J. Plant Physiol. 157.

395-403 (2000)
Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp
JOURNAL OF
PLANT PHYSIOLOGY
Remedying the iron-deficient maize plants grown at lower than the
optimal temperature and irradiance by new synthetic macromolecular
iron-chelating agents
Milena Ignatova" George Ignatov
h
, Valya Vassileva
2
, Nevena Manolova" Ilia Rashkov
1
1 Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2 Institute of Plant Physiology Acad. M. Popov, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., BI. 21, 1113 Sofia, Bulgaria
Received February 17, 2000 . Accepted June 21, 2000
Summary
The efficacy of Fe
3
+ complexes of polyethers having chelating terminal S-quinolinol (SOOH) residues
for remedying the iron-deficient maize plants at temperatures of 20/16 C and photosynthetic photon
flux density (PPFo) 500-600 I1mol m-
2
S-1 was evaluated by their effect on the fresh and dry weight,
pigment content (total chlorophyll and carotenoids), chlorophyll fluorescence, and photosynthetic
activity of maize plants. In order to estimate the effect of the oligomer nature of the polyethers with
SOOH group attached to the oxyethylene chain at different positions of aromatic ring, tests on
chlorotic plants were also performed with Fe
3
+ complexes of low-molecular-weight ligand SOOH,
and mixtures of commercial polyethers with isopropylamino end-groups (Jeffamines ED) and SOOH
(Jeff/SOOH). The efficacy of Fe
3
+ chelates of synthetic chelator ethylenediaminetetraacetic acid and
FeCI
3
6H
2
0 for remedying the chlorotic maize plants under the same conditions was also tested. At
temperatures of 20/16C and PPFo 500-600I1molm-2s-1, the Fe
3
+ chelates of polymers with SOOH
groups attached at 5-position were the most effective for remedying the iron-deficient maize plants
compared to the other tested Fe
3
+ complexes. It was found that the plant remedy process was sen-
sitive to lowering of the temperature and PPFD. The remedy of chlorotic maize plants supplied with
investigated Fe
3
+ complexes at 20/16C (day/night) and PPFo 500-600I1molm-2s-1 retarded 4-fold
compared to that observed at 30/25C and PPFo 1100-1300I1molm-2s-1. We concluded that the
longer period for remedying the iron-deficient maize plants at lower than the optimal temperature
and PPFo is probably due to the fact that, under these conditions, the amount of oMA released from
roots is several times lower, and the efficiency of the high-affinity system for uptake of Fe
3
+ -PS is de-
creased as compared to that at optimal temperature and PPFo.
Key words: iron-deficiency - maize plants - polymeric iron-chelating agents - polyethers - remedy -
lower than the optimal temperature and photosynthetic photon flux density.
* E-mail correspondingauthor:ignato@bio25.bas.bg.
0176-1617/00/157/04-395 $15.0010
396 Milena Ignatova et al.
Abbreviations: DMA 2'-deoxymugineic acid. - EDTA ethylenediaminetetraacetic acid. - Fa minimal
fluorescence. - Fm maximal fluorescence. - Fv variable fluorescence. - MA mugineic acid. - PPFD
photosynthetic photon flux density. - PS phytosiderophore. - 800H 8-quinolinol. - Jeff/800H mix-
ture of commercial polyethers with isopropylamino end-groups Jeffamines ED and 800H. - OO-P"o-
00 tetraethyleneglycol bis-(8-quinolyl) ether. - HOO-P-OOH modified with 5-chloromethyl-8-quinoli-
nol hydrochloride Jeffamines ED.
Introduction
Maize, like other iron-deficient graminaceous plants, was clas-
sified as a strategy II plant that acquires Fe by the release of
phytosiderophore (PS) to the rhizosphere and by the induction
of a high-affinity system for Fe
3
+ -PS transport across the
plasma membrane of root cells (Marschner et al. 1986, 1989,
Marschner and R6mheld 1994). It has been found that the type
of PS released by maize is 2'-deoxymugineic acid (DMA)
(Mori and Nishizawa 1987).
Chelators addition to the soils or release by the roots facili -
tates the movement of metal ions to the root surface (Lindsay
1974). The iron supplied from microbial siderophores and
synthetic chelators is taken up as Fe
3
+ -PS by barley, corn,
and probably also by graminaceous plants after ligand ex-
change with free PS (Yehuda et al. 1996). It has been sug-
gested that Fe
3
+ complexes of microbial siderophores and
synthetic chelators with effective ligand exchange with free
PS are good iron sources for Graminaceae.
Water-solubility of the metal ion complexes of synthetic
chelators is a prerequisite for plant nutrition. Previously, we
have synthesized water-soluble macromolecular chelating
agents in which 8-quinolinol (800H) derivatives are attached
to hydrophilic chain of oxyethylene units, and found that their
Fe
3
+ complexes are water-soluble (Rashkov et al. 1993, An-
gelova et al. 1995, Manolova et al. 1998). These properties
are highly promising for their use in providing iron nutrition to
plants.
