Barbara Malewicz, Teresa Kentzer, Alicja Kosakowska, Danuta

Barbara Malewicz, Teresa Kentzer, Alicja Kosakowska, Danuta

OCEANOLOGY NO IX (X979)
99
BARBARA MALEWICZ
P olish A cadem y of Sciences
Institute of Oceanology — Sopot
TERESA KENTZER
U niversity of Gdańsk
Institute of Biology — Gdańsk
ALICJA KOSAKOWSKA
DANUTA GĘDZIOROWSKA
Polish A cadem y of Sciences
Institute of Oceanology — Sopot
THE INFLUENCE OF GIBBERELLIC ACID ON THE IRON UPTAKE BY
THE BALTIC PHYTOPLANKTON
C o n t e n t s : 1. Introduction, 2. M aterials and m ethods, 3. Results and discussion;
Streszczenie; References.
The influence of gibberellic acid (GA3) upon the iron uptake and
prim ary production in B altic phytoplankton and in standard phytoplankton cultures, grown on synthetic media and n atu ral sea w ater, has been
investigated.
Iron uptake was m easured by radio isotope technique. The p rim ary
production and biomass w ere exam ined by carbon isotope m ethod and by
the determ ination of chlorophyll respectively.
In synthetic media GA3 m arkedly increased Fe uptake by phytoplank­
ton b u t only at th e restricted range of pH 6—6,5; Most probably this
stim ulation is due to the form ation of assimilable Ga3-iron complex.
The grow th stim ulation effect of GA3 resu lts probably from the increas­
ed uptake of iron by phytoplankton.
In n atu ral sea w ater GA3-iron m ixture, depending on the tim e and
place of sampling, stim ulates inhibits or has no influence on phytosyn­
thesis and biomass production. The possible factors interfering w ith GA3
action in n atu ral environm ents have been discussed.
1. INTRODUCTION
Phytoplan'kton gowth in sea w ater is often restricted by the lowered
availability of some trace elements, the uptake of which depends on m any
factors [8, 13]. Especially im portant for phytoplankton developm ent is
iron (9) but, there exist only little inform ations about its distribution in
the Baltic sea [2, 3]. It w as found th a t the am ount of iron in Baltic sea
w ater is m arkadly low er than th a t used for the cultivation of phytoplank­
ton [1, 13]. The effective concentration of sea w ater iron is still low er
than its total contents because it appears in various chemical forms and
only some of them are assim ilable for phytoplankton. Iron is assim ilated
by algae as ferric ions most probably com plexed w ith some natu ral organic
compounds contained in sea w ater (so called siderochromes). The ratio
of assim ilable and nonassim ilable iron in Baltic sea w ater has not yet been
established.
The ra th e r high contents of iron in phytoplankton cells, in reg ard
to very low concentration of assim ilable iron in the sea w aters, is obtain­
ed in the resu lt of cellular active accum ulation processes against the
concentration gradients. The factors facilitating such an accum ulation
play an im portant role in the developm ent of phytoplankton.
We have observed th at gibberellic acid, an im portant p la n t grow th
regulator [11, 12] found also in Baltic w aters (Kentzer, unpublished data),
m arkedly stim ulates the developm ent of phytoplankton in the iron con­
taining m edia [9]. The results obtained point to the enhancem ent of iron
uptake ra th e r th a n to the direct stim ulation of m etabolism by this p lan t
hormone. In the present paper m ore detailed studies on the m echanism
of the above phenom enon are presented. The influence of gibberellic
acid upon the grow th and iron uptake by phytoplankton has been investi­
gated.
2. MATERIALS AND METHODS
The m easurem ents w ere carried out from A pril till Novem ber 1974.
Samples of sea w ater and phytoplankton w ere taken at ten stations in
the southern Baltic (Fig. 1). Surface and bottom sea w ater was sampled
Fig. 1. Location of the sarapling stations
Ryc. 1. Rozm ieszczenie stacji pobrania prób
by m eans of a polyethylene bathom eter. The sea w ater im m ediately
afte r sam pling was filtered through 25 (im nylon net and afte rw ard s
through 0.45 M-m S artorius m em brane filter in order to separate the sus­
pended m atter. Before the experim ents the sea w ater was stored at low
tem perature (below 10°C). Some experim ents w ere done using artificial
sea w ater [4] and P r a t’s m edium [1], The suspension of phytoplankton
was prepared for experim ents as follows: the n atu ral phytoplankton w as
collected by m eans of standard plankton net or by bathom eter. A fter
separating from the Zooplankton by filtration through a 100 um net and
decantation from the inorganic m atter, the phytoplankton was suspended
to a concentration corresponding to 1 mg of dry m a tter in 1 ml of sea
w ater. The phytoplankton suspension (1 ml) was added to 100 ml sea
w ater-sam ples. Q ualitative and q u an titativ e analyses of phytoplankton
were made for each suspension. In experim ents both n atu ral phytoplank­
ton and some pure cultures such as Chlorella vulgaris B eijerinck and
D ictyosphaerium pulchellum [1] w ere used. The pure cultures w ere used
for com parative purposes and the m ethod of th eir inoculation was descri­
bed previously [9].
