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RELATION OF H-ION CONCENTRATION OF TISSUE FLUIDS TO
THE DISTRIBUTION OF IRON IN PLANTS
R. A. INGALLS AND J. W. SHIVE
(WITH TEN FIGURES)
Although iron is an essential element for plant growth, it is not always
present in an available form for assimilation by the plant. Factors influ-
encing the availability of iron are not well understood. Previous (^) investi- gations of the iron problem have dealt mainly with a study of the external
medium in which the plants were grown, with less emphasis attached to
the (^) study of (^) the conditions existing within the plants themselves.
PATTEN and MAIN (18) found that iron was precipitated from solution
in varying degrees from pH 3.5 to 6.0, practically all being precipitated at
6.0 and above, thus rendering it unavailable for absorption by the plant.
This was also brought out by HOPKINS and WANN (10). They found diffi-
culty in growing plants in a medium of pH 6.0, due to the fact that iron
was removed by adsorption on calcium phosphate which gradually pre-
cipitated as the solution became alkaline, a physical chemical effect capable of influencing iron availability within the plant as well as in culture media. The fact that lack of (^) available iron is not entirely due to the conditions
existing in the culture media may be shown by reference to the work of
APPEL (2). He found that buckwheat plants were less sensitive to changes
in reaction of culture media than were corn plants, corn requiring much more iron than buckwheat. Little (^) difficulty, therefore, was experienced in growing buckwheat without chlorosis in solution cultures in which corn suffered from lack of (^) available iron. After a study of the internal condi- tions existing within the plant, presented in the following pages, an expla- nation of this phenomenon (^) may be attempted.
LOEHWING (13) has reported that plants grown in a medium high in
lime display peculiar iron (^) immobility characterized by copious precipita-
tion in the roots. He states that the lime reduces the sap acidity to the
point of interference with (^) internal iron mobility. Lime injury involving
chlorosis has been reported for corn by MAZE' (16), for pineapples by GILE
(5), for rice by GILE and CARRERO (6), for pears by MILAD (17), and for
citrus fruits by LIPMAN (22).
In addition to the work cited, some work has been reported on the (^) H-ion concentration of tissue fluids. (^) HAAS (8) (^) made studies of actual and total acidities and of the total alkalinity of a number of plants of agricultural importance, together with a study of the influence of liming the soil upon 103
PLANT PHYSIOLOGY
these (^) acidities. In this connection he reports that in ten out of fourteen cases the addition of lime was followed by a decrease in actual acidity of the plant juice, (^) which seems to point to the fact that plant juices are in- fluenced to some degree by the nature of the medium in which they are grown. Other points of interest in his study of (^) plant juices were as fol- lows: (1) the presence of a hydrogen-ion concentration gradient for tissue fluids which is not always in the same direction in different species or in the stems and leaves of the same species; (2) an increase in acidity with
age increase in the plant, and (3) that illumination tends to decrease the
acidity of the plant. This point was further brought out by GusTAFSON
(7). He worked with Bryophyllum calycinum, determining the pH of its
juice at various intervals throughout the day and the early part of the
evening. He found that the acidity of the juice decreased during the day
and increased again at night, but if the plant was kept in the dark con-
tinuously for^24 hours or more, the acidity did not continue to increase
but began gradually to decrease, as the food supply was used up in respi-
ration. The highest (^) acidity was reached at 10 A. M. while the lowest was
obtained at 4 P. M. The experiment was carried out during both clear and
cloudy days, and it was found that the same general results were obtained
on both types of days but to a different degree.
CLEVENGER (4) found a similar nocturnal change in pH of the tissue
extract of cowpeas due to change in light intensity. He also pointed out
that the pH varied with temperature, being higher at high temperatures
than at low temperatures. ATKINS (3) made a study of the variation in
the juices of different plants and found a range in pH from 1.4 to 8.0, but
only in^ rare^ cases^ did^ he^ find^ any above^ 7.0.
