 2009)
Rosmarinic acid
Z.noltii
noltii
Z.
Preparation
of the
extracts
Z. marina: 2 soussous-espè
espèces
The ability of Zostera spp. to produce allelochemicals has been tested in laboratory using
bioassays. Aqueous or methanolic extracts from leaves of Zostera marina and Z. noltii were
assayed to determine their allelopathic effects on A. catenella.
1.10
Materials and Methods
A. catenella was grown in batch cultures using ESAW medium under appropriate light (µmol photons.m-2 s-1)
and temperature (20±1°C) conditions.
Methanolic and aqueous extracts were prepared with Zostera leaves from the Bay of Arcachon and the Thau
lagoon. They were analyzed for both the identity and quantity of phenolics present.
To test allelopathic affect of Zostera species, target species was cultivated in culture medium with different
concentrations of the extracts. The inhibition tests were conducted in 6-well sterile plates (72 h). The nutritive
medium was brought entirely at T0 time. A. catenella was added to each well (inoculation concentration of 8001000 cells.ml-1) and the growth was monitored by direct microscopic counts of cells. The effect of the crude
extracts on the rate of photosynthesis II (PS II) in A. catenella was assessed by sensitive fluorimetric
measurement of chlorophyll fluorescence of photosystem II (PS II) using PhytoPhyto-Pam.
ALY(4h)
0.95
0.035%
0.90
0.85
***
0.80
0.75
0.07
%
0.70
ESAW
DMSO
0,035%
0,07%
0,13%
0,27%
cumulative, time and concentration dependent.
ZA
ZA
RA
RA
Flav onoids
Flav onoids
100
100
60
60
40
40
20
20
Zn AZn AD-M
D-M
Zn A-FZn A-FM
M
Zn ThZn ThF-M
F-M
Zm AZm AD-W
D-W
Zm AZm AD-M
D-M
0.13%
Chemical analyses showed that Zostera leaves contain large amounts of
phenolics.
phenolics Rosmarinic acid (RA; 75-105.36 mg.g-1 dry weight) and flavonoids
(Fl; 7.02-82.87 mg.g-1 dry weight) presented the higher concentrations, while
Zosteric acid (ZA; 0.17-1.3 mg.g-1 dry weight) was always the minor phenolic
acid. Z. noltii contains the higher amounts of phenolics (65.51-188.59 mg.g-1
dry wt). Z. marina was less rich in phenolic acids, with a maximum of 58.52
mg.g-1 dry weight for fresh leaves sampled in Arguin (Arcachon).
80
80
Zn AZn AD-W
D-W
Control
/DMSO
1.00
120
120
0
ALY(72h)
Photosynthetic efficiency (reported to the control in Y axis) of A.
catenella is negatively affected by Zostera extracts.This effect is
-1
-1
Phenolic
Phenoliccontent
contentofofthe
theextract
extract(mg,g
(mg,g dry
drywt)
wt)
0
ALY(24h)
1.05
Zm AZm AF-M
F-M
0.27%
Photos showing heavy morphological
anomalies affecting A. catenella cells
exposed to increasing amounts (0.030.27%) of Zostera extracts.
Zm ThZm ThF-M
F-M
Extracts obtained from detrital and fresh leaves of Z. marina and Z. noltii strongly inhibited the
growth of A. catenella at very low concentration whatever the extracts. Alexandrium cells showed
severe morphological anomalies when exposed to the extracts. Significant reduction of their
photosynthetic activity was systematically observed after 4h of exposure, which became more marked
as time went on.
Species
A long-term inhibition was observed without the need of continuous addition of extracts,
suggesting that the allelochemicals produced by Zostera are stable in the medium. The significant
amounts of phenolics contained in the extracts could be responsible of the inhibition effects observed.
Z. noltii
This is the first report of the allelopathic potential of Zostera species against a dinoflagellate.
Considering the deleterious impacts of HAB on public health and economic resources, the
allelochemicals from Zostera detritus may be considered as potential candidates to mitigate the effects
of Alexandrium blooms on aquaculture.
Z. marina
Plant material
detrital leaves
Extraction Method
Extract code*
IC50 (mg.L-1)
Water, rt
Zn A-D-W
199.7
MeOH reflux
Zn A-D-M
79.8
Fresh leaves
MeOH reflux
Zn A-F-M
36.1
Fresh leaves
MeOH reflux
Zn Th-F-M
39.6
detrital leaves
Water, rt
Zm A-D-W
239.5
MeOH reflux
Zm A-D-M
92.2
Fresh leaves
MeOH reflux
Zm A-F-M
45
Fresh leaves
MeOH reflux
Zm Th-F-M
36
ACHAMLALE S., REZZONICO B., GRIGNON-DUBOIS M. (2009a) - Rosmarinic acid from Zostera detritus from Arcachon lagoon. Food Chem., 113: 878-883.
ACHAMLALE S., REZZONICO B., GRIGNON-DUBOIS M. (2009b) -Evaluation of detritus as a potential new source of Zosteric acid. J. Appl. Phycol., 21: 347-352.
COLLOS Y., VAQUER A., LAABIR M., ABADIE E., LAUGIER T., PASTOUREAUD A. (2007). Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon. Harmful Algae, 6, 781-789.
GROSS E.M., HILT S., LOMBARDO P., MULDERIJ G. (2007) - Searching for allelopathic effects of submerged macrophytes on phytoplankton - state of the art. Hydrobiologia, 584: 77-88.
HAGMANN L., JÜTTNER F. (1999) - Fischerellin A, a novel photosystem-II-inhibiting allelochemical of the cyanobacterium Fischerella muscicola with antifungal and herbicidal activity. Tet. Lett., 36: 6539-6542.