Recently, the efficacy of Fe
3
+ complexes of two types of
macromolecular chelators for remedying the iron-deficient
maize plants was evaluated (Ignatova et al. 2000 b). In the
first polymeriC chelating agent, denoted as OO-P-OO, the
end-groups are bound with a polyether chain through an
ether bond at 8-position in quinol inol moiety. In this case, com-
plexation with metal ions may proceed through the ether bond
oxygen atom and the nitrogen atom from the quinolinol ring.
QO-P-OQ
o-0-(CH2CH20 )nCH2CH20{)
<ON NO
In the second type of macromolecular chelator, HOO-P-
OOH, 8-quinolinol moiety is bound at 5-position to a polyet-
her chain:
ND HOQ-P-QOH <ON

in order to leave the hydroxyl group and nitrogen atom free
for complexation with metal ions. The efficacy of these chela-
tors was compared with that of the synthetic chelator ethyl-
enediaminetetraacetic acid (EDTA), low-molecular-weight lig-
and 800H, mixture of commercial polyethers with isopropyl-
amino end-groups (Jeffamines ED) and 800H (Jeff/800H)
or FeCI
3
' 6H
2
0. We have found that at temperatures of 30/
25 C (day/night) and photosynthetic photon flux density
(PPFD) 1100-1300 m-
2
s-1, the chlorotic maize plants
supplied with Fe
3
+ complexes of investigated synthetic chela-
tors recovered for 4 days of iron re-supply. It was also
shown that the Fe
3
+ chelates of HOO-P-OOH polymers were
the most effective for remedying the iron-deficient plants
compared to the other tested Fe
3
+ complexes. Under the
same conditions, the efficacy of Fe
3
+ -OO-P-OO and Fe
3
+-
EDTA was lower in comparison to the complexes of HOO-P-
OOH.
It has been found that for various iron-deficient graminace-
ous speCies the diurnal rhythm of PS release is different. The
secretion of PS from iron-deficient barley and wheat roots has
been found to occur within a specific period of 4 to 6 h ,
usually beginning about 2 h after the onset of light (Takagi et
al. 1984, Marschner et al. 1986, Zhang et al. 1991). Yehuda et
al. (1996) have shown a constant rate of PS release in corn
during 14 h of growth under light. It has been found that the
temperature rather than a light signal is the trigger for the ini-
tiation of PS mugineic acid (MA) release (Mori 1994). Taking
into account the investigations of these authors, the present
work studied the influence of lower than optimal temperatures
of 20/16 C (day/night) and PPFD 500-600 m-
2
s-' on
the efficacy of Fe
3
+ chelates of HOO-P-OOH and OO-P-OO
for remedying the chlorotic maize plants. The effect of the
type of bonding between the ligand and the polymer carrier,
its complexing ability, and polymer chain length on remedy-
ing the iron-deficient plants was also investigated.
Remedying the iron-deficient maize plants at low temperature and irradiance 397
Materials and Methods
Materials
Commercial polyethers having isopropylamino end-groups (Jeffamine
ED-600, Jeffamine ED-900 and Jeffamine ED-2000), 8-quinolinol
(800H), EDTA (disodium salt), tetraethyleneglycol bis-(8-quinolyl)
ether (00-P
110
-OO), and FeCI
3
6H
2
0 were purchased from Fluka
and used as received.
Preparation of HOQ-P-QOH polymers
Modified polyethers were prepared by interaction of Jeffamines ED
(600, 900 or 2000) and 5-chloromethyl-8-quinolinol hydrochloride as
previously described (Manolova at al. 1998). The mOlecular weight
characteristics for HOO-P-OOH are as follows: Mn (Vapour pressure
osmometry, chloroform, 45 'C) 1050, 1110, and 1900, respectively for
HOO-P
600
-OOH, HOO-Pgoo-OOH, and HOO-P
2000
-OOH. The subscript
number denotes the molecular weight for the commercial Jeffamine
ED used as starting material in the syntheses of HOO-P-OOH. The
average degree of substitution calculated from 1H NMR spectra
(given in respect to four OOH-substituents per chain) is 80, 42, and
43 % respectively.
ligand exchange experiment
The ligand exchange of the investigated Fe
3
+ complexes (with HOO-
P-OOH, 00-P
110
-OO, 800H, or EDTA) was studied by UV-VIS spec-
trometry (Carl Zeiss - Jena, Germany) at 20 C. The UV-VIS spectra of
an aqueous solution containing a ligand and Fe
3
+ in 1 : 1 mole ratio
were monitored before and after addition of the second ligand, in the
same concentration (2mmoI/L) as the first ligand, in order to evaluate
the competitive complexation. Fe
3
+ solutions were prepared with
FeCI
3
6H
2
0. The ionic strength was maintained at 0.1 mol/L with KCI.
Determination of stability constants of the Fe
3
+
complexes of HOQ-P-QOH and QO-P-OQ
The stability constants of Fe
3
+ complexes of HOO-P-OOH and OO-P-
00 polymers were studied potentiometrically using a reference silver-
silver electrode and a platinum electrode, at a temperature of 20'C
and ionic strength 11 = 0.1 (KCI), as recently described (Ignatova et al.