The purpose of this investigation has been to determine first, the influ-
ence of day to night variations in light intensity upon the hydrogen-ion
concentration of the plant tissue fluids of a number of different species;
second, to study the relation between hydrogen-ion concentration of the
plant tissue -fluids, as influenced by variation in light intensity, and the
"filterable" or soluble iron content of the plant tissues; third, to study
the relation between hydrogen-ion concentration of the plant tissue fluids
and the total iron content of the (^) plant tissues; and (^) finally, to (^) study the influence of these internal factors^ upon the^ iron^ mobility, its distribution,
and ability to function in the plant tissues.
Experimental methods
Plants used in the first^ part of this^ investigation were^ grown in sand
and solution cultures to insure as uniform a medium of growth as possible.
It was later^ found, however, that^ quite similar results^ were^ obtained with
PLANT PHYSIOLOGY
cultures, iron was supplied to the plants in the^ form^ of^ a^ freshly^ prepared ferrous sulphate solution containing 0.1 mg. of iron per cc. of solution. In the early stages of growth, 0.1 cc. of the iron solution containing 0. mg. of iron per cc. was added to 1000 cc. of nutrient solution. This amount, however, was increased and sometimes reduced during the later stages of growth, as the external conditions^ and the^ requirements^ of the plants made this necessary. Seeds used in these experiments were germinated between moist^ filter- papers and then transferred to a germinating net as described by SHIVE (21) When the seedlings were 4 cm.^ tall^ they^ were^ carefully^ selected for^ uni-
formity of size and vigor and transferred to their respective media,^ ten
plants being used in each sand culture and three in each solution culture. To prepare the material for the extraction^ of the^ tissue,^ it was cut into
small pieces and placed in test tubes. These were then plugged with paraf-
fined cork stoppers plunged into a^ salt-ice mixture^ and frozen as^ quickly
as possible in order to prevent any appreciable chemical change before
freezing. In^ all cases^ duplicate^ samples were used.^ Preparatory^ to ex- pressing the tissue fluids, the test tubes containing the samples were placed in tepid water and the material allowed to thaw. This^ usually^ required from five to ten minutes. At this stage, the material was removed from the tubes and the juice extracted by means of a small screw press. Pre-
cautions were taken to prevent the tissue fluids from coming in contact
with anything except glass surfaces.
pH determinations were made^ electrometrically^ by^ means^ of^ the^ hy-
drogen electrode and a type K Leeds and Northrup potentiometer. About
5.cc. of juice were used at each determination. It^ was^ placed^ in^ a^ short
Pyrex tube and hydrogen was allowed to bubble through until^ a^ constant
potential was^ attained.^ Electrodes^ were^ cleaned and^ platinized^ before
making determinations and^ again frequently^ throughout^ the^ experiments.
They were also checked against a standard acetate solution of^ known^ pH
value.
Samples of green plant tissues of the various species studied, from
which the moisture^ content^ and^ total^ iron^ content^ of^ the^ plant^ were^ deter-
mined, were^ taken^ at^ the end^ of^ each^ experimental^ interval^ (usually^ at^ the
end of two^ hour^ intervals) throughout a^ day and^ night period.^ For total
iron determinations, the^ plant tissues^ were^ dried^ in^ an^ oven^ at^ about 850^ C.
for 48 hours and at 1000 C.-102' C.^ for^24 hours.^ They were^ then^ ground
to a^ powder with^ a^ pestle and^ mortar^ in order that uniform^ samples^ might
be obtained. The material^ was^ then^ placed in^ a^ desiccator^ over^ night, after
which duplicate samples of^ 0.1^ gm. each^ were^ weighed out^ and^ placed in
Pyrex test^ tubes.^ Iron^ analyses^ were^ made^ according^ to^ a^ method^ described
INGALLS AND SHIVE: DISTRIBUTION OF IRON IN PLANTS
by WONG (26). It consisted in completely digesting the weighed sample
with 1 cc. of concentrated iron-free sulphuric acid, allowing the contents of
the tube to cool for about 20 seconds and then adding to it a ten per cent.
solution of sodium chlorate. This solution was added carefully drop by
drop and allowed to run down the side of the tube to prevent excess (^) spat-
tering when the solutions came in contact with one another. Boiling was
then continued until white fumes (^) appeared. The content of the tube (^) was clear and colorless when oxidation was completed.
