Plant
Physiol. (1969) 14, 1457-1460
Factors Related to Iron Absorption by Enzymically
Isolated Leaf Cells'
Seshadri Kannan2
Department of Horticulture, Michigan State University, East Lansing Michigan
Received March 31, 1969.
Abstract. The rate of Fe absorption by cells enzymically isolated from tobacco leaves is
corre'ated with the age of the leaves from which the cells are derived4 The cells obtained
from younger leaves absorb Fe more rapidly than those from older ones. Ca inhibits Fe and
Mn absorption. Fe and Mn are mutually antagonitic in their absorption by leaf cells. Ca
enhances the inhibition of Mn absorption by Fe, but reduces the inhibition of Fe aibsorption
by Mn. The affinity constant for Fe absorption by leaf cells is low. The chelate EDDHA
(ethylenediamine di (o-hydroxyph-nylacetate) comp)eLitively inhibits Fe absorption.
Ioni uptake by variotus plant parts, incltlding
leaves, hias been the sul)ject of muclh researclh in
recent y-ears (9,13, 18, 19, 22). Howvever, absorption
of ions by leaves has received scant attention colimpared to roots. It has been slhowvni that leaves and
leaf tis tuCs actively accumulate iolns fromi the ambient
solution (7, 19). Living cells have been isolated
enzy1mcalllv fromii leaves, stems anid roots (7,23) and
the nmechanisms of ion uptake lhave been sttudied at
tlle celluilar level (7, 11). Fur.hermore, it has been
demonstrated that lighlt, succinate and bicarbonate
as a source of CO.! for phlotosynitlhesis promote Rb
and pho lphate ul)take by leaf cells. Metabolic inhibi.ors like NaN, and DNP inhibit ioll uptake by
cells (7). Absorption of Fe by tobacco leaf cells
has been slhoNvii to be metabolic (11). Stuldies of
some factors affeciting Fe absorption by enzymically
isolatcd leaf cells are reported lierein.
Materials and Methods
Plant AMaterinls. Tobacco (ATicotiana tabacutit
L., Bturlev type) vas growvn in a greenhlotuse and
cells isolated from le aves of different positions on
the plant wvere tised to sttudy the effects of leaf age.
All other experimeicnits vere carried out with cells or
discs obtained fromii fuilly expan(de(d green leaves.
Procedure for Isolationz of Cclls. The proceduire
wvas essentiallv the samne as that described by Zaitlin
(23) and has been outlinied in detail (7, 11). Leaves
' Aichigan Agricultural Experiment Station Journal
Article No. 4867.
21PIresenit address: Biology Division, Bhabha Atomic
Research Centre, Bombay 85, India.
wvere sliced into 2 to 3 mm2 sections, and treated
with enzyme solution. The isolation mixture contained nmineral salts and organic constituents (16).
Pectinase (1 % w/v) was added and the pH adjusted to 6. Isolated cells were washed with cold
0.3 M sucrose and suspended in it at 3' until used.
For eaclh experiment, cells were freshly isolated and
used within 48 hr.
Procedutre for lncubating Leaf Cells and Leaf
Discs. The inicuibation mixture contained per 10 ml,
50 mg dry weight equivalent of cells, 1170 ,umoles
sucrose, 100 jutmoles tris-maleate (pH 6.4) and 5
/An-oles "9Fe labeled FeSO4 or "4Mn labeled MnSO4
unless otlherwise indicated. The incubation medium
for studies of ion uptake by leaf discs was of the
same composition as above, but a volume of 20 ml
instead of 10 ml was used. It contained besides,
4 leaf discs (0.8 cm2) weighing about 100 mg, and
10 ,umoles of isotopically labeled FeSO4, MnSO4,
ZnSO0 cr RbCl. The flasks containing the incubation miixture were slhaken in a reciprocating waterbatlh in the lighlt (ca. 500 ft-c) at 20 ± 2°.
M1casuireinent of Ion Uptake. At the end of the
absorption period, cells in 2 ml of the incubation
miixture were collected on weighed filter paper under
mild suction. washed twice with non-labeled 0.1 niM
soltution of FeSO4 or MnSO4 and finally with 0.3 M
sucrose. Leaf discs were removed from the incubation meditum and rinsed in non-labeled 0.1 mM solution of the respective salt for 2 min and finally in
deionized water. Air dry samples were radioassayed
in a gamma well scintillation counter. Significant
differences between triplicate samples were establislied statistically (2). The data presented in the
table and figtures were derived from separate experiments and are comparable within an experiment but
not between experiments.
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1457
Copyright © 1969 American Society
of Plant Biologists. All rights reserved.