2000a). Experiments were carried out by the addition of an aqueous
solution of ligand (varying the ligand concentration from 1.61lmo1/L to
170llmol/L) to a 25 mL aqueous solution of FeCI
3
' 6H
2
0 with a con-
centration of 431lmo1/L. The potential of the platinum electrode was
measured with a potentiometer MV 870 digital Pracitronic, Dresden,
Germany, with a precision of lOO mV. The pH values were meas-
ured with a pH meter (OP-265 with glass electrode, Radelkis, Buda-
pest, Hungary), with a precision of 0.01.
Plant material and growth conditions
Seeds of Zea mays L. cv. Kneja 611 were surface sterilized with 70 %
ethanol (v/v) for 60min, rinsed four times with distilled water, and then
germinated on wet filter paper at 25 C. After 7 days, the seedlings
were transferred to l2 L plastic growth pots (two plants per pot) con-
taining iron-free nutrient solution. The nutrient solution had the follow-
ing composition (in mmol/L): Ca(N0
3
l2' 4H
2
0 (3.0), MgS0
4
' 7H
2
0
(0.5), KH
2
P0
4
(lO); micronutrients according to Hoagland and Arnon
(1950). Plants were grown in a naturally illuminated greenhouse at 201
16'C (day/night) and PPFD 500-600 Ilmol m-
2
S-1. The nutrient solu-
tion was continuously aerated, changed every 3 days, and the pH
maintained at 6.0.
Supplementation of iron-deficient maize plants with
Fe
3
+ complexes
Control plants were grown on nutrient solution with a supplement of
0.18 mmol/L Fe
3
+, added as Fe-EDTA (treatment 1). Iron-deficiency
symptoms were well expressed 22 days after sowing. The chlorotic
plants were divided into eleven groups. Nine of them were placed
into a nutrient solution containing ferric complexes: Fe-EDTA (treat-
ment 2); Fe-HOO-P600-00H (treatment 3); Fe-HOO-Pgoo-OOH (treat-
ment 4); Fe-HOO-P2000-00H (treatment 5); Fe-00-P110-00 (treatment
6); Fe-mixture of Jeffamine ED-600 and 800H (Jeff600/80) (treatment
7); Fe-mixture of Jeffamine ED-900 and 800H (Jeff900/800H) (treat-
ment 8); Fe-mixture of Jeffamine EO-2000 and 800H (Jeff2000/80)
(treatment 9); Fe-800H (treatment 10). The concentration of 800H
(free or attached) was the same as in treatments 3-10. Another group
of chlorotic plants was transferred to a nutrient solution supplied with
0.18 mmol/L FeCI
3
' 6H
2
0 (treatment 11). A group of chlorotic plants
remained in an iron-free nutrient solution (treatment 12).
The Fe
3
+ complexes were prepared by mixing the aqueous solu-
tions of EDTA, HOO-P-OOH polymers, 800H, or Jeff/800H with Fe
3
+
solutions of a molar ratio ligand: iron of 1 : 1 and an iron concentration
of 0.18 mmol/L. Fe
3
+ solutions were prepared with FeCI
3
6H
2
0. All
treatments included ten replications. The measu.rements of fresh
weight, chlorophyll content, and photosynthesis were made four
times at intervals of 4 days, starting 22 days after seeds were sown.
For pigment and photosynthetic measurements, the most recently
matured re-greening leaf (fourth leaf acropetally numbered) was
used.
Root and shoot growth parameters
Shoots and roots were harvested at various times after iron re-supply
and their fresh weight was measured immediately. The shoots and
roots were oven-dried at 80'C to a constant weight for determination
of their dry weight. Data were expressed as means SE from five in-
dependent experiments.
CO
2
exchange measurements
Net photosynthetic CO
2
uptake of maize leaves was measured using
a portable photosynthesis apparatus (LI 6000, Li-Cor, Lincoln, NE,
USA). Plant leaves were placed in a 0.25 L chamber. Ouantum flux
density was 1100-1300 Ilmol m-
2
S-1 photosynthetic active radiation.
Fluorescence measurements
Chlorophyll fluorescence was measured at room temperature using
fluorimeter PAM 101 (H. Walz, Germany). Minimal fluorescence Fo
(chlorophyll fluorescence when all photosystem II reaction centres
are open) was determined by applying a weak modulated light
(O.4llmol m-
2
S-1) for at least 30 min to leaves previously kept in dark.
39S Milena Ignatova et al.
Maximal fluorescence in dark-acclimated leaves (Fm) was induced by
a short pulse (0.7s) of more than 4000Ilmolm-2s-1. Variable fluores-
cence (Fv) was calculated as Fm-FQ. The data of FvlFm (maximal pho-
tochemical yield of photosystem II in dark-acclimated leaves) were
also presented.
Determination of leaf pigment composition
After extraction with 80 % acetone, the chlorophyll and carotenoid
content was analysed according to Arnon (1949).