KEARNS KD, HUNTER M.D. (2000) - Green algal extracellular products regulate antialgal toxin production in a cyanobacterium. Environ. Microbiol., 2: 291–297.
Acknowledgments
This work was supported
by the SUDOE program
(ECO-LAGUNES project)
and the Region of Aquitaine.
Les substances allélopathiques
chez Zostera marina
et Zostera noltii
Micheline GrignonGrignon-Dubois
Directrice de Recherche au CNRS
[email protected]@phyvalbio.u-bordeaux1.fr
Zostera dans les mers du globe
Z. noltii
espè
espèce essentiellement Europé
Européenne : Atlantique,
Atlantique,
Iles britaniques,
britaniques, Mer Baltique,
Baltique, Méditerranné
diterrannée, Mer
Noire, Mer Caspienne,
Caspienne, Mer d’Aral.
Aral.
Petit peuplement au Maroc et en Mauritanie
Z. noltii
Zostera marina
sur la plupart des rivages europé
européens, la mer Noire
et une partie des rivages asiatiques. Peuple
également toutes les côtes d’
d’Amé
Amérique du Nord.
2 soussous-espè
espèces: Z. marina Linnaeus et Z.
angustifolia (Hornem.)
Hornem.)
Z. marina
Les herbiers constituent des écosystèmes remarquables, qui présentent un fort intérêt
écologique, patrimonial et économique.
Espèces ingenieurs peuplant les zones côtières :
contribuent à la structure physique des milieux littoraux (filtrent la colonne d’eau, stabilisent les sédiments, créent
une zone tampon le long de la côte.
formations végétales clés: niveau de production primaire et biodiversité très élevé,es ce qui les classe parmi les
écosystèmes les plus productifs de la planète.
Allélopathie
(allelon du grec "mutuel" ou "réciproque" et pathos "souffrance "
Interaction entre organismes par l’intermédiaire de molécule chimiques,
généralement des métabolites secondaires, capables d’affecter la croissance, la
santé, la biologie ou le comportement d’une autre espèce. Ces signaux peuvent
être inhibiteurs ou stimulants.
Les composés allélopathiques sont libérés dans
l’air ou le sol (plante terrestre), l’eau ou le sédiment (plante aquatique).
Allélopathie en milieu marin : peu étudiée
deux types de substances allélochimiques :
les allomones, qui procurent un avantage
à l’organisme qui les émet (substances
défensives)
et les kairomones, qui procurent un
avantage à l’organisme qui les perçoit.
Potentiel
élopathique de
Potentielall
allélopathique
deZostera
Zosteramarina
marina
(données
(donnéesrelatives
relativesàà Z.
Z.marina
marinad’Amérique
d’Amériquedu
duNord)
Nord)
Extrait aqueux de feuilles :
Inhibition de la croissance de micro-algues et de bactéries:
P. G. Harrison, A. T. Chan, Marine Biology 61, 21-26 (1980)
Contrôle de la croissance microbienne et de la consommation par les amphipodes:
P.G. Harrison, Marine Biology 67, 225-230 (1982).
<0>
Rôle possible des phénoliques dans la résistance à Labyrinthula zosterae, agent
pathogène primaire de la « wasting disease ».
R. N. Buchsbaum, F. T. Short, D. P. Cheney, Aquat. Bot. 37, 291 (1990).
L. H. T. Vergeer, A. Develi, Aquat. Bot. 58, 65 (1997).
<0>
Caractérisation de l’acide Zostèrique (acide phénolique sulfaté) ayant des propriétés
antifongiques et antibactériennes.
J.S. Todd, R.C. Zimmerman, P. Crews, R.S. Alberte, Phytochem. 34,401 (1993).
Applications : agrochimie, antifouling
Un constat : pas de bloom d’Alexandrium dans les zones abritant des
herbiers à Zostère denses : Bassin d’Arcachon
Hypothèse de travail : les Zostères sécrètent dans le milieu
des substances allélopathique capables d’inhiber le développement
d’Alexandrium catenella
Bloom:
division
exponentielle
Stratégie :
Extraire et identifier ces substances
Evaluer leur potentiel allélochimique
Communication chimique inter-espèce - stratégie de défense
extraction / isolation
processes
Seagrasses
MeOH
MeOH/H2O
Methanolic extract
Aqueous methanolic
extract
AcOEt
n-BuOH
Phenolics
Phenolics
sugars
Continuous
extraction
Inhibition de la croissance d’espèces microphytoplanctoniques
Alexandrium catenella
cell C.gracilis /mmm3
Chaetoceros gracilis
Cel/ ml
1000
BR-2173.I.s
2000
800
600
Témoins
Témoins
1750
1500
400
200
1250
1000
Z. noltii
0
0
1
2
3
4
jours
Z. noltii
750
1%
0.8%
0.6%
500
0.4%
0.2%
0.1%
0.05%
ACT3
DMSO
250
0
cell. C.gracilis/mm3
Témoin
4
20
40
80
600
500
400
300
200
100
0
100 mg/l
T0
48H
72H
Heures
BR-2097.I.s
Ce l/ml
Témoins
24H
5000
1,34%
1,07%
0,8%
0,54%
0,27%
0,13%
0,07%
ACT3
DMSO
Eau MilliQ
4500
4000
3500
Z. marina
3000
2500
0
2
4
6
Témoins
2000
jours
1500
Z. marina
1000
Témoin
20
60
160 mg/l
500
0
Heure s
T0
Coll. G. Arzul,
Arzul, Ifremer
24H
48H
72H
Coll. E. masseret & M. Laabir (UM2)
Tous les extraits montrent un fort pouvoir
inhibiteur à faible concentration, y compris ceux
préparés à partir des détritus.
