Plant Phxysiol. (1967) 42, 15-19
Absorption and Translocation of Foliar-Applied Iron
J. L. Eddings and A. L. Brown
Department of Soils and Plant Nutrition, University of California, Davis, California 95616
Received August 15, 1966.
,Summary. The absorption of 59Fe3+ by the leaves of various plant species was
studied. Stomata were fotund to play a major role in foliar absorption when leaves
were totally submerged in treatment solutions, and a correlation was found to exist
between stomatal area and absorption. Day treated leaves absorbed much more
than did night treated leaves. The use of a surfactant markedly increased absorption.
Translocation from treated leaves was demonstrated and was found to vary with
species.
The application of various materials to plant
foliage has become an everyday occuirrence in
modern agriculture. Many of these materials exert
their desired effect by remaining on the exterior
of the foliage, but at least 2 of them, namely
growth regulators ( including herbicides) and nutrients, are effective only when they penetrate into
the leaf itself. Leaf cuticle presents a major
barrier to the penetration of these materials, but
appears to be slowly permeable to both polar and
apolar substances (5). In reviewing the conflicting
literatuire on cuticuilar and stomatal uptake of foliar
applied materials, Currier and Dybing (5) conclude that '. . . both stomata and cuticle may be
involved, with the stomatal component varying
widely duie to a complex of factors influencing
stomatal opening." The major problem remaining
unanswered is the relative importance of each path
under specified conditions.
It is well established in the literature that surfactants increase the effectiveness of herbicides.
The effect of surfactants on nutrient uptake is not
so clear, however. Some investigators report no
effect oIn uiptake due to the presence of a suirfactant (1), some report a decrease (7), and others
report an increase (7, 8). In reviewing the literature, one arrives at the concltusion that factors not
always well explained or considered in specific
articles play a major role in whether or not surfactants increase, decrease, or have no effect on
nutrient uptake. Such factors are A) method of
application, B) nature of the nutrient being sttudied,
C) nature of the surfactant being studied, D) reactions between the nutrients and surfactants (stuch
as complex formation or precipitation), E) characteristics of the plant species, and F) the method
of assessing uptake (quantitative measurements or
growth responses).
If stirfactants promote the uptake of foliarapplied stubstances, as they do for herbicides and
as some investigators report they do for nutrients,
a specific mechanism or mechanisms for this promotion must exist. To date, 2 authors (5, 10) have
summarized these possible mechanisms as A) improving coverage, B) decreasing or removing air
films between solution and leaf surface, C) reducing
interfacial tension between relatively polar and
apolar submicroscopic regions of the cuticle, D)
acting as cosolvents or solubilizing agents in cuticular penetration, E) increasing or inducing stomatal entry, F) increasing plasmalemma permeability
by stimulation or toxicity, G) increasing apoplastic
movement to the plasmalemma-cell wall interface,
H) acting as humectants to retard drying of the
solution or I) interacting directly with the other
applied stubstance.
Iron has classically been considered a relatively
immobile ion in plants and iron deficiencies have
been attributed to this immobility (4). More recent
work has showni that iron is at least moderately
mobile in plants (2, 3) and that a good degree of
correlation exists between the chlorophyll content
of leaves and their iron content (9). Foliar-applied iron has been shown to be redistributed from
the leaf to which it was applied to yotung expanding
leaves and to regions of meristematic activity (3, 6).
The purpose of this investigation was to determine, under well defined conditions, the stomatal
component of foliar absorption and the subsequent
mobility of foliar absorbed iron.
Materials and Methods
Plant Cultutre. Four plant species or varieties
were selected for study, namely red kidney bean
(Phaseolus vulgaris, L. var. Red Kidney), small
white bean (Phaseolus vulgaris, L. var. Small
White), sorghum (Sorghum vulgare, L. var.
RS610), and tomato (Lycopersicum esculentum, L.
Improved). The plants were germi-
var. Pearson
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15
16
PLANT PHYSIOLOGY
natedl on cheesecloth moistende(d with tap water anid
transplante(l to aerate(l Johnsoin's soluition. They
were then grown in a greenhouise for 30 (laYs,
suifficient time for several matu.re, fully expan(led
leaves to develop. Uni form leav es were selectedl
for treatment.
