Download Light-Dependent Iron Transport into Isolated Barley Chloroplasts

Plant Cell Physiol. 38(1): 101-105 (1997)
JSPP © 1997
Light-Dependent Iron Transport into Isolated Barley Chloroplasts
Naimatullah Bughio, Michiko Takahashi, Esturo Yoshimura, Naoko-Kishi Nishizawa and Satoshi Mori
Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1
Bunkyo-ku, Tokyo, 113 Japan
Translocation studies of 59Fe(IH)-epihydroxymugineic
acid in intact barley plants revealed that Fe transport from
leaf veins to mesophyll cells is light-regulated. Similarly, Fe
absorption studies with isolated chloroplasts showed that
the Fe influx is light-dependent whereas its efflux occurred
in the dark.
ed not only in roots but also in the xylem sap (256 mM in
Fe-deficient plant) and leaves of barley (Mori et al. 1987),
and 2-deoxymugineic acid in xylem sap (210mM in Fedeficient plant), phloem sap (not detectable in Fe-deficient
plant but 2.26 mM in Fe-sufficient plant) and leaves in rice
(Mori et al. 1991), there is no evidence to show whether Fe
cotransports with MAs in leaf veins or not. It is speculated
that some Fe must be circulating as Fe(III)-MAs in the
veins since almost equimolar amounts of Fe and deoxymugineic acid exist in the phloem sap of Fe-sufficient rice
plants (Mori et al. 1991). Also, environmental conditions
in phloem appear favourable for the transport of MAs in
the shoot: phloem pH is greater than 8 and among natural/
synthetic Fe-chelators, MAs have the highest chelating activity under alkaline conditions (Takagi 1991). Prior to this
report, no information was available on the short term Fe
transport in plant shoots. The problem has been addressed
in the past but the use of other chelators such as Fe-citrate
or Fe-EDTA restricts the rapid absorption and translocation of Fe (Takagi et al. 1984), therefore the immediate response by shoots to the change in their environment is too
slow to be detected. Similarly, the use of FeCl3 alone in
quantitative Fe uptake experiments has a limitation. It is
well known that under neutral pH conditions FeCl3 is immediately transformed into insoluble Fe(OH)3 gels which adsorb to the cell wall, cell membranes and envelopes of cell
organella, and therefore can not be used in Fe transport
studies. Finally, environmental factors which may regulate
the transport of Fe(III)-MAs in plant shoots have not been
studied.
Key words: Autoradiography — Barley — Chloroplast —
59
Fe(III)-epihydroxymugineic acid — Iron-fluxes.
The pioneering work of Takagi (1972, 1976) paved the
way for the establishment of two iron acquisition systems
in the plant kingdom, strategy-I (Romheld and Marschner
1986a) and strategy-II (Romheld and Marschner 1986b).
Strategy-I is typical for dicots and monocots except grasses
and is characterized by increased root Fe(III)-reducing
capacity. On the other hand, strategy-II is associated with
the roots of graminaceous plants. In this system, natural
chelators (MAs) are released from the roots, bind with
sparingly soluble Fe(III) in the medium and convert it to
water soluble Fe(III)-chelates, thus transporting Fe into the
root cells via a highly specific transport system for Fe(III)MAs (Takagi et al. 1984, Mihashi and Mori 1989). MAs
consist of six compounds: mugineic acid, avenic acid, 3-hydroxymugineic acid, 3-epihydroxymugineic acid (epiHMA),
2'-deoxymugineic acid and distichonic acid. The biosynthetic pathway of the MAs has been established (Mori 1994)
and methionine has been recognized as a precursor (Mori
and Nishizawa 1987). MAs not only play a vital role in
enhancing availability of sparingly soluble Fe but are also
responsible for its rapid absorption by roots of the plant.
The Fe uptake rate in rice roots (Takagi et al. 1984) and
barley roots (Romheld and Marschner 1986a) is increased
100-1,000 fold when Fe is supplied as Fe(III)-MAs compared to Fe(III)-EDTA or Fe(III)-Desferal.