Potassium sulphocyanate, 5 cc. per sample, was used as an indicator,
and the contents made to a known volume with distilled water. The red
color produced by the indicator varied in depth according to the amount
of iron present. Each sample containing the unknown was compared in a
Duboseq calorimeter against a standard iron solution. This solution was
prepared by dissolving 0.7 gm. of recrystallized ferrous ammonium sulphate
in about 50 cc. of distilled (^) water. To this was added 20 cc. of 10 per cent. sulphuric acid and then sufficient one-tenth (^) normal potassium permanganate
solution was added to just oxidize the ferrous salt completely. It was then
diluted to 1 liter. This solution contained 0.1 mg. of iron per cc.
To both the standard and the unknown iron solution to be determined
was (^) added 0.25 (^) cc. of dilute (30 per cent.) nitric acid. It was found that the addition of this solution prevented the reduction of iron (^) from the ferric
to the ferrous form, thus preserving the color of the test and standard solu-
tions without (^) change for a sufficient length of time to compare with accuracy at least twelve unknowns against the same (^) sample of the standard.
Since it was impossible to remove all the moisture by pressure from the
plant tissue, some means had to be adopted by which total (^) filterable iron
could be determined on the basis of dry plant tissue. This was accom-
plished as follows:
The tissue for which iron determinations were to be made was cut into
rather small pieces and thoroughly mixed. One sample was taken from
the mixed tissue and frozen in order to extract the tissue fluid. Iron
analyses were made on 1 cc. of (^) the extracted filtered fluid according to the method previously (^) described. The remainder of the plant tissue was
weighed, dried, and the moisture content determined. From this, the
amount of moisture per gram of dried plant tissue was calculated.
The quantitative (^) iron analyses on 1 cc. samples of the filtered tissue
fluid, together with the determinations of moisture and the content of solid
material in the filtrates, furnished the necessary basis for the calculation of
the iron per gram of dry material in the plant (^) tissues in question.
INGALLS AND SHIVE: DISTRIBUTION OF IRON IN PLANTS
available iron, thus causing considerable loss in production, while under
the same conditions other plants appear to suffer no injury from lack of
available iron. It is reasonable to assume, therefore, that an internal
mechanism renders small quantities of iron mobile in the plant and avail-
able to the chlorophyllous cells.^ It was with these points in mind that the
present investigation was undertaken.
The influence of light intensity on the^ H-ion^ concentration^ of^ plant tissue
fluids The data obtained from the experiments conducted for the purpose of showing the effect of light intensity on the hydrogen-ion concentration of plant tissue fluids are presented in tables I and II. In the first column of each table is given the time at which pH (^) determinations of tissue fluids (^) were made during a period of 24 hours. In (^) the succeeding columns are given the average pH values of juices of stems and leaves of the plants indicated at the (^) head of the respective columns. Each value represents the average of two or more determinations. The results given in table I are all from plants with thin leaves, while those of table II deal with thick-leaved, fleshy (^) plants. Figure 1 shows the graphs plotted from the data as given in table I, rep- resenting the course of change in the pH values of the stem and leaf juices of (^) buckwheat plants during a period of 24 hours. The experiment from which these data were obtained was carried out in the spring of the (^) year, the
TABLE I PH VALUES OF TISSUE FLUIDS OF STEMS AND LEAVES OF BUCKWHEAT, CLOVER AND RuMex PLANTS AT TWO HOUR INTERVALS
BUCKWHEAT CLOVER (^) Rumex TIME__ STEMS LEAVES STEMS (^) LEAVES LEAVES
9: 00 (^) A. M. 4.615 5.047 5.596 5.968 4. 11: 00 A. MA. (^) 4.531 5.139 5.697 5.934 4. 1: 00 P. M. (^) 4.804 5.342 5.883 6.272 4. 3: 00 P. MA. 4.726 5.333 5.868 6,238 4. 5: 00 P. AM. 4.767 5.376 5.985 6.342 (^) 4. 7: 00 P. MA. 4.757 5.351 5.951 6.340 4. 9: 00 P. M. 4.446 5.300 (^) 5.866 6.255 (^) 4. 11: 00 P. M. 4.548 5.190 (^) 5.765 6.002 (^) 4. 1: 00 A. AM. (^) 4.497 5.139 5.783 5.951 (^) 4. 3: 00 A. M. (^) 4.353 5.089 5.714 5.968 4. 5: 00 A. MA. 4.447 (^) 5.021 5.630 5.850 4. 7: 00 A. M. 4.454 4.920 (^) 5.613 5.850 4.