Application Techniquc. Applicatioin of the
radioactive iron was accomplishedl by total stubmersion of the plant leaf into 100 ml of aquleouis soluition containing 59FeCl3 with an activity of 1 Mc/ml.
The total iron concentration of the soluition was
3.25 X 10-4 Fe3+ and the soluition had a pH of 2.
Calcullations from the soluibility produtct of
Fe(OH), indicate that at pH 2, 1.0 Ai Fe3+ can be
present in soluition. Althouigh calclulationis based
solely on the soluibility produict may not be entirely
correct (primarily sinlce other ions suich as FeOHI2+,
Fe((OH ).,, ancd Cl- also existed in this solultion),
the calculations are acculrate enouigh to convince
one that precipitation of iron couldk not occulr at
this pH and this concentrationi.
In addition to 59FeClI, the plus suirfactant sollition contained 0.10 % Silr-Ten (sodiuim dioctylsuilfosuiccinate, formerly VTatsol OT, a commercial
stirfactant of American Cyanamid). The plus
suicrose, 0.5 Ai, was included in anl attempt to limit
the effect of the night treatment to a condition of
closedl stomata; in other words, to provide an energy
source for energy requiiring activities.
The leaves were treated bv suibmersion for
various time periods, after which they
wvere removed and immediately washed in a soltution of
0.5 N H,Cl containing a (letergent (Dreft, a Procter
and Gamble producLt) for ahouit 30 secondls, followed
by 2 rinses in (listille(d water.
This rather drastic
washing technique was employed in order to remove
all or most suirface adsorbe(l iron from the leaves
after treatment. Treatment periods of the dav and
night were selected to have comparable temperatilres (21F 2°). Day treatments were made between
9 and 11
and inight treatments between 10 and
12 PAt.
Absorption Stuidies. The first phase of the iron
absorption stud(lies was (lesigne(i to assess the uiptake
of "9Fe3+ into intact leaves of 2 plant species, red
kidney bean acnd( sorghuim, from soluitionis of 59FeCl,
alone, 59FeCl., pluis suirfactant, an d 59FeCL. plus
suirfactant and( suicrose, in the (lay ain(l at night.
Suibmersion time was fifteeni minuites for all treatments. In the case of the beans, the terminal
leaflet of the trifoliate leaf was submerged to the
point of petiole attachment with the entire petiole
remaining attached to the leaflet. After treatment
andl washing, the petiole was detachedl and( (liscarded. In the case of the sorghuim, the terminal
one-half of the blade was submerged, and after
treatment and washing, the basal one-half of the
leaf was (letache(I an(l discar(le(l. Restults of this
experiment are presentedl in figJre 1. Each graprh
represenits the average of 2 replicate leaves.
59 fe only
0 59 Fe plus surfactant
59 Fe plus surfactont plus sucrose
I
*' 6400
5600
>- 4800
, 4000
m
0
O
3200
0
O
z
o
1600
800
360
w
320
U.
w~~~~~~~~~~
iE
2400
_E
4
I
I
I
I
280
4
W
240
ti
4200
0
°
160
,
120
U)
80
cN
z
I
I
A
0
L-
0
4
AM
w
Or
12
-J
I0
8
0
6
Ul
4
2
IL
z
D3
0
0
U
DAY
NIGHT
DAY
NIGHT
Fi(;. 1. Foliar tuptake of 9'Fe as affected hx solutioni
coniposition, (lday xs night treatmenit, anid plant species.
Results are expressed 3 wvaxs to show the correlationi between stomatal area and uptake.
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EDDINGS AND BROIWN-UPTAKE AND TRANSLOCATION OF 59FE
The second phase of the absorption study involved an experiment designed to show the uptake
of 59Fe3+ from a solution of FeCl3 plus surfactant
as a function of time. In this experiment 4
plants were used, namely red kidney bean, small
white bean, sorghum, and tomato. Treatment time
is defined as the time of submersion of the leaves
in the experimental solution prior to washing. The
sorghum and beans were submerged in the manner
previously described and the tomato leaves in a
manner similar to that for beans. Results of this
experiment are presented in figure 2. Each point
on the graphs represents an average value of three
replicate leaves.