Leaf area measurement
The leaf area was measured using an Mk2 area meter (Oelta-T De-
vices Ltd. Cambridge, UK) connected to a TC7000 Series Camera
(Burle Industries Inc., Lancaster, PA, USA).
Results
Effect of Fe
3
+ complexes on the pigment content
The Fe
3
+ complexes may be divided into two groups accord-
ing to the rate of accumulation of total chlorophyll in the
leaves of chlorotic maize plants supplemented with
Fe
3
+ chelates at 20/16 'c and PPFD 500-600 J.l.mol m-
2
S-1.
Fe
3
+ -HOO-P-OOH, Fe
3
+ -SOOH, and Fe
3
+ -Jeff/SOOH in-
creased the total chlorophyll content up to the control value
after S days of iron re-supply, for the next S days the treated
plants showed higher chlorophyll content than the controls
(Figs. 1 A and 2 A). Chlorophyll accumulated more slowly in
the leaves of chlorotic plants supplied with the second group
of chelates (Fe
3
+ -EDTA and Fe
3
+ -OO-P110-00). The total
chlorophyll content in the plants treated with these com-
plexes was equal to the controls on the 12th day of iron re-
supply, and slightly higher than the controls on the 16
th
day.
The total chlorophyll content in the chlorotic and control
plants did not significantly change within the investigated pe-
riod of 16 days (Figs. 1 A and 2 A). In 3S-day-old plants, the
chlorophyll and carotenoid levels in all treatments were
higher as compared to the controls (Fig. 3 A). The total
chlorophyll content was highest in the leaves of plants
treated with Fe
3
+ -HOO-P
600
-OOH (143 % of the control value)
or with Fe
3
+ -HOO-P
2000
-OOH (144 % of the control value). In
the cases of plants supplied with Fe
3
+complexes of HOO-
Pgoo-OOH, Jeff/SOOH, SOOH, and FeCI
3
' 6H
2
0, the total
chlorophyll content was 132 % to 136 % of the control value.
Chlorophyll level was lowest in the iron-deficient plants
treated with Fe
3
+ -EDTA (116 % of the control value) and Fe
3
+-
OO-P11O-00 (119 % of the control value). As shown in Fig.
3 A, carotenoid content in the leaves of plants supplemented
with Fe
3
+ -HOO-P
600
-OOH, Fe
3
+ -HOO-Pgoo-OOH, and Fe
3
+-
HOO-P
2000
-OOH was the highest (165 %, 160 %, and 166 % of
control, respectively). In the leaves of plants grown on an
iron-free medium, the total chlorophyll and carotenoid con-
Figure 1. Changes in total chlorophyll content (A), in photosynthesis
(8) and in fresh weight (e) of maize plants supplied from the 22
nd
to
38
th
day with 0.18 mmol/L Fe
3
+ chelates. Experimental conditions: nu-
trient solution pH 6.0, temperature 20/16'C (day/night), PPFD 500-
600 Ilmol m-
2
S-1. Chlorotic plants treated with Fe
3
+ -HOQ-P
600
-QOH
(e), Fe
3
+ -800H (T), Fe
3
+ -EOTA (_), control plants (A), and chlorotic
plants (grown during the experiments in iron-free medium) (0). Vert-
ical bars represent the means SE, n = 5. FW, fresh weight.
tents was the lowest (S % and 31 % of the control value, re-
spectively).
Effect of Fe
3
+ complexes on the photosynthetic
intensity
The rate of photosynthesis at temperatures of 20/16 'c (day/
night) and PPFD 500-600 J.l.mol m-
2
S-1 was the highest after
treatment with chelates of HOO-P-OOH (Figs. 1 B, 2 Band
3 B). Photosynthetic intensity of chlorotic plants supplied with
Fe
3
+ -HOO-P-OOH reached the control level after 6 days of
iron re-supply and higher levels for the next 10 days. The
Remedying the iron-deficient maize plants at low temperature and irradiance 399
:c
+
s
l:[

.... Cl
.2 Cl
E
() .......
r
'" '",
'iii <)I
CIl E
ON
>()
s-
_ 0
o E
:::!.
0.. .......
l:
Cl
om
1:
ca
'"
Q.
CIl
....
.9 u.
3
2
0
30
20
10
0
50
40
30
20
10
00
A
B
C
22 24 26 28 30 32 34 36 38
Plant age (days)
Figure 2. Changes in total chlorophyll content (A). in photosynthesis
(8) and in fresh weight (C) of maize plants supplied from the 22
nd
to
3S
th
day with 0.1S mmol/L Fe
3
+ chelates. Experimental conditions: nu-
trient solution pH 6.0. temperature 20/16'C (day/night), PPFD 500-
600 Jlmol m-
2
S-1. Chlorotic plants treated with HOO-P
600
-OOH (e).