PLANT PHYSIOLOGY
6 .0 -I-
- 8
5.6 (^) Clover-
7 a.m. 11 3 p.m.^7 11 3 p.m.
FIGS. 1 (upper) and 2 (lower). Graphs representing^ the^ course^ of^ change^ in^ pH values of leaf and stem juices of buckwheat and clover plants during a 24-hour experimental interval.
pH
PLANT PHYSIOLOGY
TABLE II
PH VALUES OF TISSUE FLUIDS OF STEMS AND LEAVES OF Sedum, (^) BryophylluM AND Tradescantia PLANTS AT TWO HOUR INTERVALS
Sedurm Bryophyllum Tradescantia TIME -_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ STEMS LEAVES STEMS LEAVES STEMS LEAVES
9: (^00) A. M .............. 5.131 5.122 3.685 3.584 4.641 (^) 4. 11: 00 A.M............ 4.970 5.038 4.311 4.125 5.241 (^) 5. 1: 00 P. M.. 5.122 5.081 4.750 4.852 5.528 (^) 5. 3: 00 P.M............. 5.376 5.292 4.835 4.818 5.461 (^) 5. 5: 00 P. M. 5.359 5.359 (^) 4.936 4.920 5.511 5. 7: 00 P. M..... 5.494 (^) 5.440 4.666 4.649 5.545 5. 9: (^00) P. M.. 5.249 5.249 4.159 4.243 5.148 5. 11: 00 P.M......M 5.224 5.198 3.753 3.719 5.351 (^) 5. 1: 00 A.M......... 5.207 5.114 3.821 3.685 5.275 (^) 5. 3: 00 A. M............ 5.087 5.080 3.618 (^) 3.516 4.666 4. 5: 00 A. M.. .... 4.869 4.945 (^) 3.652 3.550 4.548 4. 7: 00 A. (^) M....... 4.784 4.911 3.516 3.347 4.599 4. 9: 00 A. M. (^) 3.449 3.483 4.954 4.
from 4.91 at 7: 00 A. M. to 5.46 at 5: 00 P. M. In this plant the pH values
of stem and leaf juices show no significant differences and the graphs rep-
resenting these values follow somewhat the same course throughout. This
appears to be characteristic of fleshy, thick leaved plants such as were here
used.
Bryophyllum, another of the thick leaved, fleshy plants, shows an ex-
treme range in the pH values of its tissue fluids from day to night, as is indi.
cated by the graphs of figure 4. This range is more than double that indi-
cated for Sedum. The maximum pH 4.92 for leaf juices occurred at 5: 00
P. M. and the minimum pH 3.34 at 7: 00 A. M., while the corresponding
maximum and minimum values for stems, pH 4.93 and pH 3.44 are shown
for 5: 00 P. M. and 7: 00 A. M., respectively. The graphs representing the
courses of pH values for stems and leaves throughout the experimental
period run quite closely together, but again there are no significant differ-
ences between stem and leaf values such as are indicated for the thin-leaved plants.