Translocation Study. The translocation study
was sdesigned to assess the mobility of the foliarly
app lied 59Fe, that is, to determine how much of the
abs(orbed 59Fe would translocate and how fast the
tratnslocation could be accomplished. Leaves of
tomLato, sorghum, and small white bean were treated
in tthe manner previously described, except that they
werre not removed from the plants. Treatment time
wass 15 minutes followed by normal washing.
leaves were removed from the plants at
Tre
17
1600-
TOMATO,
wL
0
8
0
800
O
0>
z
0
o
0
D
20
SM^ALL WHITE BEAN
n_
40
60
TIME - Hours
80
100
FIG. 3. Translocation of 59Fe from treated leaves as
a function of time after treatment.
various times after treatment and analyzed for their
59Fe content. The results of this investigation are
shown in figure 3. The sorghum determinations
represent averages of 2 leaves, the tomato and small
white bean values are averages of 3 leaves.
Stontatal Cou-nts and Measurentents. Stomata
were counted and measured by the silicone rtubber
impression technique (11). The silicone rubber
used was a General Electric product, RTV-11, a
self-leveling liqulid. The silicone rubber was catalized with Nuocure 28 (a product of Heyden
,00
Newport Chemical Corporation) which cautses the
SORr ,
silicone rubber to cure in about 2 mintutes. The
/
sO"^roX
W.
catalized silicone rubber was poured onto the leaf
allowed to harden for about 3 minutes,
!0o
then peeled off. The negative leaf impression th,s
woo- * //obtained was then painted with Duco Cement (a
product of DuPont) and the cement allowed to
REDKIDNEYBEAN
harden. The resultant positive leaf impression was
SALL WHITE BEAN
oo
*
a transparent, non-elastic, flexible prodtuct that was
100
40
60
80
mounted on glass microscope slides and observed
0
20
with a light microscope. The stomata were clearly
.
TOMATO=
visible and they were measured and counted.
SOo
2280
Leaf Area and Stomatal Area Calculations.
c
Since all radioactive couinting data obtained was on
a dry weight basis, conversions of dry weight data
9
to leaf area data and then to stomatal area data
140 e/e /were performed in the following manner:
ALL WHITE BEAN
REDKIDNEYBEAN
A) Leaf weight per unit leaf area was determined by measuring leaves with a planimeter, then
________________________________________
obtaining their dry weight by drying to constant
60
20
40
100
80
o
weight at 700 and calculating a leaf weight to area
TOMATO
SORGHUM
lo.
ratio. The total leaf area includes both leaf sutrE
faces
since during submergence both were exposed
8
/i^ELL//
/BEAN/
ODKIONEY BE^N, ^
to solution.
Qe
B) Stomata were counted and measured for
/ 4X
6
both leaf surfaces of the 4 plants studied and stoc
/;/<matal area per unit leaf area was calculated (the
4area of a stoma was calculated from the equlation
2. i/>for the area of an ellipse, A=3 ab, where a and b
are the semiaxes of the ellipse, since an open stoma
40
10 0
is approximately elliptical in shape).
20
60
0
0
TIMSE-minutes
The values determined and used to make these
conversions are presented in table I.
FIG. 2. Foliar uptake of 59Fe as a function of subRadioactive Counting Technique. For assaying
mea rsion time. Again results are expressed 3 ways to
the 519Fe content of treated leaves, the leaves were
sho>w the correlation between stomatal area and uptake.
~ated
//surface,
nU
-i
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18
PLANT PHYSIOLOGY
Table I. Some Leaf Characteristics of 4 Plants Studied Necessary for Conversion of 59Fe Uptake from Dry Weight
Basis to Leaf Area and Stomatal Area Bases
Plant Species
Red kidney bean
Small white
bean
Sorghum
Tomato
Mg dry
wt per
cm2
leaf
area
3.04
2.89
Stomata
per cm2,
lower
surface
(X 10-4)
4.0
4.0
Stomata
per cm2,
upper
surface
(X 104)
.10
.34
Stomata
per cm2,
both
surfaces
(X 10-4)
4.1
4.3
Average
size of
individual
stoma
Average
area of
individual
stoma
(,u)
(/2)
2 X 7
2 X 7
11
11
2 40
3.23
1.6
5.3
1.10
.78
2.7
6.1
5 X 15
3 X 12
59
28
dried at 700 to constant weight, ground, weighed
into planchets, and ashed at 5500 until a white ash
was obtained. The ash was then plated onto the
bottom of the planchets (to give a uniform counting
sturface) by suspending the ash in a solution of
ethanol and detergent, in the planchet, and allowing
the solution to evaporate. This procedure was repeated several times in order to obtain a uniform
plating. The samples were then counted with a
thin window Geiger-Mueller tuibe in standard
fashioin.