1458
PLANT PHYSIOLOGY
Table I. Effects of Ca on Fe and Mn Absorption by
Tobacco Leaf Cells
The incubation mixture contained per 10 ml, 50 mg
dry weight equivalent of cells, 1170 /Amoles sucrose, 100
umoles tris-maleate (pH 6.4) and 5,umoles 59Fe-labeled
FeSO4 or 54Mn-labeled MnSO4, besides CaSO4 as a
variable. The cells were incubated for 4 hr in the light
at 500 ft-c and at 200.
Concn.
of CaSO4
Absorption rate
Fe
Mn
than that of Mn on Fe uptake. While Ca partially
reverses the inhibition of Mn on Fe uptake, it enhances the inhibitory effect of Fe on Min uptake.
A hyperbolic relation between absorption rate of
Fe and concentration (Fig. 4) suggests a saturation
phenomenon, that is, some type of carrier-mediated
transport. The inhibition of Fe absorption by
EDDHA (ethylenediamine di(o-hydroxyphenylacetate) is probably of a competitive nature (Fig. 4,
insert).
nanomnoles/mg cells"4 hrl
17.4a
5.2'
M
Control
2 X 104.
5 X 10-4
18.3
8 X 10 4
17.0O
10-8
8.3b
2 X 10 8
4.8b
10
4.9a
3.6'
2.8b
Discussion
Ion uptake by an intact leaf is a multistep procew
(22) involving adsorption on the surface, penetration
...I.
Within each column, mneans followed by different letters are significantly different at odds of 19:1.
cn c
6
Results
Fe absorption decreases with increasing age of
leaves from which cells are isolated (Fig. 1). The
absorption of Fe and Mn by leaf cells is inhibited
by Ca (table I). The inhibitory effect of Ca is also
observed on the absorption of Fe, Mn, and Zn by
leaf discs (Fig. 2). However, Ca does not inhibit
Rb absorption by leaf discs.
Interactions of Fe and Mn in their absorption
and the role of Ca on Fe-Mn uptake are portrayed
in Fig. 3. These data suggest that absorption of
Fe and Mn is mutually antagonistic. The inhibitory
effect of Fe on Mn absorption is much greater
'n
-J
0
0
S
z4
100
~
v
0w
~
0 0.05
KM
.5
5.0
ca CONCENTRATION (mM)
FIG. 2. Effects of increasing levels of Ca on the
absorption of Fe, Mn, Zn, and Rb by tobacco leaf discs.
TheLnoes
incubation mixture contained in 20 ml, 2340
sucrose, 200 ,umoles tris-maleate (pH 6.4), 4 leaf discs
(0.8 cm2) weighing about 100 mg, and 10 ,ffmoles of
isotopically labeled FeSO4, MnS04, ZnSO4 or RbCI.
The leaf discs were incubated for 4 hr in the light at
500 ft-c and at 20°.
cr6
0
z
z*
w0
F u 2
'
a-
2
4
6
10
12
8
14
LEAF NUMBER BEGINNING FROM APEX
FIG. 1. Effects of tobacco leaf age (designated by
leaf number from apex to base) on the absorption of
Fe by leaf cells. Regression equation, y = 4.01 --0.12x,
r = -0.83 at P = 0.01. The incubation mixture contained per 10 ml, 50 mg dry weight equivalent of cells,
1170 ,umoles sucrose, 100 umoles tris-maleate (pH 6.4)
and 5 /Amoles 59Fe-labeled FeSOV. The cells were incubated for 1 hr in the light at 500 ft-c and at 200.
through the cuticle and subsequent absorption by
leaf cells. Using cuticles and cells enzymically iso
lated from leaves, it has been possible to study the
mechanisms of penetration of ions through cuticles
and absorption by cells, separately (2).
Living cells enzymically separated from leaves
and suspended in solution offer a good system for
physiological studies and are subject to easy manipulation. Furthermore, these cells are suited for short
term studies of the mechanisms of ion uptake at the
cellular level. It has been shown earlier that
enzymically isolated cells are physiologically active
and that the results obtained with this system are
real. Responses to different treatments are clearly
discernible (7, 11).
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Copyright © 1969 American Society of Plant Biologists. All rights reserved.
KANNAN-FE ABSORPTION BY
0j 100.@
Fe/+Mn4Ca
O
z
t
,
80
o
zO
60
-e/0r,oAsc
Fe+Mn
0
/)
Mol
C O 601
20
0
V)Z
40.
I-.
~~
(I M
20
CO
~
~~~~nF
b
60RTIO
mnol
~
COC
4Z I
z
0
.05
wegh6H
,umoles
tequvaen e ofcls
510,mlsscoe 10
b.n4Fe-nlao
1459
ISOLATED LEAP CELLS
coincide with differences in ion transport (15). The
existence of biochemical gradients in young and old
tobacco tissues has been recognized (12). A critical
evaluation of the role of these phenomena on ion
uptake process would be quite revealing.