The rate of the iron uptake by the phytoplankton cells was deter­
m ined by using 59Fe. To 100 m l of the artificial sea w ater samples or to
the samples of P r a t’s m edium 1 ml of the 59Fe solution was added. T he
radioactivity of the Fe-solution was 25 nCi/ml. In each com bination one
of the samples studied was enriched w ith 0.1 m l of gibberellic acid (GA3>
solution. The final concentration of GA3 in these samples was 10~10 mM/L
The samples w ere incubated a t optimal tem p eratu re and in light condi­
tions. A fter 1 hour 10 ml portions of these samples w ere filtered b y
S artorius m em brane filters to isolate the phytoplankton cells. The rem ain­
ing phytoplankton sedim ent was w ashed twice w ith 2 ml artificial sea
w ater, and tested for the activity of 59Fe by using the G eiger-M üller
counter.
The effects of exogeneously applied GA3 w ere studied in short and
long-term experim ents. The resu lts of short action of GA3 w ere estim ated
by m easuring the p rim ary production afte r 4 hours of phytoplankton
incubation. The carbon isotope 14C m ethod developed by Steem ann Niel­
sen was used for m easurem ents of the prim ary production [7, 14]. The
samples of the phytoplankton suspended in the sea w ater enriched w ith
a gibberellin-Fe m ixture (0.02 mM Fe/1 1 and 1 X 10~7 mM GA3/1) w ere
incubated in the illum inator a t a constant tem p eratu re for 4 hours. The
tem perature was kept at the same value as it had been found in the sea
a t the tim e the phytoplankton was collected. For com parison the p rim ary
production of three control samples was investigated a t the same tim e
and the same conditions:
I — for th e p h y to p la n k to n su sp en d ed in th e sea w a te r e n ric h ed w ith
0.02 mM Fe/1.
II — for the phytoplankton suspended in the sea w ater enriched w ith
1 X 10-7 mM GAa/l.
I ll — for the phytoplankton suspended in ’’p u re” sea w ater.
In all experim ents the phytoplankton samples w ere incubated in
continuous light w ith in tensity of about 4000 lux.
The long-term influence of GA3 on the biomass production of the
phytoplankton was m easured by estim ation of the contents of the photo­
synthetic pigm ent — chlorophyll a, afte r 8 days of incubation. Samples
of 400 m l sea w ater containing the phytoplankton suspension and the
control samples w ere prepared as described above, b u t the am ounts of
gibberellic acid and iron used w ere higher in this case. 6 X 10-7 mM/1 of
GA3 and 0.05 mM/1 of iron w ere added. In addition the w ater sam ples
w ere enriched w ith n u trien ts solution in am ounts required for the optimal
grow th during a long-term incubation [6], The samples w ere earated for
12 hours a day and illum inated continously. A fter incubation analyses of
chlorophylls and carotenoids w ere made [15], The filtering procedure and
analyses of th e photosynthetic pigm ents w ere carried out according to
Calberg and Gargas [5, 7], The phytoplankton biomass was m easured as
dry m atter.
3. RESULTS AND DISCUSSION
Table 1 presents the results of the influence of exogeneously applied
GA3 on the 59Fe uptake by phytoplankton in synthetic media. These
experim ents Showed th a t gibberellic acid used in the very sm all am ount
of 1 0 '10 mM/1 was able to increase th e Fe uptake by the phytoplankton
m arkedly b u t only at a restricted range of the pH value of 6 or 6.5 In a
n eu tral or alcaline m edium no or negligible effects w ere observed.
