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.
var. Pearson

RS610), and tomato (Lycopersicum esculentum, L. Improved). The plants were germi-

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.
m AM

59 fe only 0 59 Fe plus surfactant 59 Fe plus surfactont plus sucrose

*' 6400
I

5600
>- 4800

, 4000
0

O
0

3200
2400
1600
800 360 320 280

iE

w~~~~~~~~~~

z
o

_E
w

I I I
ti

U.
4
W

240
4200

160
120 80

cN

U)
z
0

I I

A
L-

4
w

Or

12

-J

I0
8 6

Ul

4
2

D3

IL
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.

EDDINGS AND BROIWN-UPTAKE AND TRANSLOCATION OF 59FE

17

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

1600wL

TOMATO,

0 8 0

800
O 0> D n_

z
0

SM^ALL WHITE BEAN

20

40

TIME - Hours

60

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

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 2 40 3.23

Stomata per cm2, lower surface (X 10-4) 4.0 4.0

1.6 5.3

Stomata per cm2, upper surface (X 104) .10 .34

1.10 .78

Stomata per cm2, both surfaces (X 10-4) 4.1 4.3

2.7 6.1

Average size of individual stoma

Average area of individual stoma

(,u)
2 X 7 2 X 7
5 X 15 3 X 12

(/2)
11 11
59 28

Stomatal area per cm2 leaf area (/2 X 10-5) 4.5 4.8
16.2 168

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

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 %

EDDINGS AND BROWN-UPTAKE AND

TRANSLOCATION OF 59FE

19

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

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.