In table I and table II are presented also data for Rurnex and Trades-
cantia, dealing with the influence of light intensity on the H-ion concentra-
tion of plant tissue fluid. These data are not here discussed, and are pre-
sented merely to emphasize the fact that acidity change with variation in
light intensity is a phenomenon common to many types of plants and occurs
INGALLS AND SHIVE: DISTRIBUTION OF IRON IN PLANTS
pH
(^7) a.m. 11 3 p.m. (^7 11) 3 a.m. 7 FIGs. (^3) (upper) and 4 (lower). Graphs representing the course of (^) change in (^) pH values of leaf and stem juices of Sedum and (^) Bryophyllum plants during a 24-hour experimental interval.
in proportion to the degree of succulency. This is further emphasized by
tests of (^) many species, the data for which are not here presented. It has long been known that the succulent plants exhibit periodic rise and fall of the acid (^) content of their juices, and the relation of this phenom- enon to the respiratory (^) processes has been the subject of extensive and thor-
113
INGALLS AND SHIVE: DISTRIBUTION OF IRON IN PLANTS
9 a.m. (^1) p.m. 4 8 12 6 a.m. FIGS. 5 (upper) and 6 (lower). Graphs representing the pH values of tissue fluids of Sedurn and Bryophyllum plants which were (^) exposed to intermittent (^) (unbroken line) and continuous (^) (broken line) periods of darkness.
during the experimental periods. Data for such a comparison are given in
table III and are shown graphically in figures 5 and 6.
It will be observed that the juices of the plants exposed to alternate light
and dark show the usual wide range in pH values, while the juices of the
plants kept in (^) continuous darkness show only very slight fluctuations which are not at all related to the light factor.
115
PLANT PHYSIOLOGY
It may be of interest here to emphasize the point that comparisons of plant tissues or tissue fluids, particularly with respect to pH values and also, as will be brought out later, with respect to soluble iron (^) content, can (^) be of little value unless the (^) samples upon which quantitative measurements are made are collected at the same time during the day or night. External con- ditions, particularly light intensity, which is subject to almost continuous fluctuation, have a pronounced influence upon these internal factors and may render any set of measurements of them useless for (^) purposes of com- parison unless careful attention is given to the collection and preparation of experimental material. Relation of pH values to soluble iron content of plant tissue fluids
It has been suggested by HOFFER and CARR (9) that the mobility of iron
and aluminum salts in plants is associated with high sap acidity, and they
have shown that under certain conditions relatively large quantities of iron and aluminum will accumulate in different parts of corn plants. It has also been shown by MARSH and SHIVE (15) that plants under certain condi- tions may become chlorotic from lack of iron in the leaves when the total iron content of the plants is excessively high. Furthermore, in view of the
fact that the iron requirement of plants is relatively high during periods
of high light intensity and low during periods of diminished light, it is of interest to determine whether or not the soluble or filterable iron in plants
bears any relation to the periodic fluctuation in the pH values of the tissue
fluids resulting from variations in light intensity. Accordingly, the soluble (filterable) iron content of tissue extracts taken at regular intervals throughout 24-hour experimental periods was deter- mined for a number of species. The manner of taking samples, preparing the extracts, and the technique employed in making the pH (^) tests and the
chemical analyses have already been described. The data are presented in
table IV. This experiment, like those previously described, was carried out
on clear days so that the plants were exposed to approximately maximum
variation in light intensity from day to night. Examination of the data of table IV brings out the fact that there is a direct (^) and very exact relation (^) between the (^) H-ion concentration of the tissue
fluids and the soluble iron content of all the species investigated. In each
species, the fluctuation in^ pH values of the plant juices with variation in light intensity is followed, in the inverse^ order, by a corresponding fluctua- tion in the soluble iron content. That (^) is, for (^) each species, low pH values
correspond with high soluble iron content and high pH values with low
soluble iron content. To bring out the exactness^ of this relation^ and to show the course of fluc-
tuation of the soluble iron^ content^ in the^ plants during a 24-hour period,
PLANT PHYSIOLOGY
pH ~~~~~~~~~~~~F pere^ mgs. g.-
5.5 E -^ -^ -^ -^ |^ |.
53 __ -.
3.5 -A 4 X.