The occurrence of stim ulation only at low er pH values in all phy­
toplankton and media studied can not be attrib u ted to the eventual ge­
neral preference of acidic conditions for the iron uptake. A lthough in
P r a tt’s m edium iron accum ulation by Chlorella vulgaris cells in the
absence of gibberellic acid is in higher pH values slightly lower th an in
acidic conditions, phytoplankton I (table 2) and D ictyosphaerium pulchellum in artificial sea w ater accum ulated iron a t higher pH m ore than
a t lower.
The stim ulatory action of gibberellic acid in acidic conditions could
thus be explained by one of the following mechanisms. E ither only a
nondissociated horm one molecule is capable of traversing the cytoplasm atic m em brane (or its uptake for some other reasons is possible only a t low
pH) and acting in a cell as general grow th stim ulant it increases the
dem and for the iron increased uptake which would be a secondary effect,
or the gibberellic acid in acidic conditions (in nondissociated form?)
complexes the iron ions thus facilitating th eir tran sp o rt across the m em ­
brane by playing the role of an iron carrier (siderochrome). In the la tter
Table
1
Tabela
1
The influence of gilbberellic acid on the uptake of 59Fe by phytoplankton
W pływ kw asu giberelow ego na pobranie 59Fe przez fitoplankton
Phytoplankton
M edium
pH of the m edium
Fitoplankton *
Środowisko
pH środowiska
C
A
B
Prat’s
6.0
' 6.5
7.0
7.5
8.0
870
885
830
750
710
1250
1190
900
730
740
artificial
sea w ater
6.0
6.5
7.0
7.5
. 8.0
630
720
750
945
1020
980
1140
790
920
1100
artificial
sea w ater
6.0
6.5
7.0
7.5
8.0
1100
1500
1870
1740
1860
1780
1920
2010
1870
1730
.
I
D
U ptake of 59Fe (c.p.m.)
P obieranie 59Fe (imp./min.)
* C h a rac te ristic of p h y to p la n k to n is given in T able 2.
A — p h y to p la n k to n w as grow n in m edium e n ric h e d w ith 5*Fe.
B — p h y to p la n k to n w as g row n in m edium e n ric h e d w ith 5#Fe and gibberellic acid.
Table
2
Tabela
2
Characteristic of phytoplankton used
Charakterystyka jakościow a fitoplanktonu
Phytoplankton
Species of phytoplankton used
Fitoplankton
Rodzaj badanego fitoplanktonu
I
Chaetoceros sp., D iatom a elongatum , N avicula lacustris, Scenedesm us acum inatus, A nabaena flos-aquae
11
Chaetoceros borealis, D iatom a elongatum , N avicula lacustris
III
Euglena, S cenedesm us acum inatus,
O scillatoria sp., M elosira jiirgensi
IV
Coscinodiscus grani, S celetonem a costatu m ,
anum
c
C hlorella vu lgaris Beijerinck
D
D ictyosph aerium pu lch ellum w ood
Ankistrodesm ut,
jalcatus,
P ed ia stru m
b o ry-
case the enhancem ent of iron uptake would be prior to th e stim ulation
of grow th resulting from th e increased contents of cellular iron.
It has been dem onstrated previously [10] th a t GA3 in n eu tral or
slightly acidic conditions and in the presence of iron strongly increases
the phytoplankton grow th b u t only at concentrations much higher
th a n those used in these studies. The concentration 10“ 10 mM/1 has negli­
gible effect on grow th promotion but is enough for the m arked stim ula­
tion of iron uptake. Therefore it seems to be more probable th a t th e
direct influence of gibberellic acid on the iron uptake e.g. by the form a­
tion of assim ilable GA3-iron complex is the prim ary effect in the phe­
nomenon of stim ulation of iron accum ulation by this plant hormone. The
final elucidation of this problem will be a subject of our fu rth e r studies.
The stim ulatory effect of gibberellic acid on the iron uptake can also
be helpful in studing the physiological effects of very low concentrations
of GA3 (e.g. in sea waters) on phytoplankton. In such concentrations
the grow th stim ulation, as negligible, is not or h ardly m easurable. On
the other hand the determ inatio of radioactive iron uptake provides a
useful tool in such studies.
The results presented in Table 1 indicate also the m arked effect of
pH itself on iron uptake by phytoplankton which probably influences the
grow th of these organisms. This factor m ight thus play a role in the
developm ent of phytoplankton in n atu ral environm ents.
A part from the dem onstration of iron accum ulation prom oting ac-
V,
VI, VII,
VIII, IX, X,
v.