Fe
3
+ -OO-P110-00 (.0,), Fe
3
+ .Jeff600/S00H (V), control plants (A),
and chlorotic plants (grown during the experiments in iron-free me-
dium) (0). Vertical bars represent the means SE; n = 5. FW, fresh
weight.
change of photosynthetic activity of chlorotic plants treated
with Fe
3
+ -EDTA and Fe
3
+ -00-P110-00 was lower compared
to that of Fe
3
+ -HOO-P-OOH (Figs. 1 Band 2 B). Exposure of
chlorotic plants to chelates of 800H or Jeff/800H provided
lower photosynthetic intensity than that of controls and of all
other investigated chelates within the period of 16 days of
iron re-supply.
In 38-day-old plants, the rate of photosynthesis for Fe
3
+
complexes of HOO-P-OOH was the highest: 187 %, 175 %,
and 173 % of the control value for HOO-P
600
-OOH, HOO-PgoO-
OOH and HOO-P
2000
-OOH, respectively (Fig. 3 B). When
chlorotic plants were treated with complexes of 800H, mix-
tures Jeff/800H, and FeCI
3
6H
2
0, the photosynthetic rate
was the lowest - 72 % to 78 % of the control value. Photosyn-
thetic activity in plants grown on an iron-free medium was 6 %
of the control value.
Effect of Fe
3
+ complexes on the plant fresh and dry
weight
The fresh and dry biomass of chlorotic plants supplied with
Fe
3
+ chelates of HOO-P-OOH at temperatures of 20/16'C
(day/night) and PPFD achieved a value
near the controls after a much longer period -16 days of iron
re-supply (Figs. 1 C, 2 C and 4). The fresh weight of iron-defi-
cient plants supplied with all other investigated chelates was
lower than that of controls. Treatment with complex of 00-
:;
'E;..
0
()
I
.,
'iIi
CD

>-
III

.c:
0-
r-
1:

'E
:g,
os
!!?
os
Oi

0
9
8
7
6
5
4
3
2
0
A
o chlorophyll (a+b)
_ carotenoids
2 3 4 5 6 7 8 9 10 11 12
Treatments
Figure 3. Effects of different ferric chelates on (A) total chlorophyll
(a+b) and carotenoids content, (8) net photosynthesis and (C) leaf
area of 3S-day-old maize plants. Treatments: 1 - control plants; 2 -
Fe
3
+ -EOTA; 3 - Fe
3
+ -HOO-P600-00H; 4 - Fe
3
+ -HOO-Pgoo-OOH; 5 -
Fe
3
+ -HOO-P
2000
-OOH; 6 - Fe
3
+ -OO-P110-00; 7 - Fe
3
+ -mixture of
SOOH and Jeffamine EO-600; 8 - Fe
3
+ -mixture of SOOH and Jeff-
amine ED-gOO; 9 - Fe
3
+ mixture of SOOH and Jeffamine EO-2000; 10
- Fe
3
+ -SOOH; 11 - FeCI
3
'6H
2
0; 12 - chlorotic plants; temperature
20/16'C (day/night) and PPFD 500-600 Jlmol m-
2
S-1. Values are
means SE; n = 5. FW, fresh weight.
400 Milena Ignatova et al.
30
A
o roots
_ shoots
-
-
25
:-
c::
'"
a.
20
.2l
-

15 0>
"cp


10 rn
Q)
...
U-
5
0
B
o roots
-
_ shoots
- 3
E
'" a.
.2l
-

Cl
2
"cp


C

2 3 4 5 6 7 8 9 10 11 12
Treatments
Figure4" Effects of different ferric chelates on fresh (A) and dry (8)
weight of roots and shoots of 3S-day-old maize plants. Treatments: 1
- control plants; 2 - Fe
3
+ -EDTA; 3 - Fe
3
+ -HOQ-P600-QOH; 4 - Fe
3
+-
HOO-Pgoo-QOH; 5 -Fe
3
+ -HOQ-P
2000
-QOH; 6 -Fe
3
+ -QO-P110-0Q; 7 -
Fe
3
+ -mi xture of SQOH and Jeffamine ED-600; 8 -Fe
3
+ mixture of
SOOH and Jeffamine ED-gOO; 9 - Fe
3
+ -mixture of SQOH and Jeff-
amine ED-2000; 10 - Fe
3
+ -SQOH; 11 - FeCI
3
6H
2
0 ; 12 - chlorotic
plants; temperature 20/16 C (day/night) and PPFD 500-600 !1mol
m-
2
S l . Values are means SE; n=5.
P
11O
-OO increased the plant fresh and dry weight compared
to that observed in plants receiving EDTA complex. The sup-
ply of the complexes Fe
3
+ -Jeff
6
oo1S00H, Fe
3
+ -Jeff
90
ofSOOH,
Fe
3
+ -Jeff
2
ooo1S00H, Fe
3
+ -SOOH, and FeCI
3
6H
2
0 did not
produce any significant differences in plant fresh and dry
weight compared with the chlorotic plants (Fig. 4).
In 3S-day-old maize plants grown on an iron-free medium,
the shoot/root fresh weight ratio was close to 1.0 (Fig. 4 A).