Most of the plant nutritional aspect of phytosiderophore (MAs) research is focussed on the elucidation of the
MAs-mediated Fe uptake mechanism in plant roots. Little
information is available on their role in Fe transport within
the foliage of the plant. Although epiHMA has been detect-
Therefore, we initially chose to investigate the influence of light on Fe transport in intact barley plants.
Autoradiographic studies were conducted with intact Fesufficient barley plants. Iron was supplied through roots as
59
Fe(III)-epiHMA and leaves were partly covered with
aluminum foil. In the covered areas of the leaves, Fe transport from the leaf veins to the mesophyll cells was drastically reduced. This suggested a regulatory role of light in Fe
transport within the plants and formed the basis of the
present investigation. This communication reports the
results of autoradiographic studies with intact barley plants
and isolated chloroplasts supplied with 59Fe(III)-epiHMA
with special emphasis on the role of light in Fe transport
into chloroplasts.
Plant culture conditions and preparation of59Fei+-epi-
Abbreviations: MAs, mugineic acid family phytosiderophores; epiHMA, 3-epihydroxymugineic acid.
101
102
Light-regulated iron transport in barley
hydroxymugineic acid—Barley plants (Hordeum vulgare
L. cv. Ehimehadaka no. 1) were cultured in modified
Kasugai's medium (Mori and Nishizawa 1987): 0.7 mM
K2SO4, 0.1 mM KC1, 0.1 mM KH 2 PO 4) 2.0 mM Ca(NO3)2,
0.5 mM MgSO4, \0 fiM H3BO3, 0.5 (iM MnSO4, 0.2 /JM
CuSO4, 0.5 nM ZnSO4> 0.01 /iM ( N H ^ O y O j , , and 0.1
/JM Fe-EDTA. Plants were grown in a growth chamber
under the following conditions: 19°C/14 h, 320^mol photons m~ 2 s~' under light and 14°C/10h in darkness. The
pH was adjusted daily to 5.5 and the nutrient solution
was renewed once a week. As reported earlier, 59Fe(III)epiHMA is absorbed more rapidly than Fe(III)-EDTA,
a
hence 59Fe(III)-epiHMA was used in absorption experiments. 59Fe(III)-epiHMA was prepared as a solution of 34
l*mol epiHMA and 6.25 /nnol 59FeCl3 (185 MBq, purchased from NEN, U.S.A.) in 1 ml 0.5 M HC1. An excess of
epiHMA was added to compensate for its photochemicaldegradation during the course of the experiment (Kamei et
al. 1993). The epiHMA was extracted and purified from the
root-washings of Fe-deficient barley (cv. Ehimehadaka no.
1) following the method of Takagi et al. (1984), and its purity was more than 90%.
Effect of darkness on Fe transport in leaves—Two
Fe-sufficient barley plants (with no chlorotic symptoms)
b
24 h
Fig. 1 (a) Autoradiogram of barley plant supplied with 59Fe(III)-epiHMA through roots and kept under light for 4 h. Numbers show
the leaf positions: 1 indicates the first leaf, 4 the newest leaf and 5 the tiller, (b) Autoradiogram of barley plant after 5*Fe(III)-epiHMA absorption. Arrows indicate the shadowed place. Upper weakly labeled root portion was not dipped in the nutrient solution during the absorption to avoid contamination of the shoots with 59Fe by air bubbling, (c) Autoradiogram of partly covered 6th leaves of two different
barley plants after 59Fe(III)-epiHMA absorption through roots: top, 12 h; bottom, 24 h. Arrows indicate the leaf areas covered with
aluminum foil during absorption. Both plants were of the same age (7 leaves).