VI, VIII, IX,
x , ^ °^ ąhc
Fig. 2. The influence of GA3 on the prim ary production of B altic w ater enriched
w ith iron ions. Num bers in circle represent the num bers of the various sam pling
stations
Rye. 2. W pływ GA 3 na produkcję pierw otną wód Bałtyku w zbogaconych w jony że­
lazowe. Num ery w kółkach oznaczają punkty pobrania prób
tivity by GA3 in sythetic m edia the question arises w hether or not such
conditions which allow gibberellic acid to facilitate the iron uptake by
phytoplankton occur in Baltic w ater. This question was the subject of some
fu rth e r studies.
In these experim ents th e short and long-term influence of the
<3A3-Fe m ixtu re added to n atu ra l Baltic w ater was investigated during
the whole period of seasonal developm ent of phytoplankton. The obtained
resu lts w ere sim ilar in the short-, as w ell as in th e long-term experim ents.
The data obtained from m easuring the p rim ary production are illustrated
in Fig. 2. The effect of the long-term influence of GA3 on the phyto­
plankton biomass (indexed by the contents of chlorophyll a) is shown
in Tables 3 and 4. On the basis of the presented experim ents it can be
assum ed th a t GA3 can prom ote the developm ent of phytoplankton (pro­
bably by the stim ulation of iron uptake) also in the n atu ra l sea w ater
conditions. N evertheless the stim ulation was not observed in all sea w ater
Table 3
Tabela 3
The effect of gibberellic acid, added to the surface sea w ater on the growth of
phytoplankton expressed by the contents of chlorophyll a
W pływ GA3 dodanego do powierzchniowej w ody m orskiej na rozwój fitoplanktonu
w yrażony poprzez pomiar chlorofilu a
Station
Stacja pobrania
prób
(see Fig. 1)
Contents of
chlorophyll a
(mg/dm3)
Phytoplankton
Surface sea w ater
Fitoplankton
(see Tab. 2)
P ow ierzchniow a woda
morska
Zawartość
chlorofilu a
(mg/dm3)
2
3
4
1
3
I
untreated
plus G A 3
plus Fe
plus G A 3 and Fe
5
I
untreated
plus GAg
plus Fe
plus G A 3 and Fe
0.16
0.13
0.19
0.13
C
untreated
plus G A 3
plus Fe
plus G A 3 and Fe
0.07
0.23
0.23
0.51
II
untreated
plus G A 3
plus Fe
plus Fe and GA3
0.03
0.04
0.07
0.10
5
7
0.15
0.16
0.35
0.48
1
2
3
4
5
II
untreated
plus GA3
plus Fe
plus GA3 and Fe
6
II
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.11
0.13
0.15
0.29
7
D
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.11
0.08
0.21
0.16
5
C
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.07
0.08
0.14
0.30
8
III
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.06
0.16
0.21
0.55
10
IV
untreated
plus GA3
plus Fe
plus GAS and Fe
0.09
0.15
0.27
0.51
2
IV
untreated
plus GAj
plus Fe
plus GA3 and Fe
0.16
0.16
0.20
0.38
D
untreated
plus GAg
plus Fe
plus GAj and Fe
0.12
0.13
0.15
0.24
2
c
untreated
plus GAS
plus Fe
plus GA3 and Fe
0.12
0.11
0.14
0.25
1
IV
untreated
plus GAj
plus Fe
plus GA3 and Fe
0.68
0.21
0.82
0.24
2
0.03
0.03
0.13
0.14
Tabela
4
The effect of gibberellic acid, added to the bottom sea w ater on the grow th of phy­
toplankton expressed by the contents of chlorophyll a
W pływ kw asu giberelow ego dodanego do przydennej w ody morskiej na w zrost fitoplanktonu obserwow any poprzez pom iar chlorofilu a
Station
Stacja pobrania
prób
(see Fig. 1)
Phytoplankton
Fitoplankton
Contents of the
chlorophyll a
Bottom sea w ater
Woda przydenna
Zawartość
chlorofilu, a
(mg/dm®)
3
4
(see Tab. 2)
1
2
I
untreated
plus GAg
plus Fe
plus GA3 and Fe
0.08
0.08
0.14
0.25
4
I
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.10
0.22
0.31
0.37
5
I
untreated
plus GAg
plus Fe
plus GAj and Fe
0.14
0.16
0.19
0.40
c
untreated
plus GAS
plus Fe
plus GA3 and Fe
0.07
0.12
0.08
0.29
4
c
untreated
plus GAg
plus Fe
plus GA3 and Fe
0.06
0.05
0.13
0.17
5
D
untreated
plus GAj
plus Fe
plus GA3 and Fe
0.21
0.21
0.30
0.50
I
untreated
plus GAg
plus Fe
plus GAS and Fe
0.28
0.31
0.32
0.33
3
3
6
1
2
3
4
II
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.13
0.09
0.16
0.37
6
II
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.07
0.06
0.32
0.16
6
D
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.10
0.09
0.39
0.24
III
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.24
0.29
0.61
0.89
8
III
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.21
0.34
0.45
0.79
9
III
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.21
0.34
0.45
0.79
IV
untreated
plus GA3
plus Fe
plus GA3 and Fe
0.15
0.25
0.24
0.59
7
8
10
sam ples exam ined. Depending on the place and tim e of sam pling GA3
stim ulates, inhibits or has no influence on the production of biomass
(Fig. 2) and on photosynthesis (Fig. 3). Results can not be correlated to the
particular sampling place or to the specific tim e of collecting the w ater
samples. N evertheless all samples in which GA3 stim ulated the biomass
production also exhibited stim ulated photosynthesis.