Corrélation
inverse
significative
entre
concentration en polyphénols et IC 50, ce qui
confirme leur rôle dans l’activité inhibitrice
observée
Zostera noltii
Zostera marina
2 50
300
p o l yp héno l s
polyphé nols
200
I C 50
2 50
IC50
200
150
150
10 0
10 0
50
50
0
0
Zn A- D- W
Zn A-M -W
Zn A-F-M
Zm A-D-W
Zm A- D- M
Zm A- F-M
Les substances phénoliques chez Zostera
Chez les plantes terrestres, les composé
composés phé
phénoliques jouent un rôle majeur dans l'interaction des
plantes avec leur environnement. Ils peuvent être soumis à d’importantes fluctuations face aux
agressions du milieu et ont de ce fait une grande importance en écophysiologie.
Objectifs : caractériser les polyphénols responsables de l’activité inhibitrice (Z. noltii et Z. marina)
Variation interspécifique
Variation saisonnière de leur production
Sont-ils tous identiques quel que soit l’herbier : varaiabilité géographique?
Sont-ils exsudés dans le milieu marin? : Zostère vivante & échouages (laisses de mer)
Impact des facteurs anthropiques sur leur production
caractérisation chimique : mise en évidence de substances bio-actives
Acide Zostérique
Acide Rosmarinique
flavonoïdes sulfatés
(antifongique,
antibactérien,
antisalissure)
anti-oxydant puissant, antiviral
anti-asthénique, anti-inflammatoire,
antimutagène, antibactérien
Production des polyphénols: variation saisonnière chez
Z. noltii (Bassin d’Arcachon)
-Suivi mensuel d’
d’un herbier pendant 3 ans (2007(2007-2010): 44 collectes, 76 extraits pré
préparé
parés et
analysé
analysés (RMN, HPLC)
Hiver
Teneurs importantes en
polyphénols tout au long
de l’année :
16000-46000 ppm/ps
Pics saisonniers : hiver et
parfois automne
fortes teneurs de diosmétine
sulfaté (Z2S) tout au long de
l’année :
7500-21000 ppm
Ac. rosmarinique (RA) très variable
selon saison: 2500-24000 ppm (pic
hivernal)
Hiver
Automne Hiver
Automne
Printemps
Eté
Eté
Production de biomasse : 30000 – 44000 T/an (ps
(ps))
Polyphenols : 1000010000-40000 ppm/ps
ppm/ps soit 300 – 1700 T/an
Extraits MeOH : ppm de subtance/plante
ZAsèche
280 nm
Extraits MeOH : ppm de subtance/plante
ZAsèche
280 nm
25000
25000
CAF 328 nm
CAF 328 nm
RA 328 nm
RA 328 nm
Z2S 350 nm
Z2S 350 nm
20000
20000
15000
15000
10000
10000
5000
5000
0
0
1
16 6/0
/0 8 /2
1 8/ 0
19 9/0200 02
/0 7 /22
1 7/ 0
14 4/1200 03
/1 0 /23
1 0/ 0
10 0/1200 07
/1 2 /27
0 2/ 0
07 7/0200 07
/0 1 /27
0 1/ 0
04 4/0200 08
/0 2 /28
1 2/ 0
10 0/0200 08
/0 3 /28
0 3/ 0
07 7/0200 08
/0 4 /28
0 4/ 0
09 9/0200 08
/0 5 /28
0 5/ 0
09 9/0200 08
/0 6 /28
0 6/ 0
06 6/0200 08
/0 7 /28
0 7/ 0
06 6/0200 08
/0 8 /28
0 8/ 0
06 6/0200 08
/0 8 /28
2 8/ 0
28 8/0200 08
/0 8 /28
0 8/ 0
06 6/1200 08
/1 0 /28
1 0/ 0
10 0/1200 08
/1 1 /28
0 1/ 0
07 7/1200 08
/1 2 /28
0 2/ 0
04 4/0200 08
/0 1 /28
0 1/ 0
09 9/0200 09
/0 2 /29
0 2/ 0
08 8/0200 09
/0 3 /29
1 3/ 0
13 3/0200 09
/0 4 /29
0 4/ 0
03 3/0200 09
/0 5 /29
2 5/ 0
28 8/0200 09
/0 5 /29
5/ 0
20 09
09
Ac zostérique( ZA): faible
teneur par rapport à RA et Z2S,
mais relativement constante
: 300-400 ppm
Variabilité géographique chez Z. noltii
Golfe du Morbihan
(56)
Bassin
d’Arcachon
(33)
Etangs
Méditerranéens:
Salses Leucate (69)
Thau (34)
Berre (13)
Ria Formosa (Pt)
Iles Baléares (Es)
Baie de Cadiz (Es)
Z. Marina : 2 sous-espèces
Variabilité géographique
chez Z. marina?
Z. marina Linnaeus et Z. angustifolia (Hornem.)
Hornem.)
Z. noltii
Zone géographique
Bassin d’Arcachon
Golfe du Morbihan
Etang de Thau
Etang de Salses
Leucate
Etang de Berre
Z. noltii
x
x
x
x
Espagne
Delta de l’Ebre
Cadiz
Baléares
x
x
x
Portugal
Ria Formosa
x
Mer
Baltique
Danemark (Baltique)
Pologne (Baltique)
x
x
USA
Californie (côte ouest)
Massachuset (côte est)
x
x
France
Z. marina
x
x
x
x
x
Z. angustifolia (Hornem.)
Hornem.) domine en Mer Baltique, tandis que Z. marina Linnaeus domine sur la faç
façade Atlantique
Herbiers marins: particulièrement
fragiles et sensibles à la détérioration de la
qualité des eaux et à la concentration
d'activités humaines
60 % de la population mondiale vit sur le
littoral. Si rien ne change : 80% attendu en
2030
Le littoral est le réceptacle de toutes les
pollutions d’origine continentale.