With the chelates of SOOH, Jeff/800H, and FeCI
3
' 6H
2
0,
which caused insignificant recovery of chlorotic plants, the
shoot/root fresh weight ratio varied from 1.0 to 1.4. Exposure
of chlorotic plants to nutrient solutions with Fe
3
+ complexes
of HOO-P-OOH and 00-P110-00 resulted in a significant in-
crease of shoot and root fresh biomass, with a shoot/root
fresh weight ratio of 1.3 to 1. 8. After treatment with complex
of Fe
3
+ -EDTA, an enhancement of shoot fresh weight was
higher compared to that of the root fresh weight, and the
shoot/root fresh weight ratio was 2.3. There were significant
differences between the shoot and the root dry biomass in all
' investigated treatments.
The fresh and dry weight of plants supplemented with
SOOH, Jeff/SOOH, and FeCI
3
6H20 was lowest (from 12 % to
20 % of the control value) compared to all other investigated
Fe
3
+ chelates. When Fe
3
+ complexes of HOO-P-OOH were
added to the nutrient solution, the fresh and dry biomass of
maize plants was the highest (from 76 % to 94 % of the control
value). After treatment of iron-deficient plants with Fe
3
+ -00-
P 110-00, the fresh and dry biomass was 74% and 69 % of the
control value, respectively, while after application of Fe
3
+-
EDTA - 56 % and 50 % of the control. Cultivation of plants in
an iron-free medium resulted in a fresh and dry weight pro-
duction of only 11 % and 7% of the control value, respectively.
Effect of Fe3+ complexes on the leaf area
The leaf area of all treatments was lower than that of the con-
trol plants within a period of 16 days of iron re-supply (Fig.
3 C). The leaf area of iron-deficient plants supplied with che-
lates of HOO-P-OOH was close to that of the control plants
(92 % to 94 % of the control value). Upon treatment with Fe
3
+-
00-P
110
-OO and Fe
3
+ -EDTA, the leaf area was 76 % and 65 %
of the control value, respectively. Chlorotic plants supplied with
Fe
3
+ complexes of SOOH, Jeff/SOOH, and FeCI
3
' 6H
2
0
showed the lowest leaf area (15-20 % of the control value).
When plants were grown on an iron-free medium, the leaf
area was 10 % of the control value.
Effect of the Fe
3
+ complexes on the chlorophyll
fluorescence parameters
The parameters of chlorophyll fluorescence F
o
, F
m
, F
v
, and FvI
Fm measured in leaves of plants were presented in Table 1.
The fluorescence parameters Fm and Fv in chlorotic plants
were lower than that in controls and in iron-deficient plants re-
ceiving Fe
3
+ complexes. Maize plants grown on an iron-free
medium showed the highest value for Fo.
The FjFm values were similar for control plants and for
chlorotic plants supplied with Fe
3
+ -HOO-P600-00H, Fe
3
+-
00-P
110
-OO, Fe
3
+ -SOOH, Fe
3
+ -Jeff/SOOH, and Fe
3
+ -EDTA.
The FjFm ratio measured in the leaves of chlorotic plants was
61 % of the control value.
Remedying the iron-deficient maize plants at low temperature and irradiance 401
Table 1. Chlorophyll fluorescence parameters: Fa, F
v
, F
m
, FvfF
m
of
leaves of maize plants (cv. Kneja 611) supplied from the 22
nd
to 38
th
day with 0.18 mmol/L Fe
3
+ complexes. Control plants were grown in a
nutrient solution supplied with 0.18 mmol/L Fe
3
+ -EDTA. Chlorotic
plants were grown on an iron-free nutrient solution. Experimental con-
ditions: nutrient solution pH 6.0, temperature 20/16 'C (day/night),
PPFD 500-600 I!mol m-
2
S-1. All values are means SE, n = 5.
Treatments Parameters
Fa %of Fv Fm FvfFm
%of
control control
Control plants 736.4 100 2787.0 3523.4 0.791 100
25.3 63.1 69.5 0.005
Fe
3
+ -EDTA 754.2 102 3269.4 4023.6 0.813 103
31.3 69.5 65.3 0.003
Fe
3
+ -HOO-P6oo-00H 740.2 101 3301.1 4041.3 0.817 103
29.6 73.1 70.8 0.001
Fe
3
+ -OO-P 110-00 749.1 102 3235.5 3984.6 0.812 103
25.1 56.7 71.9 0.004
Fe
3
+ -Jeff600/800H 798.7 108 3175.1 3973.8 0.799 101
36.1 67.8 69.3 0.002
Fe
3
+-800H 787.8 107 3170.9 3958.7 0.801 101
33.5 59.5 79.3 0.005
Chlorotic plants 891.0 121 832.5 1723.5 0.483 61
47.1 23.5 27.5 0.001
Discussion
In the present work, the efficacy of the new synthetic macro-
molecular iron-chelating agents for remedying the iron-defi-
cient maize plants at temperatures of 20/16 'C (day/night)
and PPFD 500-600 Ilmol m-
2
S-1 has been investigated. The
changes in the growth and photosynthetic parameters of
3S-day-old maize plants, as well as in dynamics (within a pe-
riod of 16 days), were also studied. In our previous paper
(Ignatova et al. 2000 b), we have shown the influence of
Fe
3
+ chelates on remedying the chlorotic maize plants at tem-
peratures of 30/25 'c (day/night) and PPFD 1100-1300llmol
m-
2
S-1. Under these conditions, chlorotic plants supplied
with Fe
3
+ -HOQ-P-QOH achieved the photosynthetic intensity
of the controls after one day of iron re-supply. The values of
leaf chlorophyll content and plant fresh weight became close
to the control values after 2 days and 4 days of iron re-
supply, respectively. The total chlorophyll content and photo-
synthesis of the plants supplied with Fe
3
+ -QO-P110-0Q, Fe
3
+-
EDTA, Fe
3
+ -Jeff/SQOH, and Fe
3
+ -SQOH became close to the
control values after 3 days and 2 days of iron re-supply, re-
spectively. The fresh weight of these plants did not achieve
the control value after 4 days of iron re-supply.