A t the p resent stage of our studies we are not able to explain the
reasons for the divergent reaction of phytoplankton in various sea w ater
samples to the action of gibberellic acid. N um erous factors can here play
a role such as: type of phytoplankton population, the physiological state
of these organisms, the varying pH of sea w ater, the presence of some
inhibitors etc. U ndoubtedly the changing pH of sea w ater m ight play a
m ajor role. F u rth e r studies are needed to clear up all these questions.
On the basic of the results presented we postulate th a t gibberellic
acid stim ulates the iron uptake, m ost probably due to the form ation of
assim ilable GA3-iron complex, and th a t this stim ulation enhances the
phytoplankton developm ent which properties m ight reflect some phe­
nomena occurring in n atu ral environm ents.
I
1 S E A W A TE R
Y ///X - CONlROLlsea
♦
5 0 ; j M F e . 6 • 10"jjM
GA 3
w a t e r * 50 pMFe )
Fig. 3. Seasonal changes in the growth reaction of phytoplankton to the exogeneously
applied GA3 (the concentration of phytoplankton cells m easured by the chlorophyll
content)
A, B, C — different periods of testing, ch a — chlorophyll a
Rye. 3. Zm iany sezonow e w reakcji wzrostow ej fitoplanktonu na działanie egzo­
gennego GA3 (zagęszczenie kom órek fitoplanktonu oznaczono na podstaw ie zaw ar­
tości chlorofilu)
S — O ceanologia N r 11
OCEANOLOGIA N r 11 (1979)
110
BARBARA MALEWICZ
P olska A kadem ia Nauk
Zakład Oceanologii — Sopot
TERESA KENTZER
U niw ersytet Gdański
Instytut Biologii — Gdańsk
ALICJA KOSSAKOWSKA
DANUTA GĘDZIOROWSKA
Polska A kadem ia Nauk
Zakład Oceanologii — Sopot
WPŁYW KWASU GIBERELOWEGO NA POBIERANIE ŻELAZA
PRZEZ FITOPLANKTON BAŁTYKU
Streszczenie
Zbadano w p ływ kw asu giberelow ego (GAS) na pobieranie żelaza i na w ielkość
produkcji pierwotnej dla fitoplanktonu bałtyckiego oraz standardow ych kultur fitoplanktonu inkubow anych w środowisku naturalnej w ody morskiej i na podłożach
syntetycznych.
Pobieranie żelaza m ierzono stosując izotop 69Fe. Produkcję pierwotną mierzono
m etodą 14C, a w ielkość biom asy określano poprzez pomiar zaw artości chlorofilu a.
Stwierdzono, że GA3 w yraźnie stym uluje pobieranie żelaza przez fitoplankton
i że zjaw isko to zachodzi w ściśle określonym zakresie pH: 6—6,5. Bardzo praw do­
podobne jest, że zjaw isko to polega na tworzeniu się przysw ajalnego kom pleksu
GAS-F e i że stym ulujący efekt GA3 na w zrost fitoplanktonu polega na zw iększeniu
pobierania żelaza przez kom órki fitoplanktonu.
W pływ GAS na pobieranie żelaza przez fitoplankton inkubow any w naturalnej
w odzie m orskiej ibył różny i zależał od sezonu i m iejsca pobrania prób. W pracy
przedyskutowano, które z czynników środowiska mogą w pływ ać w istotny sposób
na działanie GA3 na fitoplankton w naturalnych warunkach.
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