Light-regulated iron transport in barley
were transferred from the culture medium to two conical
flasks (200 ml) each containing 100 ml of aerated (Fe-free)
nutrient solution and 58 nM 59Fe(III)-epiHMA. One plant
was allowed to absorb 59Fe(III)-epiHMA without masking,
while the middle part of the leaves of the other were
covered on all sides with a strip of aluminum foil (3 cm
wide), gently pressed it into position to stop the incidence
of light. After 4 h of 59Fe(III)-epiHMA absorption, the
plants were pressed, dried and autoradiographed. Results
showed that without masking, Fe was transported to all
plant tissues (Fig la). Meristematic regions, new leaves,
newly forming tiller and root tips accumulated a relatively
greater amount of Fe compared to other tissues. Iron accumulation depended on the age of the leaves; radioactivity
was strongest in the tiller (No. 5) or the newest leaf (No. 4)
and weakest in the oldest leaf (No. 1). Within individual
leaves, veins as well as intervenal spaces (mesophylls) were
observed to have been radiolabeled. However, Fe transport
in the covered areas of leaves, indicated by arrows (Fig. lb)
was drastically reduced, the covered areas being labeled
only in their veins. Similar studies were conducted for a
longer absorption time (12 or 24 h). Unexpectedly, the
longer absorption period could not restore normal Fe supply from the leaf veins to the mesophyll cells in the covered
areas of the leaves (Fig. lc). This suggested that the radial
Fe transport in the leaf from xylem to apoplasm or from
apoplasm to mesophyll cells is light regulated.
Chloroplast is the site of chlorophyll biosynthesis in
which both light and Fe play an important role (Wettstein
et al. 1995). More than 90% of the plants' Fe is located in
the chloroplast (Terry and Abadia 1986), where it is stored
as ferritin in stroma, found in many iron proteins such
as cytochromes and ascorbate peroxidase (Miyake et al.
1993), and detected in non-heme iron such as ferredoxin
etc. We, therefore, anticipated that the chloroplast in mesophyll cells would be the most suitable organelle for the
study of light-dependent Fe transport.
Isolation of chloroplasts—The chloroplasts were isolated according to the modified method of Asada et
al. (1973). Fresh barley leaves (80 g) were homogenized
with 160 ml of an extraction medium (0.33 M sorbitol, 10
mM MgCl2, 2 mM EDTA, 0.5 mM KH2PO4 and 50 mM
HEPES; pH 7.0, adjusted with KOH). The homogenate
was filtered through a double layered miracloth and centrifuged at 1,000 xg for 1 min at 0°C. The supernatant was
collected and centrifuged at 2,000xg for 10 min at 0°C.
All subsequent centrifugations were performed at 0°C
unless otherwise mentioned. The pellet was resuspended in
the extraction medium. A 5 ml chloroplast suspension was
overlayed on a sucrose density gradient in a 50 ml glass centrifuge tube which contained 10 ml each of 60%, 45%, and
20% sucrose (prepared in the extraction medium). In all,
four tubes were prepared. The tubes were centrifuged at
2,000 x g for 30 min. The chloroplasts were harvested from
103
the 45% sucrose layer with a Pasteur pipette and suspended
in the extraction medium at a ratio of 1 : 5 (v/v). The
suspension was centrifuged at 2,000xg for 10min. The
supernatant was discarded and chloroplasts were collected
and made up to the required volume with a reaction buffer
(1 : 100, culture solution without Fe : extraction medium).
Time course of Fe absorption by chloroplasts under
dark and light—A chloroplast suspension (0.5 ml) was
transferred into 2 ml Eppendorf tubes kept on ice. Each
sample was supplied with 59Fe(III)-epiHMA stock solution
to give a final concentration of 0.062 mM. The tubes were
then placed in 25 ml transparent glass test tubes and laid
horizontally on a flat bed reciprocal shaker with a 3 cm
stroke length and at a speed of 120 strokes min" 1 . The samples were incubated on the shaker in light (320 txmo\ photons m" 2 s"') at 19°C. The reaction was stopped after
appropriate time by covering the Eppendorf tubes with
aluminum foil and burying them in ice for 3-4 min. After
the addition of 1.5 ml reaction buffer, the samples were
shaken gently and centrifuged at 2,000xg for 5 min. The
supernatant was discarded. This process was repeated by
adding 2 ml reaction buffer and the pellet was then assayed
for 59Fe on an auto well gamma scintillation counter (ARC
300; Aloka, Japan). For absorption studies in the dark, all
processes were identical except that the Eppendorf tubes
and glass tubes were covered with aluminum foil.