5 a.m. 8 1 P.m. 5 12 FIGS. 7 (upper) and 8 (lower). Graphs representing the^ course of variation in^ pH values and soluble iron content of Seduin and Bryophylluntl plants during a 24-hour experimental interval.
the data for two species of^ fleshy succulents^ (Bryophytlum^ and^ Sedum) and for two species of^ thin-leaved plants showing^ a^ relatively^ low degree of succulency (tomato^ and^ buckwheat) have been plotted^ to form the graphs of figures 7 and 8, and 9 and 10. The pH values and the values for the soluble iron content for each of these species are plotted together to form a pair of^ graphs with common abscissas,^ the^ ordinates^ on^ the^ left indicating
118
INGALLS AND SHIVE: (^) DISTRIBUTION OF IRON IN PLANTS
pH values, those on the right expressing soluble iron content (mg. per
gram of dry plant tissue). To avoid intersecting of the graphs, the ordi- nates on the right are written in the inverted order. The lower graph for each species (figs. 7 and 8) shows (^) the usual course of
pH change during a 24-hour period and this, in every case, is almost dupli-
cated by the inverted graph representing the (^) course of fluctuation in (^) the soluble iron content during the same period, thus indicating an intimate relation between pH values of tissue fluids and that portion of iron in the plant which may be regarded as the (^) active fraction, on the reasonable as-
sumption that filterable iron here considered is mobile, readily available, and
capable of functioning in the plant processes. Another significant and important relation is here indicated. A com-
parison of the data for Bryophyllurm with those for Sedum (fleshy suc-
culents, figs. 7 and 8) brings out the fact that the juices of the former show relatively low pH values, ranging between 3.48 (^) and 4.90, with a relatively very high content of filterable iron, ranging between 0.0861 and 0.1071 mg. per gram of dry tissue; while the juices of the latter show higher pH (^) values, 4.94 (^) to 5.43, with a correspondingly much lower content of filterable iron, ranging from 0.0399 to 0.0536 mg. per gram of dry tissue during a 24-hour period. A comparison of the data for (^) tomato and buckwheat (thin-leaved plants with relatively low degree of succulency, figures 9 and 10) shows this relation in an equally definite manner. Of the four species graphically considered, the tomato shows the highest pH values, varying between 5.
and 6.15, and the lowest content of filterable iron, fluctuating between
0.0314 (^) and 0.0237 mg. per gram of dry tissue, during an experimental period of 24 hours. This relation holds for all the species the data for which are presented in table IV. Furthermore, the relation holds also for different organs of the same plant, as (^) between stems and leaves, when these organs show considerable (^) difference in the pH values of their juices. This is clearly shown by the data in table IV for stems and leaves of buckwheat, tomato, asparagus and soybeans. The significance of this relation will be further considered in the (^) following section. It might be well here to (^) suggest that from the data thus far presented it appears that those plants in which the pH values of the tissue fluids lie close to (^) the precipitation point of iron (about 6.0) show greater fluctuation in the filterable iron content from day to night than do those plants in which the pH values of the tissue fluids lie considerably below the precipitation point of iron. This is probably to be inferred from the fact, as will (^) be brought out later, that in plants of the latter type a high percentage of the total iron is in the soluble form, and under such conditions small fluctuations in this fraction might be expected. However, this is merely put forth as
INGALLS AND SHIVE: DISTRIBUTION OF IRON IN PLANTS
TABLE V
AVERAGE PH VALUES, TOTAL AND SOLUBLE IRON CONTENT OF VARIOUS PLANTS OVER A 24-HOUR PERIOD
LEAF STEM
PLANT FE,^ PER TISSUE^ GRAM OF^ DRY^ FE, PER^ TISSUEGRAM^ OF^ DRY
ACIDITY TOTAL SOLUBLE ACIDITY TOTAL SOLUBLE pH nlg. w11g. pH^ fg. mg. Bryophyllum ......... 4.04 0.137 0. Rumex (whole plant) 4.24 0.219 0. Buckwheat .......5.........5.10 0.269 0.0516 4.58 0.082 0. Sedum (whole plant) 5.16 0.278 0. Tobacco 5.74 0.325 0. Tomato 5.87 0.297 0.0277 5.38 0.089 0. Asparagus .5.87 0.373 0.0256 5.54 0.111^ 0. Soybeans .6.01^ 0.469^ 0.0246^ 5.79^ 0.142^ 0. Clover (^) ............................... 6.10 (^) 0.571 0.0281 5.78 0.214 0.