It has been shown that Ca is essential for mainitaining the integrity of the selective absorption
mechanism for K, Rb, and Na (5), and the apparent
permeability of cell membranes (20). Ca, especially
at higher -concentrations, inhibits Fe as well as Mn
uptake by leaf cells i(table I). Effects of Ca on ion
uptake seem to vary with the plant and ion species.
Uptake of K by maize and soybean roots is differentially affected by Ca (8). Elzam and Hodges (3)
on the basis of their findings, have recently emphasized the hazards involved in applying results obtained with 1 or several plant species to all species.
The inhibition of Fe absorption found herein is not
unique with leaf cells, but has been reported for Fe
uptake by roots (14).
The inhibitory effect of Ca on Fe and Mn uptake
by leaf cells is analogous to that on Li absorption
by plant roots (4, 6). It has been suggested that
Ca modifies the root surface in some way as to
provide an efficient barrier to Li *(6). This does
not explain how this barrier could be specific for Li,
but not for K. On the other hand, Ca may have a
selective action on the hypothetical ion-carrier
mechanism and thus render specific carrier sites
non-functional. Ca may reduce the effective pore
diameter of membranes or free-space polymers and
of Fe lby leaf clls,aticeasings concentedrat ions
ofF sO4
yeanuat( increashring
Cainthiaed absorptio of Mnwrcells
(Fe
conenlgtratioS0 oft- anSOd 20m0adCa1.m)
Th Ie inualtio mixur
pv
oeer10m, 50tshgwdry
conontie
weigh
of
1
170
chellbs,
equivsofalen
100
strctra difrneMreivleglesf
n Fesucrose,
upae
5Felabee
iler ad GeO4ord
Fmoes
utrismaeate (pH 6.4,
MSO4, beside sbtheanterer ing ionsias
5Mn-a(9observed
iniatedabsovbe. Thre ceplls wer incuated
neor l hrain
n h
e
thle lightatn50 oft- adi aoti200.acs
Stutoufoldiafrene
apition
Rltcesponse ofde clorti leave
of Fe saltsi arknow(21dei mayHoevrdapotan soingti
av iale Keamoimur andiit Good
Fegarptake ar notfrec
.
sustneselik leucyine
mals.n(9) obervedthat orgni
lediaves
ioared
aboredf moell raplysb
nearthmoe bhase
Si, mM
FIG. 4. Rate of absorption of Fe (V) as a function
of FeSO4 (S). (Insert) Lineweaver-Burk plot of Fe
absorption by leaf cells in the presence and absence of
EDDHA. (0.1 mM). The incubation mixture contained
per 10 ml, 50 mg dry weight equivalent of cells, 1170
Recently, differences in thle ultrastructure of the leaf ,umoles sucrose, 100 ,umoles tris-maleate (pH 6.4), and
cells of young and old branches of Moiurn have been
59Fe-labeled FeSO4 as a variable. The cells were inin these from
demonstrated and changesDownloaded
ultrastructures
hr in the light at 500 ft-c and at 20°.
forby1www.plantphysiol.org
on July 31, 2017 - cubated
Published
cutice
fole eve.Srctural
differencesar
inl
l di ;
pae
Copyright © 1969 American Society of Plant Biologists. All rights reserved.
14606PLANT I'HYSIOLOGY'
thus restrict the entry of large hvdrated ions (6, 18).
This could also be true for polyvalent ions like Fle
and Mn.
The interference of Fe absorption bv otlher cations
is one of the causative factors for Fe clhlorosis (14).
The results (Fig. 3) slhow that the interference of
Mn on Fe absorption is at the cellular level. Fe and
Mn which are chemically related appear to be muiiltually, but not equally competitive. This plhenonmellon
of mutual competition also exists betwveen Ca and Sr
in. their uptake and distributtion in plants (1). F;e
and Mn are probably absorbed by a common meclhanism which appears to have a higlher affinity for Fe
than for Mn. It has been shown that Ca is indispensable to the unimpaired fuinctioning of selective
K-Rb transport (5). In Fe-Mn transport also, Ca
appears to favor Fe absorption in preference to Amn.
This effect is however different from the inhlibitory
effect of Ca on Fe or Mn absorption when present
alone.
It has been shown that Fe absorption bv leaf
cells and also by intact leaves is reduced by EDDHIA
(10) and this inhibition appears to be competitive
(Fig. 4, insert). The nature of carrier-mediated
cation transport appears to be complex. It is postulated that these carriers bind ions by a process of
chelation similar to the binding of functional miietals
by enzymes.