The results of this time course study were in complete
agreement with those of the autoradiographic studies.
Iron absorption by the illuminated chloroplasts was much
higher than those which had been kept in darkness. Iron uptake increased from 0.028 nmol Fe (mg Chi)" 1 at 0 min to
0.186 nmol Fe (mg Chl)~l at 60 min in the light whereas in
darkness it remained almost constant [around 0.023 nmol
Fe (mg Chi)"1] over the same period (Fig. 2). Iron absorption increased until 60 min after the commencement of the
experiment and then it formed a plateau. This may be due
to the exhaustion of low molecular weight substrates which
0.25
0.20
.0.15
0.10
0.05
0.00
Fig. 2 Time course of Fe absorption by isolated chloroplasts
under light and dark at 19CC. Bold line indicates Fe absorption in
darkness and thin line represents the absorption under light. Each
value is the mean of 3 repeats. The chloroplasts were separated
from 50-d-old plants.
104
Light-regulated iron transport in barley
are vital for maintaining the viability of the chloroplasts.
These substrates might have been lost during isolation of
chloroplasts due to high osmolarity of the sucrose density
gradient. The reliability of the experiment was considered
ideal as the treatments were replicated three times and the
experiment as such was performed four times. The amount
of light-mediated Fe absorption at 19°C was twice the
amount measured at the lower temperature of 0°C (data
not shown).
Effect of light-dark cycles on Fe fluxes in chloroplasts
—After the addition of 59Fe(III)-epiHMA, samples were
treated with light, darkness or light-dark cycles. The experiment was replicated three times. Illuminated chloroplasts
accumulated higher amounts of Fe as compared to those
kept under continuous darkness or treated with alternate
light-dark cycles (Fig. 3). Iron initially absorbed during 20
min of irradiation was seen to leave the chloroplasts (efflux)
when they were kept in darkness for 20 min (Fig. 3; see
from 20 min to 40 min time interval indicated by the bold
line). Re-illumination for another 20 min (from 40 min to
60 min) restored the initial status, demonstrating photoreversibility of the reaction. These light stimulated Fe influxes and dark induced Fe effluxes were neither light nor
dark-triggered (on-off) reactions, rather they were light or
dark dependent reactions since both needed continuous
light or continuous darkness. However, the preceding experiments did not identify the dominant site of light dependent Fe transport in chloroplast, i.e., thylakoidal membrane or chloroplast envelope. We therefore conducted
experiments with intact and osmotically shocked chloroplasts.
Effect of light on Fe absorption by disrupted chloroplasts—To rupture the chloroplast envelope, 1.0 ml reaction buffer (without sorbitol) was added to 0.5 ml freshly
0.6
isolated chloroplasts and kept on ice for 10 min under dark
conditions. 59Fe(III)-epiHMA was added to the chloroplast
suspension which was then incubated for 30 min, one under light and the other under dark conditions. The samples were washed with reaction buffer (without sorbitol)
and centrifuged at 120,000xg for 12h at 2°C on an
ultracentrifuge (Optima TL™ Ultracentrifuge, Beckman,
U.S.A.). After another washing the pellet was assayed for
59
Fe. The intact chloroplasts were treated similarly to serve
as a control.
Under light conditions, thylakoidal membranes and intact chloroplasts showed differing Fe absorption activities.
The thylakoidal membranes absorbed only about 25% of
the total Fe accumulated by intact chloroplasts under light
(Fig. 4). Similar results were obtained in the case of dark
treated samples (Fig. 4). In another similar experiment,
chloroplasts received an osmotic shock for 1 h or 2 h
followed by 30-min 59Fe(III)-epiHMA absorption under
light. About 20% of the total Fe incorporated into chloroplasts was localised in the thylakoidal membranes (data not
shown) with the remaining 80% of absorbed Fe probably
residing in the stroma. The results of the two experiments
are in complete agreement with each other. These findings
suggest that even though Fe absorption by the thylakoidal
membrane is light-regulated, the chloroplast envelope is
the dominant site of light-regulated Fe(III)-epiMA absorption. The results also confirm that most of the chloroplasts
used in the previous light/dark experiments were intact.