The results obtained from the previous and the present
study show that at 20/16 'C and PPFD 500-600 Ilmol m-
2
s-1,
the remedying of chlorotic maize plants supplied with tested
Fe
3
+ chelates was less pronounced and was retarded 4-fold
compared to that at 30/25 'C and PPFD 1100-1300llmol
m-
2
S-1. At temperatures of 20/16 'C (day/night) and PPFD
500-600 Ilmol m-
2
S-1, Fe
3
+ complexes of HOQ-P-QOH com-
pletely remedied the iron-deficient plants more rapidly and
their efficacy was more pronounced compared to the other
treatments. The efficacy of Fe
3
+ -HOQ-P600-QOH, Fe
3
+ -HOQ-
Pgoo-QOH and Fe
3
+ -HOQ-P
2000
-QOH complexes was almost
equal and was influenced by the polyether chain length of
these polymers. The efficacy of Fe
3
+ -EDTA and Fe
3
+ -QO-
P
110
-OQ was lower and less pronounced than that of Fe
3
+
chelates of HOQ-P-QOH. The complexes of SQOH and Jeff/
SQOH had the lowest efficacy on remedying the iron-defi-
cient plants compared to the other tested chelates. It has
been found that in animal cells, 8QOH chelates penetrating
across the plasma membrane have a toxic effect and cause
cellular damage (Kontoghiorghes 1988). It may be proposed
that the efficacy of chelates of 8QOH or Jeff/8QOH in reme-
dying the chlorotic maize plants is due to direct transport
across the plasma membrane of plant roots. However, the
toxic side effect of those chelates caused the lower efficacy
in remedying the chlorotic plants, resulting in decreased pho-
tosynthetic activity and plant biomass accumulation, despite
high pigment content. The fresh weight of iron-deficient
plants supplied with Fe
3
+ -8QOH or Fe
3
+ -Jeff/8QOH during
the 16 days period at 20/16 'c and PPFD 500-6001lmol m-
2
s-
1
increased only a 1.5- to 1.8-fold in comparison to the chlorotic
plants. Previously (Ignatova et al. 2000b), we found that at 30/
25 'c and PPFD 1100-1300 Ilmol m-
2
S-1 the fresh weight of
iron-deficient plants, treated with Fe
3
+ -8QOH or Fe
3
+ -Jeff/
8QOH, increases 2.4- to 2.7-fold compared to the chlorotic
plants within a period of 4 days. Therefore, at a lower temper-
ature and PPFD, the harmful effect of Fe
3
+ complexes of 8QOH
or Jeff/8QOH was more pronounced.
It has been shown that the parameters of chlorophyll fluo-
rescence (Fa, F
v
, Fm and FvlFm ratio) are sensitive to the
stress environmental factors (Ogren 1988, Bolhar-Norden-
kampf et al. 19S9, Lanaras et al. 1993). In our experiments,
an increase in Fa value and a decrease in FvlFm ratio, meas-
ured in the leaves of chlorotic plants, indicates damage to
photosystem II. In the case of iron-deficient plants, supplied
with Fe
3
+ complexes, and grown at 20/16 'c and PPFD 500-
600llmoi m-
2
S-1, the parameters of chlorophyll fluorescence
did not change Significantly compared to the same param-
eters at optimal temperatures and PPFD. This indicates that
decreasing the temperature by 10 'C and lowering the PPFD
from 1100-1300 Ilmol m-
2
S-1 to 500-600 Ilmol m-
2
S-1 does
not cause a stress effect on the growth of the remedied
chlorotic maize plants.
According to Buyer and Sikora (1990) and Yehuda et al.
(1996), the rate and effectiveness of Fe uptake from synthetic
chelators and microbial siderophores in graminaceous plants
402 Milena Ignatova et al.
is controlled by the following factors: concentration of the
complex Fe
3
+ -ligand, stability constants of the various lig-
ands able to form a complex with Fe
3
+ or with other com-
petitive metals, kinetics of ligand exchange between Fe
3
+-
chelating compound and free PS, amount of PS released,
and efficiency of the Fe
3
+ -PS uptake system.