Régression des herbiers
marins
perturbation
de l’ensemble de
l’écosystème côtier
Régression des herbiers
Régression des herbiers
marins
perte de
biodiversité
Herbier : environ 150 espè
espèces
Sédiment nu ∼ 20
La biodiversité augmente la capacité
des écosystèmes à réaliser différentes
fonctions écologiques, qui sont à
l’origine des services écosystémiques
(bénéfices directs ou indirects
l’homme).
La Directive Cadre sur la Stratégie pour
le Milieu Marin (DCSMM ; 2008/56/CE) établit une
politique communautaire visant à atteindre ou
maintenir un « bon état écologique » du milieu
marin au plus tard en 2020.
Deux types de descripteurs sont utilisés
pour qualifier les herbiers marins: leur superficie et
les
caractéristiques
démographiques
et
biométriques des populations. Ils permettent de
mettre en évidence l’évolution des milieux, mais
pas d’anticiper les destructions d’habitats, alors
qu’il serait primordial d’agir à un stade précoce.
En matière de dysfonctionnement, le
lien causes-effets est difficilement identifiable à
l’échelle macroscopique.
Parallèlement à ces descripteurs, nous
proposons d’utiliser un outil « bio-marqueur
capable de décrire l’état de la plante à l’échelle
moléculaire.
L’objectif est de pouvoir détecter au
niveau du métabolisme, les signes avant-coureurs
de la régression pour permettre une gestion
préventive de ses effets dévastateurs.
algues vertes opportunistes, halophytes envahissantes:
les poisons des lagunes
Quelques exemples de stress
subi par les herbiers
Impact des facteurs anthropiques sur la production
des polyphénols (Zostera noltii, Bassin d’Arcachon)
Taussat
Germ
3
2
Germ
Apparition de zones de
« dépérissement » :
Vert, devient noirâtre, puis rougeâtre
1
Comparaison Germanan/Taussat
Germanan/Taussat
- échantillons du 28/05/09
ppm/plante sèche
Dépérissement : perte des polyphénols
45000
total flavo
40000
total ac phenol
35000
flavo+ac phenol
30000
25000
20000
dépérissement
15000
10000
5000
0
Germ 1
vert
Germ 2
vert
Germ 3
vert
Germ 2:
rouge
Germ 2
noir
Taussat
vert
Dépérissement : perte des défenses chimiques
Impact des facteurs anthropiques sur la
production des polyphénols (Zostera noltii, Arcachon)
Effet de l'accum ulation d'algues vertes sur la teneur en polyphénols
35000
30000
25000
Impact d’
d’algues nitrophiles (Cladophora
(Cladophora
agaegropila et Enteromorpha intestinalis)
intestinalis)
sur les herbiers à Z. noltii (site
d’Arcachon):
L’accumulation
d’
vertes
sur
d’algues
l’herbier conduit à la diminution du
contenu phé
phénolique des feuilles de
Zostè
Zostère.
20000
15000
10000
5000
0
Absence algues - Présence algues Présence algues Présence algues
repousse herbier - Feuilles vertes
- Feuilles
- Feuilles
intermédiaires
marrons
Impact des facteurs anthropiques sur la production des polyphénols
(Zostera noltii, étang de Salses-Leucate)
L'apport excessif de nutriments (azote, phosphore) est directement
directement
toxique pour les herbiers, mais plus grave, il stimule la croissance
croissance
des épiphytes et des algues flottantes macroscopiques et
microscopiques. Cela se traduit par une diminution de la quantité
quantité de
lumiè
lumière solaire pouvant atteindre les feuilles des plantes, ce qui
réduit leur photosynthè
photosynthèse et donc leur production primaire.
primaire.
7
Effet des épiphytes sur la teneur en polyphénol chez Zostera noltii
45000
40000
35000
4
6
3
2
5
1
non epiphyté
épiphyté
30000
25000
20000
15000
10000
5000
0
2
3
4
5
6
7
Quantité
Quantité d’épiphytes
’épiphytes sur les feuilles: 6>>3>2=5>>7
Les épiphytes (du grec έπί
«sur», φυτόν «végétal»;
littéralement «à la surface
d'un végétal») sont des
plantes qui poussent en se
servant d'autres plantes
comme support.
Impact des facteurs anthropiques sur la production des polyphénols
Baltique
(Z. marina, Mer Baltique)
Klutz (De)
Sopot (Pl)
Mφ
φn (Dk)
8000
7271
7000
6000
phénoliques : total
5000
4000
3000
2878
2000
Effets
conditions climatiques
Pollution
1283
384
1000
0
Mon klint
30/09/05
Mon setge
30/09/05
Baltique
Sopot
BA 30/10/05
Arcachon
Mon klint : peu de rejets
Mon Setge : rejets industriels à proximité de l’herbier
Sopot : pollution importante
Les herbiers marins au cœur d’un projet européen
Gestion environnementale des zones
lagunaires à vocation aquacole- Eco-lagunes
2009-2011 :
France – Espagne – Portugal
7 partenaires
Les objectifs:
Démontrer qu’
qu’une bonne gestion environnementale des milieux
aquatiques lagunaires, en pré
préservant la biodiversité
biodiversité, peut garantir le
développement d’
d’une activité
activité économique durable
Harmoniser les outils de suivi des herbiers
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Un herbier en dé
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d’une lagune malade
AVANT DE COMMETTRE
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Phenolic fingerprints of Zostera noltii
from Atlantic and Mediterranean coasts
Micheline Grignon-Dubois and Bernadette Rezzonico
UMR 5805, EPOC, Université Bordeaux 1, 351, cours de la Libération, 33405 Talence cedex, France
One of the most interesting and fast developing fields in phytochemistry is chemical taxonomy, also called biochemical systematic. Some of the
most useful chemical markers are the secondary metabolites. They have been demonstrated to play an ecological role, being involved in (pathogenic)
interactions between plants and microorganisms or in communication with a symbiont. In the interaction of a plant with its abiotic and biotic
environment, secondary metabolites provide protection against abiotic stress (such as ozone, UV light, cold, drought, heavy metals, or nutrient
deficiency) or selective defense against herbivory and pathogen infection.