Results
and Discussion
It is apparent from figure 1A that the addition
of the surfactant caused a large increase in iron
uptake in both the sorghum and the red kidney
bean leaves during the day. An increase also occturred due to surfactant for the sorghum during
the night. One should note from figure 1A that
on a dry weight basis, sorghum leaves take up a
muich larger amount of "9Fe where the surfactant
was added in the day treatments than do the bean
leaves. The question naturally arises as to why
these 2 species behave so differently with respect to
uptake when grown under the same conditions and
treated in the same manner.
Figure 1B presents the uptake data on a total
leaf area basis rather than a dry weight basis.
Again sorghum and bean do not absorb like amounts
of iron.
In figure 1C the uptake data is presented on a
stomatal area basis, and we see that a good agreement now exists between the sorghum and bean
leaves for the day-plus surfactant treatments. This
agreement is strong evidence for stomatal uptake.
The fact that the agreement exists only between
day-plus surfactant treatments is easily explained.
Night treatments should not agree on a stomatal
area basis since the stomata are closed at night.
The day-minus surfactant treatments probably reflect some stomatal entry since they are higher than
the night-minus surfactant treatments and the agreement between the 2 species is certainly better on a
stomatal area basis than on either a dry weight
Stomatal
area per
cm2 leaf
area
(/2 X 10-5)
4.5
4.8
16.2
168
basis or a leaf area basis. The day-plus surfactant
plus sucrose treatments are depressed somewhat
from the day-pluis surfactant treatments. This depression may be explained as being the result of an
increase in solution viscosity due to the sucrose.
The depression is more pronounced in the bean
leaves than in the sorghum leaves since the beani
stomata are much smaller than the sorghum stomata, a fact resulting in a higher perimeter to area
ratio or a higher resistence to the mass flow of a
viscous solution through the stomata. An alternative explanation for the sucrose depression might
be that the sucrose caused the plasmolysis of the
guard cells and subsequently the closure of the
stomata, although to affect this closure would probably require longer than the 15 minute treatment
time.
Figture 2 deals with the uptake of 59Fe3+ as a
function of time of submersion. Figure 2A presents the uptake data on a dry weight basis, figure
2B on a leaf area basis, and figure 2C on a stomatal
area basis. The most important point to be realized
from these graphs is the good agreement between
species for iron uptake as a function of time on a
stomatal area basis (fig 2C). The essentially linear
rate of uptake for the first 30 to 40 minutes followed by a sharp decrease in rate is highly suggestive of a mass flow mechanism. The sharp decrease
in uptake rate may occur due to the filling of the
sub-stomatal chamber with treatment solution. The
poorer agreement between species as suibmersion
time increases can be explained as an expression
of internal leaf characteristics, such as the size of
the sub-stomatal chamber and the arrangement of
the mesophyll cells suirrounding the chamber.
In figure 2A and 2B, uptake by the 2 bean
varieties is essentially the same. Also, the sorghum
and tomato curves are very similar to each other.
The reason for these similarities is found in table
I. The 2 bean varieties have similar stomatal areas,
as do the sorghum and tomato, or in other words,
a correlation exists between stomatal area and
uptake.
The graphical description of the translocation
of 59Fe from treated leaves as a ftunction of time
presented in figure 3 shows that from 25 to 60 %
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EDDINGS AND BROWN-UPTAKE AND
of the applied iroin was translocated from the treated
leaf, depending upon the plant species in quiestion:
plant species is shown to be an important consideration when assessing the mobility of iron. It is
also of interest that most of this loss occurred in
the first 50 hoturs after treatment. It would appear
that the foliar-appliedl iron was, to varying degrees,
mobile.