Effect ofDCMU on light-induced Fe transport—The
chloroplasts were dosed with different concentrations of
DCMU. The samples were then incubated for 10 min with
59
Fe(III)-epiHMA under light. The Fe absorption by chloroplasts was inhibited almost completely by DCMU (1 x 10"5
M) under light (Fig. 5). This suggests that Fe absorption by
chloroplasts depends on the linear electron transport in thylakoids or the ATP generated in them. The possibility that
the Fe absorption is signaled by light which is mediated by
o.s
0.8
I
0.6
I Light
Dark
0_2
Chloroplasts
Fig. 3 Time course of Fe absorption by separated chloroplasts
showing Fe influx under light and Fe efflux in darkness at 19°C.
Two sets of the samples were incubated under continuous light or
continuous dark for different time periods and the third one received 20-min light-dark cycles. The chloroplasts were separated from
40 day old plants.
Thylakoid
Fig. 4 Iron absorption by thylakoidal membranes under light
and dark. After 10 min osmotic shock the disrupted chloroplasts
were treated with Fe(III)-epiHMA and incubated for 30 min
under light and dark conditions followed by ultracentrifugation
(see text). The "thylakoid" component includes chloroplast envelopes, etc. Each value is the mean of 3 repeats.
Light-regulated iron transport in barley
105
studies are warranted t o answer these questions.
This work has been supported by CRES (Core Research for
Evolutional Science and Technology), Japan Science and Technology Corporation (JST) (S.M). We thank Professor Kalyan Singh,
Banaras Hindu University, Varanasi, India and Dr. S. Klair, JSPS
fellow, Department of Applied Biological Chemistry, Division of
Agriculture and Agricultural Life Sciences, The University of
Tokyo, Tokyo, Japan for reviewing and correcting the manuscript.
o.o
Fig. 5 Effect of DCMU on Fe absorption by chloroplasts under
light. The chloroplasts were incubated for lOmin or Omin at
19°C. Unfilled bars indicate Fe absorption by chloroplasts kept
under light whereas filled bars represent Fe absorption in darkness. All values are means of 3 repeats.
chromoproteins such as phytochromes awaits investigation.
In view of the above findings, it is concluded that the
Fe fluxes in chloroplasts are regulated by light and dark.
This phenomenon is probably responsible for the results of
our autoradiographic studies in which dark treatment interrupted Fe transport from leaf veins to the mesophyll cells.
Since the mesophyll chloroplasts are a strong Fe sink, high
Fe demand by illuminated chloroplasts will strongly influence Fe transport into mesophyll cells from apoplast in
the leaf. Conversely, Fe efflux from chloroplast may be constitutively occurring. Prior to this experiment, such effluxes
have not been reported. The measurement of efflux of the
previously absorbed Fe under light (light-induced Fe influx) was possible because of the use of labeled Fe.
The next question is the regulatory mechanism of light
dependent Fe influx into the chloroplast. To our knowledge, there is no information concerning the Fe acquisition
mechanism by chloroplasts from cytoplasm in plants. Do
chloroplasts possess a similar Fe uptake mechanism as the
root cells of Strategy-I or Strategy-II plants (Romheld and
Marschner 1986a, b)? Are there different chloroplasts in
graminaceous and nongraminaceous plants with regard to
Fe acquisition, and is this related to their different Fe acquisition mechanisms in roots? In other words, (1) Is there
an Fe 3+ -reductase in the chloroplast envelope?, (2) Is there
an Fe 2+ -transporter in the chloroplast envelope as in the
cell membrane of E. coli (Kammler et al. 1993) and in the
cell membrane of Arabidopsis (Eide et al. 1996)?, and (3) Is
there a specific transporter for 59Fe(III)-epiHMA in the
chloroplast envelope of graminaceous plants? Further
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