Phenolics are of a great importance in seagrass-environment relationships due to their involvement in the response of the plant to stress. Only a
few studies have investigated the concentration of phenolics in Zostera noltii, aside from our papers related to rosmarinic and zosteric acid. Our
ongoing studies have shown that the phenolics contained in Z. noltii from the bay of Arcachon and Thau lagoon (France) significantly inhibited the
growth of microphytoplancton spp. like Chaetoceros gracilis and Alexandrium catenella (1,2).
Documenting the presence of phenolics in living tissues of Z. noltii, and how these compounds vary in abundance between seagrass meadows
across large geographical scales and growing in different habitats conditions is crucial to understanding the landscape-level adaptability of the plant
to environmental factors..
Zostera noltii Hornem.
Hornem. (Zosteraceae, common name dwarf eelgrass, synonym
Zostera nana (Roth) is an important species of eelgrass occurring along European
and North African coasts.
Classification:
Empire
Eukaryota
Kingdom Plantae
Subkingdom Viridaeplantae
Phylum
Magnoliophyta
Class
Monocots
Order
Potamogetonales
Family
Zosteraceae
Genus
Zostera
Z. noltii bed
Bay of Arcachon
Gulf of Morbihan
France
Thau lagoon
Salses Leucate
Berre
Spain
Portugal
seasons
W, Sp, Su, Au
Su, Au
W, Sp, Su, Au
Su
Su
collections
24
2
4
2
1
Ebro
Cadiz
Balearic island
Su
Sp, Su
Su
1
2
1
Ria Formosa
Su
1
Z.
Z. Noltii
Noltii sampling
sampling sites
sites
Gulf
Gulfof
ofMorbihan
Morbihan
(Fr)
(Fr)
Bay
Bayof
of
Arcachon
Arcachon
(Fr)
(Fr)
French
French
Mediterrannean
Mediterrannean
lagoons:
lagoons
lagoons:
lagoons::
SalsesSalses
SalsesLeucate(69)
(69)
Salses--Leucate
Thau
Thau(34)
(34)
Berre
Berre(13)
(13)
Z. noltii distribution
W = winter, Sp = spring, Su = summer, Au = autumn
Ria
RiaFormosa
Formosa(Pt)
(Pt)
Identifying features
•Leaves grass green in colour.
•Leaf tips blunt and emarginate (notched), becoming indented in older leaves.
•Leaves 6 -30 cm long, 0.5-1.8 cm wide with 3 irregularly spaced veins.
•Leaf sheath short, 0.54 cm, and open with two membranous flaps.
•Reproductive shoots lateral. Seeds, 1.5-2 mm long (excluding style), white, and smooth.
•Rhizome 0.5-2 mm thick with 1-4 roots per node; fibre bundles in the innermost layers of the outer cortex.
Like most of Zostera sp. this species may exhibit morphological variation depending on location,
tidal zone and age of plant (Phillips & Menez, 1988).
Balearic
Balearicislands
islands
(Es)
(Es)
Bay
Bayof
ofCadiz
Cadiz(Es)
(Es)
Zosteric acid
Rosmarinic acid
Z. noltii leaves were collected from eight seagrass beds
representative of three types of coastal ecosystems:
four mesotidal bays on the Atlantic coast (France,
Portugal, Spain),
three Mediterranean coastal lagoons (France),
an open-sea Mediterranean Zostera bed (Spain).
Methanolic and aqueous extracts were prepared from the
plant material, and analyzed for both the identity and
quantity of phenolics present using NMR and HPLC.
These techniques has been proven to be method of choice
for secondary metabolites profiling of plant material. They
are non-destructive and can be performed at micro- scale
on samples of 10-50 µg.
350
PDA-280nm
2029 1.052 mg/ml dmso/H2O 4/1
PDA-350nm
2029 1.052 mg/ml dmso/H2O 4/1
Retention Time
PDA-328nm
2029 1.052 mg/ml dmso/H2O 4/1
350
Retention Time
Retention Time
ApiS
300
Chemotype = a chemically distinct entity in a
plant or microorganism with differences in the
composition of the secondary metabolites.
300
Bay of Cadiz
250
250
mAU
200
mAU
200
DiosS
150
31,6
150
7-sulfatedsulfated-apigé
apigénin chemotype - (ApiS)
ApiS)
Cadiz, Ria Formosa
100
50
39,6
40,6
30,5
31,2
27,3
27,7
28,1
28,9
18,3
9,8
10,2
11,0
7,9
8,5
50
33,0
34,0
9,1
100
0
0,0
0
2,5
5,0
7,5
10,0
12,5
15,0
17,5
20,0
22,5
25,0
27,5
30,0
32,5
35,0
37,5
40,0
42,5
45,0
Minutes
Zostera noltii : 2 chemotypes
500
PDA-280nm
2035 sol 1.042 mg/ml dmso/H2O 4/1
RetentionTime
PDA-350nm
2035 sol 1.042 mg/ml dmso/H2O 4/1
DiosS
High performance liquid chromatography (HPLC) combined with diode array detection (DAD) was used for both
qualitative and quantitative analyses of the extract composition. As expected on the basis of NMR data, the HPLC
flavonoid profiles were largely dominated by a single product, which was respectively eluted at 31.6 min (λmax, 336
nm; Cadiz, Faro) and 33.0 min (λmax, 347 nm; all the other sites). Minor flavonoids were also found and identified
to luteolin-7-glucose (λmax, 349 nm), and luteoline-7 sulphate (λmax, 349 nm). In addition, small amounts of
apigenin-7-glucose (λmax, 335 nm) were detected in the samples from Cadiz. All the UV absorptions are in
agreement with the literature. These assignments were supported by HPLC comparison with standards and LC-ESIMS analysis in positive mode. In particular, the mass spectra clearly show for all the flavonoids detected, the [M+1]
molecular peak and the characteristic ion peak at [M+1-80] for sulphated flavonoids or [M+1-162] for glucoflavonoids.