Conclusions
Stomata tunidotubtedly play a major role in the
tuptake of ferric ion uinder the conditions presented
here, the conditions being total submersion of detached leaves into an aqueous soltution with a stirfactant present. If total submersion of a plant leaf
in a soluitioin does not differ greatly from total
spray coverage of a plant leaf with a soluition,
stomatal uiptake should also be of major importance
for the uptake of sprayed materials. Short term
submersion of leaves probably does not differ greatly
from spray coverage, the one exception beinig that
sutbmersioin affects both leaf sturfaces whereas
sprayiing may or may not. We have shown that
stomatal tuptake is of short dturation, even tunder
conditioins of total stubmersion; however, conditions
favorinig stomatal tuptake following a spray application are also of short duration and stomatal tuptake ceases whein the spray solution dries. Reports
of long term uptake are complicated by a lack of
differentiation between absorption, adsorption, precipitation, and translocation. On the other hand,
materials not subject to precipitation and which do
not dry rapidly, suich as herbicidal petroleuim derivatives or materials with oily carriers, probably remain in the liqutid state long enouigh for cuiticular
penetration to l)ecome important.
Critics of the stomatal luptake theory state that
penetration of solutions into substomatal chambers
does not constituite absorption, sinlce the material
in qtlestion has not entered the symplastic system
of the plant. However, penetration of materials
into the cuticle or through the cuiticle and into
epidermal cell walls does not constituite absorption
for the same reason.
WN'e have shown that translocatioin of foliarapplied iroin varies with species. This variation
may be explained by the stomatal and veinal patterns of the 3 species. The stomata of the
grass species are alligned in regullar longituidinal
rows interspersed with veinal tissue so that the
dlistanice from an individual stoma to conduicting
TRANSLOCATION OF 59FE
19
elements is never very great. On the other hand,
the broadleaf species have a palmate veination pattern and a random distribultion of stomata so that
the distance from an individual stoma to conduicting
tissuie varies greatly; in other words, the pathway
to conducting tissuie in broadleaf species is more
tortulouis than in grass species. As the iron passes
through the stomata, it is probably absorbed by the
parenchyma cells suirrouinding the suibstomatal chambers and symplastically translocated to the phloem,
and the greater the number of cells throuigh which
it mulst pass, the smaller the amouint eventually
reaching the phloem.
Literature Cited
1. BARRIER, G. E. AND W E. LooMis. 1957. Absorption and .ranslocation of 2,4-dichlorophenoxyacetic
acid and 32P by leav es. Plant Phvsiol. 32: 225-
31.
2. BRANTON, D. AND L. JACOBSON. 1962. Iron transport in pea plants. Plant Physiol. 37: 539-45.
3. BROWN, A. L., S. YAMAGUCHI, AND J. LEAL-DIAZ.
1965. Evidence for translocation of iron in plants.
Plant Physiol. 40: 35-38.
4. BROWN, J. C. 1956. Iron chlorosis. Ann. Rev.
Plant Physiol. 7: 171-90.
S. CURRIER, H. B. AND C. D. DYBING. 1959. Foliar
penetration of herbicides review and presenit status.
Weeds 7: 195-213.
6. DONEY, R. C., R. L. SMITH, AND H. H. WIEBE.
1960. Effects of various levels of bicarbonate,
phosphorus, and pH on the translocation of foliarapplied iron in plants. Soil Sci 89: 269-75.
7. FISHER, E. G. AND D. R. WALKER. 1955. The apparent absorption of phosphorus and magnesium
from sprays applied to the lower surface of McIntosh apple leaves. Proc. Anm. Soc. Hort. Sc.
65: 17-24.
8. GUEST, P. L. AND H. D. CHAPMAN. 1949. I1vestigation on the use of iron sprays, dusts, and soil
applications to control iron chlorosis of citrus.
Proc. Am. Soc. Hort. Sci. 54: 11-21.
9. JACOBSON, L. AND J. J. OERTLI. 1956. The relationship between iron and chlorophyll content in
chlorotic sunflower leaves. Plant Phvsiol. 31:
199-204.
10. SARGENT, J. A. 1965. The penetration of growtli
regulators into leaves. Annn. Rev. Plant Physiol.
16: 1-12.
11. ZELITCH, I. 1961. Bioclhemical control of stomIlatal opening in leaves. Proc. Natl. Acad. Sci. 45:
1703-08.
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