Mechanisms of ion uptake could be niore conveniently studied with leaf cells (18) especially for
investigating the liglht dependence of ion fluxes in
higher plant leaf tissues. With furtlher refinenments
in the techniques of isolation and incubatioln, experiments could be carried out with isolated cells that
are comparable to those with isolated cliloroplasts
and mitochondria.
absorption by barley roots. Proc. NatI. Aca(l.
Sci. U. S. 49: 634-92.
6. JACOBSON. L., D. P. MOORE. AND R. J. HANNAPEL.
1960. Role of calciunm in absorption of monovalent
cations. Plant Physiol. 33: 332-58.
7. JYUNG, W. H., S. H. WITTWER AND Al. J. BUKOVAC. 1963. Ion uptake by cells enzymically isolated from green tobacco leaves. Plant Physiol.
40: 4C0-14.
8. KAHN, J. S. AND J. B. HANSON. 19;7. The effect
of calcium on potassium accumulation in corn and
soybean roots. Plant Physiol. 32: 3'2-16.
9. KAMIMURA, S. AND R. N. GOODMAN. 1964. Influence of foliar characteristics on the absorption
of a radioaclive model comp^und by apple leaves.
Phvsiol. Plantarum 20: 911-19.
10. KANNAN, S. AND S. H. WITTWER. 1963. Effects
of chelation and urea on iron absorption by intact
leaves and enzymically isolated leaf cells. Plant
Plhysiol. 40: xii.
11. KANNAN, S. AND S. H. WITTWER. 1967. Absorp
tion of iron by enzymically isolated leaf cells
Physiol Plantarum 20: 911-19.
12. LAVEE, S. AND A. W. GALSTON. 1953. Structural,
physiological and biochemical gradients in tobacco
pith tissue. Plant Pliysiol. 43: 1760-63.
13. LEGGETT, J. E. 1963. Salt absorption by plants.
Ann. Rev. Plant Physiol. 16: 333-46.
14. LINGLE, J. C., L. 0. TIFFIN, AND J. C. BROWN.
1963. Iron uptake-transport of soybeans as influenced by other cations. Plant Physiol. 33: 71-76.
15. LiUTTGE, U. AND G. KRAPF. 1953. Die Ultrastructur
der Blattzellen junger und alter Afniiwi-Sprosse
und ihr Zusammenhang mit dzr Ionenaufnalhme.
Planta 81: 132-39.
16. MURASHIGE, T. AND F. SKooG. 1962. A revised
medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Platntarum 15:
473-97.
Acknowledgnment
17. OLAND, K. AND T. B. OPLAND. 1956. Uptake of
magnesium by apple leaves. Physiol. Planitarum
The author is grateful to the Rockefeller Founidation
9: 401-11.
and the United States Atomic Energy Commission for
18.
C. B. 1963. len absorption in Atriplex
OSMOND,
their financial support, and to Dr. S. H. Wittwer for
leaf tissue. Australian J. Biol. Sci. 21: 1119-30.
hiis valuable guidance and encouragement during the
tudy.
19.- RAINs, D. W. 1968. Kinetics and energetics of
light enhanced potassium absorption by corn leaf
tissue. Plant- Physiol. 43: 394-400.
Literature Cited
20. VAN STEVENINCK, R. F. M. 1965. The significance of calcium on the apparent permeability of
1. COLLANDER, R. 1941. Selective absorption of cacell membranes and the effects of substitution with
tions by higher plants. Plant Physiol. 16: 691other divalent ions. Physiol. Plantarum 18: 54-69.
720.
21.
WALLACE, A. 1965. Micronutrient deficiencies in
. DUNCAN, D. B. 1955. Multiple range and mulplants and their correction with chelates. Agr.
tiple F tests. Biometrics 11: 1-42.
Sci. Rev.- 3: 1-7.
3 ELZAM, 0. E. AND T. K. HODGES. 1967. Calcium
22.
S. H. ANb M. J. BUxOVAC. 1969. The
W1rrwER,
inhibition of potassium absorption in corn roots.
of
nutrients through leaf surfaces. Handuptake
Plant Physiol. 42: 1483-88.
buch der Pflanzenerniihrung I. Springer-Verlag.
4. EPSTEIN, E. 1960. Calcium-lithium competition in
In press.
absorption by plant roots. Nature 183: 705-06.
23. ZAITTIN, M. 1959. Isolation of tobacco leaf cells
5
EPSTEIN, E., D. W. RAINS, AND 0. E. ELZAM.
capable of supporting virus multiplication. Nature
of dual mechanisms
1963. Resolution Downloaded
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1002-03.
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