Our results were confirmed by acid hydrolysis of the crude extracts, which led to apigenin (Cadiz, Ria Formosa) or
diosmetin (all the other sites). In addition, authentic samples of the 7-sulphated-flavonoids were synthesized by
sulfation of luteolin, apigenin and diosmetin with TBAHS. Comparison of the NMR, MS and UV spectra and HPLC
retention time allows unambiguous identification of the sulphated flavonoid content of Z. noltii from the different
seagrass beds.
R = SO3H
Flavonoids content of Z, noltii (mg.g-1 dry wt, calculated as diosmetin equivalent): comparison Cadiz/Arcachon
350
300
7-Gluco
300
250
40,6
200
35,2
35,5
31,6
28,9
25,0
200
27,3
27,8
250
DiosS
7-sulfated
total
Lu-
Api-
Lu-
8.41
0.17±
± 0.01
0.56±
± 0.01
1.15±
± 0.07
9.26
0.15±
± 0.01
0.89±
± 0.01
0.44±
± 0.01
11.32
0.47±
± 0.02
tr
1.63±
± 0.09
12
16.78
0.61±
± 0.01
tr
1.57±
± 0.08
-
150
27,3
13,1
27,8
150
mAU
ApiS
mAU
Chemical analyses showed the sampled seagrasses leaves
contain flavonoids and phenolic acids including rosmarinic acid
(RA), and zosteric acid (ZA) (3-5). RA and ZA were found at all the
locations. The concentration of RA is seasonally dependent (2.75105.36 mg.g-1 dry weight), while the variations in the ZA remains
low (300-400 µg.g-1 dry weight).
The three types of phenolics are present in methanolic extracts,
while water allows selective extraction of flavonoids and ZA.
450
400
Bay of Arcachon
50
33,0
9,3
100
13,1
100
50
0
2,5
5,0
7,5
10,0
12,5
15,0
antioxidant,
antioxidant, antiantiinflammatory,
inflammatory, antiviral,
antimicrobial
500
PDA-328nm
2035 sol 1.042 mg/ml dmso/H2O 4/1
400
0,0
antifongic,antibacteri
al,
al, antifouling
RetentionTime
RetentionTime
450
350
ApiS
flavonoïds
17,5
20,0
22,5
25,0
27,5
30,0
32,5
0
35,0
37,5
40,0
42,5
7-sulfatedsulfated-diosmé
diosmétin chemotype - (DiosS)
DiosS)
Arcachon, Morbihan, Balearic islands,
islands, French
Mediterranean lagoons
ApiS
DiosS
± 0. 0.64±± 0.01
5.08±
15± 0. 0.64±± 0.01
6.85±
45,0
Minutes
± 0.
8.43±
15
±0
13.41±
Lu
unsubstituted
Api
0.63±
± 0.01
0.18±
± 0.01
-
0.48±
± 0.01
0.21±
± 0.01
-
0.51±
± 0.01
-
0.28±
± 0.01
0.30±
± 0.01
-
0.89±
± 0.02
Dios
.32
Apigenin sulphate had never been reported for Z. noltii before. Based on these data, our results showed that Z. noltii grown in the Bay
of Cadiz and in Ria Formosa are chemically distinct from specimen grown in the other sites. This is the first report of a possible
chemotype for a marine magnophytes. Understanding the geographic variation of Z. noltii sulphated flavonoid content and its possible
link with ecological factors is now under progress.
1. M. Grignon-Dubois, G. Arzul, Results to be published.
2. M. Laabir, M. Grignon-Dubois, P. Cecchi, B. Rezzonico, F. Rieuvilleneuve, E. Masseret, in : Proceeding of the 4th mediterranan symposium
on marine vegetation. El Asmi, S., Langar, H., Belgacem, Tunis, 251 pp. 2010, 187-188.
3. S. Achamlale, B. Rezzonico, M. Grignon-Dubois, Food Chem. 113, 878-883 (2009).
4. S. Achamlale, B. Rezzonico, M. Grignon-Dubois, J. Applied Phyc., 21, 347-352 (2009)..
5. M. Grignon-Dubois, B. Rezzonico, T. Alcoverro, Estuarine, Coastal and Shelf Science, under press.
Acknowledgments
This work was supported by the
SUDOE program (ECO-LAGUNES
project) and the Region of
Aquitaine.
Effect of environmental stress on the phenolic content of
Zostera noltii
Micheline Grignon-Dubois and Bernadette Rezzonico
UMR 5805, EPOC, Université Bordeaux 1, 351, cours de la Libération, 33405 Talence cedex, France
Seagrass meadows are susceptible to coastal environmental impacts and can serve as early warning systems of ecosystem degradation. Zostera
noltii Hornemann is an important species of eelgrass occurring along European, North Africa, Mediterranean, Black Sea and Azov Sea coasts. The
coastal habitats favoured by Z. noltii are under increasing threat from coastal development, pollution and other forms of human disturbance. The role
of excess epiphyte, macroalgae or phytoplankton growth in shading of seagrass leaves and negatively affecting seagrass health is generally agreed
to be a prevalent mechanism in seagrass decline worldwide.