The concentration of the investigated synthetic ligands in
experiments carried out at temperatures of 20/16 C and
PPFD 500-600 Ilmol m-
2
S-1 was the same as that of syn-
thetic ligands used in the experiments carried out at temper-
atures of 30/25C and PPFD 1100-1300 Ilmol m-
2
S-1. From
the ligand exchange experiments at two different tempera-
tures, 30C and 20C, it was found that EDTA was exchanged
by 8QOH, but HOQ-P-QOH and QO-P-OQ did not exchange
EDTA. It was also observed that HOQ-P-QOH exchanged
QO-P-OQ from their Fe
3
+ complexes. The reported stability
constant for Fe
3
+ chelates of DMA is logp = 33.3 (Murakami et
al. 1989), of EDTA is 10gP = 25.1 (Bottari and Anderegg 1967),
and of 8QOH is logP3 = 36.9 (Martell and Smith 1974). Pre-
viously (Ignatova et al. 2000 a), the stability constants of
Fe
3
+ -HOQ-P
600
-QOH (logP1 = 14.16, logP2 = 18.57), Fe
3
+-
HOQ-Pgoo-QOH (logP1 = 14.07, logP2 = 17.88), Fe
3
+ -HOQ-
P
2ooo
-QOH (lOgP1 = 14.51, logP2 = 19.34), and Fe
3
+-QO-P
110
-
OQ (logP1 = 13.52, logP2 = 17.62) were determined potentio-
metrically. The apparent stability constants of the studied
Fe
3
+ complexes at pH 6.0 and ionic strength 11 = 0.1 were
calculated from the relationship: P'M'L' = PML/<XM<XL, where P'M'L'
is the apparent stability constant, PML is the stability constant,
<XM at pH 6.0 was calculated to be 5.01 x 10
5
. We presumed
that the proton dissociation constants of HOQ-P-QOH and
QO-P-OQ polymers were equal to proton dissociation con-
stants of the free ligand 8QOH (pK1 = 4.99, pK2 = 9.66, 25C,
11 = 0.1) (Smith and Martell 1975). Taking into account the pro-
ton dissociation constant of 8QOH, the <XL coefficient (Ring-
bom 1963) at pH 6.0 was calculated to be 5.02 x 10
3
. From
the reported proton dissociation constants of DMA pK1 =
3.19, pK2 = 8.25 and pK3 = 10.0, (Murakami et al. 1989), the
<XL coefficient (Ringbom 1963) at pH 6.0 was calculated to be
1.79 x 10
6
. The calculated values of apparent stability con-
stants at pH 6.0 and 11 = 0.1 were: 10gP' = 21.35 for Fe
3
+-
DMA; 10gP' = 19.4 for Fe
3
+ -EDTA; logP3' = 27.5 for Fe
3
+-
8QOH; logP1' = 4.12, logP2' = 8.22 for Fe
3
+ -QO-P110-0Q;
10gP{ = 4.76, logP2' = 9.17 for Fe
3
+ -HOQ-P600-QOH; 10gP{ =
4.67, logP2' = 8.48 for Fe
3
+-HOQ-P
goo
-QOH; 10gP{ = 5.11,
logP2' = 9.94 for Fe
3
+ -HOQ-P2000-QOH. From apparent stabil-
ity constants of tested Fe
3
+ chelates and from results of the lig-
and exchange experiments it follows that the stability of the
Fe
3
+ complexes of DMA and investigated chelating agents at
both temperatures decreases in the order: 8QOH > DMA >
EDTA> HOQ-P-QOH > QO-P-OQ. Because of the similar or-
der of ligand exchange at both temperatures, the observed
efficiency of the investigated Fe
3
+ complexes for remedying
the iron-deficient plants at both temperatures also changes in
the same order: Fe
3
+ -HOQ-P-QOH > Fe
3
+ -QO-P-OQ > Fe
3
+-
EDTA > Fe
3
+ .Jeff/8QOH Fe
3
+ -8QOH. Therefore, the con-
centration of the synthetic chelators, the apparent stability
constants of ligands, complexing with Fe
3
+ and kinetics of
ligand exchange between Fe
3
+ -synthetic chelators and free
DMA did not change at different temperatures and PPFD in
the present and previous (Ignatova et al. 2000 b) studies. The
longer period for remedying the iron-deficient maize plants at
20/16C and PPFD 500-6001lmol m-
2
S-1 is probably due to
the fact that under these conditions the amount of DMA re-
leased from roots is several times lower and the efficiency of
the high-affinity system for uptake of Fe
3
+ -PS is decreased as
compared to that at 30/25 C and PPFD 1100-1300 Ilmol
m -2 s -1. This assumption is in accordance with the sugges-
tions that initiation of PS release depends on temperature
and light intensity (Takagi et al. 1984, Marschner et al. 1986,
Zhang et al. 1991, Mori 1994, Yehuda et al. 1996).
Acknowledgements. Financial support from the Bulgarian National
Science Fund is gratefully acknowledged (Contract CH-818).
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