Plants are provided with a large arsenal of chemicals that are utilised as defensive mechanisms. It is of interest to develop and validate
biomarkers based on these chemical defences for monitoring conservation status and ecotoxicological impact in seagrass meadows. In this context,
phenolic compounds constitute good candidates for providing insights into water quality. Our previous work have decrypted the phenolicfingerprints of Zostera spp. and highlighted the potential of polyphenols as indicators of the coastal lagoon environmental quality. Documenting the
presence of those compounds in living tissues, and how they vary in abundance in presence of environmental stress would be helpful to understand
how human activities influence marine communities
th African coasts.
Little attention has been directed towards understanding the effects of
environmental variables on the phenolic content of Z. noltii.
Viridaeplantae
Study sites
In this study, we have compared the phenolic content of healthy green
leaf tissue of Z. noltii to specimens under the pressure of environmental
stress:
the accumulation of opportunistic macro-algae,
the excessive development of epiphytes,
the recurrent apparition of withered patches in Z. noltii beds.
Potamogetonales
Z. noltii distribution
Bay of
Arcachon
SalsesSalses-Leucate
The study was conducted in the Bay of Arcachon (French Atlantic coast) and at Salses-Leucate (French Mediterranean coastal lagoon).
Leaves of Z. noltii were collected in the seagrass beds. Methanolic and aqueous extracts were prepared from the plant material, and analyzed for
both the identity and quantity of phenolics present using NMR and HPLC.
Bay of Arcachon : effects of invasive species and blooms of opportunistic macroalgae
(mostly Enteromorpha and Cladophora spp.)
Zostera noltii in the Bay of Arcachon
Invasive species: S. anglica has rapidly expended displacing native species like Z. noltii
Algal mats induce highly
unfavourable conditions
Zostera noltii bed,
richest habitat with
and biodiversity,
reduced in the inner
Seagrasses of the genus Zostera are widespread around the world. In
Europe, there are two representatives, Zostera noltii Hornem and Z. marina.
Both are present in Arcachon Lagoon, France, especially Z. noltii which
constitutes 95% of the largest Zostera bed in Europe.
Extensive meadows of Z. noltii are found in the intertidal zone, covering about
40% of the lagoon
Loss of seagrass
Effects of stressors on the production of polyphenols
Germ
2
1
Germ
Blooms of Enteromorpha and Cladophora spp. occur all along
the year leading to algal mats, which induce highly
unfavourable conditions for Zostera noltii
Recurrent apparitions of
withered patches in Z. noltii
beds
3
Comparison Germanan/Taussat
Germanan/Taussat
28/05/09
ppm/plante sèche
Withering : decrease of the phenolic content
45000
total flavo
40000
total ac phenol
35000
flavo+ac phenol
bare sediments
Suffocation by accumulations of opportunistic macromacro-algae
(Zostera noltii,
noltii, Bay of Arcachon)
Arcachon)
Taussat
which represents the
regard to productivity
is being drastically
part of the bay
Effet de l'accum ulation d'algues vertes sur la teneur en polyphénols
35000
The effects of macroalgae
accumulations and withering
were investigated in Arcachon
bay (French Atlantic coast).
30000
25000
detrimental effects of
macroalgal mats on
the phenolic content
20000
15000
10000
5000
0
Absence algues - Présence algues - Présence algues - Présence algues repousse herbier Feuilles vertes
Feuilles
Feuilles m arrons
interm édiaires
30000
25000
20000
Withered patches
15000
10000
5000
0
Germ 1
vert
Germ 2
vert
Germ 3
vert
Germ 2:
rouge
Germ 2
noir
Taussat
vert
Withering : loss of the chemical defences
Nutrient enrichment encourages rapid growth of epiphytic algae which
which may
cause severe shading of Zostera noltii
Zosteric acid
Rosmarinic acid
flavonoïds
SalsesSalses-Leucate
..
Samples were collected on August 29th 2009 at 7 stations (S1-S7)
providing a large gradient of physico-chemical and environmental
parameters. After collection, the seagrass material was sorted and
divided into all green leaves (green bars) without visible epiphytes and
leaves still loaded with epiphytes and macroalgae (yellow bars).
7
antifongic,antibacteri
al,
al, antifouling
antioxidant,
antioxidant, antiantiinflammatory,
inflammatory, antiviral,
antimicrobial
Effect of epiphytes on the phenolic content (Zostera noltii leaves; ppm/dw)
50000
6
non epiphyté
40000
4
In all cases studied, the phenolic content of impacted specimens was found
significantly lower than for the healthy ones.
épiphyté
30000
3
20000
2
10000
0
5
1
Phenolic content in “green”
specimen is always higher than in
epiphyted (yellow), showing the
negative effect of epiphytes. The
highest values were found at S7
(northern part of the lagoon),
which is less eutrophicated than
S3 and -6 (Ifremer, 2010).
2
3
4
5
6
7
Amount of epiphytes on the leaves:
leaves: 6>3>2=5>>7
These results show that these environmental stresses negatively affect the
metabolism of Zostera noltii, and that polyphenols are good indicators of Zostera
health conditions.
Acknowledgments
This work was supported by the SUDOE program (ECO-LAGUNES
project) and the Region of Aquitaine.
Spatial variability in the phenolic content of Zostera spp from Salses-Leucate lagoon
M. Grignon-Duboisa, B. Rezzonicoa and L. Fonbonneb
a.UMR Environnements et Paléoenvironnements Océaniques (EPOC), Univ. Bordeaux 1, PhyValBio, F 33405 Talence Cedex
b. RIVAGE Salses-Leucate, Mairie de Leucate, rue du Dr Sidras, F 11370 Leucate
Mediterranean lagoons are highly productive areas representing more than 50% of the coastline in LanguedocRoussillon (South of France). Many of them suffer from eutrophication and concomitant deterioration of water quality.
Seagrass meadows are susceptible to coastal environmental impacts and can serve as early indicators of system-wide
degradation. It is of interest to develop and validate chemical biomarkers for monitoring conservation status and
ecotoxicological impact in Zostera meadows. In this context, phenolic compounds constitute good candidates, which may
provide insights into water quality. They play several important functions in plants, and they are common in marine
ecosystems. Documenting the presence of those compounds in alive tissues, and how they vary in abundance becomes
crucial to understand how human activities influence marine communities. To do this we analyzed the abundance of
specific phenolic compounds (rosmarinic (RA), caffeic (CAF), zosteric acids (ZA) and flavonoids (F)) of Zostera spp. from
different sites located across the Salses Leucate lagoon (Achamlale et al., 2009a, b).
Z. Noltii (Zn)
Z. marina var. angustifolia
(Za)
Zn (A)
Mediterranean sea
Sampling sites
7
Batch
&
sites
The Salses-Leucate lagoon
(42.8°N 3.0°E, France)
S1
S2
S3
S4
S5
S6
S7
-
SL2A-Zn
-
SL4A-Zn
SL5A-Zn
SL6A-Zn
SL7A-Zn
6
4
3
Zn (B)
-
SL2B-Zn
SL3B-Zn
-
SL5B-Zn
SL6B-Zn
SL7B-Zn
Za (A)
SL1A-Za
-
SL3A-Za
-
SL5A-Za
-
-
Za (B)
SL1B-Za
-
SL3B-Za
-
-
-
5
2
1
Materials and Methods
Samples were collected at Salses-Leucate
lagoon (42.8°N 3.0°E, France) on August 29th 2009 a t
7 stations (S1-S7) providing a large gradient of
physico-chemical and environmental parameters.
After collection, the seagrass material was sorted
and divided into all green leaves (A) without visible
epiphytes and leaves still loaded with epiphytes and
macroalgae (B). This led to 11 samples of Z. noltii
and 5 samples of Z. marina var. angustifolia (Table
1). Methanolic extracts were prepared from the plant
material, and analyzed for both the identity and
quantity of phenolics present using NMR and HPLC
(Table 1, data are expressed in mg (gdw-1) of dry
matter of Zostera).
The phenolic profile was largely dominated by flavonoids whatever the species, but the concentration mean
values are greater in Z. noltii (20 mg.g-1) than in Z. marina var. angustifolia (6 mg.g-1 dw). The proportions remain
constant with Z. marina var. angustifolia, for which only traces of RA, ZA and CAFF were found. In contrast, important
variations of the amount of RA were observed with specimen (A) of Z. noltii : 42, 31 and 39% respectively at S4, -6 and -7,
but only 9% at S2 and -3.
Concentrations in specimen A are always higher than in B, showing the negative effect of macroalgae and
epiphytes, which were particularly abundant at S3 and S6. Interestingly, the lowest values were found for the specimen B
from these two stations. The highest values were found at S7 (northern part of the lagoon), which is less eutrophicated
than S5 and -6 (Ifremer, 2010). The lowest content with Z. marina var. angustifolia was found at S1, an artificial channel
affected by pollution and physical disturbance due to the passage of boats. Understanding the specific functional role of
each of these compounds is now in progress.
Rosmarinic acid
RA
CAFF
ZA
F
Species
Z. noltii
Sites
S2
Phenolic proportions (% of the
total)
Total
mg.g-1
dw
F
RA
ZA
CAFF
2-A-Zn
88
9
1
1
19.84
2-B-Zn
93
4
1
2
15.35
S3
3-A-Zn
89
9
1
1
21.98
3-B-Zn
94
3
2
1
8.742
S4
4-A- Zn
56
42
1
1
27.83
S5
S6
S7
Z. marina
var angustifolia
Extract
codes
S1
S3
S5
This preliminary study highlights the
potential of polyphenols as indicators of the
coastal lagoon environmental quality
120%
F
RA
ZA
CAFF
100%
80%
5-A-Zn
81
15
3
1
25.54
5-B- Zn
88
7
3
1
16.45
6-A- Zn
66
31
2
1
24.86
6-B- Zn
79
15
5
1
2.76
7-A- Zn
59
39
1
1
41.57
7-A- Zn
77
20
1
2
16.20
1-A- Za
98
1
1
0
6.02
1-B-Za
99
0
1
0
2.42
3-A-Za
92
3
1
3
9.17
3-B-Za
94
2
1
4
6.80
40
5-A-Za
97
1
1
1
5.92
35
60%
40%
20%
0%
-20%
2-AZn
2-BZn
3-AZn
3-BZn
4-AZn
5-AZn
5-BZn
6-AZn
6-BZn
7-AZn
7-AZn
1-AZa
1-BZa
3-AZa
3-BZa
5-AZa
1-BZa
3-AZa
3-BZa
5-AZa
Total phenolic (mg/g dw)
45
30
Acknowledgments This work was supported by
the SUDOE program (ECO-LAGUNES project)
25
20
15
10
ACHAMLALE S., REZZONICO B., GRIGNON-DUBOIS M. (2009a) - Rosmarinic acid from beach waste:
Isolation and HPLC quantification in Zostera detritus from Arcachon lagoon. Food Chem., 113: 878-883.
ACHAMLALE S., REZZONICO B., GRIGNON-DUBOIS M. (2009b) - Evaluation of detritus as
a potential new source of Zosteric acid. J. Appl. Phycol., 21: 347-352.
IFREMER (2010). Réseau de Suivi Lagunaire du Languedoc-Roussillon : Bilan des résultats 2009.
Rapport RSL-10/2010, 321 pp.
5
0
2-AZn
2-BZn
3-AZn
3-BZn
4-AZn
5-AZn
5-BZn
6-AZn
6-BZn
7-AZn
7-AZn
1-AZa