The data for the various species presented in table V are (^) arranged in
the ascending order of average pH values of the different species. The data
were obtained by collecting samples of each species at regular intervals
(two or^ four hour intervals) throughout twenty-four hour periods on^ clear
days. The various measurements were made on these samples and the cor- responding (^) values obtained for each species were then averaged; so that each value in the table represents the average of the values obtained over a twenty-four hour period. It will be observed that the total iron (^) content increases in the different species as the pH value of the tissue fluids increases: that is, high total iron in any given species corresponds to high pH value of the tissue fluids and low total iron (^) with low pH values. Thus, Bryophyllun with the low average pH value of 4.04, shows also a relatively low total iron (^) content of 0.137 mg. per gram of dry tissue; while clover with a pH value of 6.10, shows the ab- normally high total iron content of (^) 0.571 mg. per gram of dry tissue. On the other hand, the soluble iron content of the different species varies in the inverse order with variation in pH values of the tissue fluids. That is, low pH values correspond to high soluble iron, and high pH values correspond with low soluble iron. Thus, (^) Bryophyllumn with a low average pH value of 4.04 shows a low (^) total iron content (0.137) but relatively high soluble iron (0.0958); while clover with a pH of 6.10 (^) shows the abnormally high total iron of 0.571 mg. per gram of dry tissue but a very low (^) content of soluble
iron (0.0281). These relations hold, not only for the different species, but
PLANT PHYSIOLOGY
also for different organs of the same plant, as between stems and (^) leaves when these organs show considerable difference in the pH values of their tissue fluids, as (^) has already been pointed out. From the foregoing considerations it is quite apparent that plants like Bryophyllum, Rumex and others with (^) relatively low pH values of the tissue fluids, absorb only very small quantities of iron, and that practically all (^) of the iron absorbed remains in a soluble form and is presumably (^) available and capable of functioning in chlorophyll production and other plant processes. But plants such as clover, soybeans, and (^) others with high pH values of the tissue fluids absorb relatively very large quantities of (^) iron, if this is available in the external medium, but only a small proportion of this remains in a (^) soluble form in the plant. Much of the total iron in plants like these is precipitated, probably along paths of translocation, and is there- fore unavailable and undoubtedly does not function in the plant (^) processes. If, for any reason, all of the iron in (^) plants of this type should become soluble at any one time, iron toxicity would probably follow and might re- sult in the death of the plant. The cause for the accumulation (^) of relatively large quantities of unavail- able iron in the tissues of plants in which the pH values of the (^) sap lie close to the precipitation point of iron, or the (^) mechanism by which this is accom- plished, is at present not clear. It may be (^) suggested, however, that through the precipitation of iron in the plant tissues this element is (^) removed from the field of osmotic activity and thereby a (^) diffusion gradient for (^) it
may be maintained from the outside to the inside of the plant, resulting in
the accumulation of relatively large quantities of non-available iron. From the data here presented, it is to be expected that (^) plants in (^) which
the pH values of the tissue fluids lie close to or above the precipitation point
of iron may yield a high total iron content, but only a small proportion of
this total iron can function in chlorophyll production and other plant ac-
tivities. This is (^) made clear, not only by chemical analyses of the tissues and
tissue fluids, but also by the fact that chlorosis is likely- to occur in these
plants from lack of available iron under slightly unfavorable conditions,
and particularly under conditions of high light intensity during periods
of which the plant sap attains its maximum pH value and the plant its
minimum value for soluble iron. On the other hand, plants in which the
pH values of the tissue fluids lie considerably below the precipitation point
of iron show relatively low total iron, but nearly all this iron can be ex-
tracted with the tissue fluid and is capable of passing through a quantita-
tive filter-paper of the highest quality. This iron appears to be quite mobile
in the plant and is uniformly distributed, as is indicated by quantitative
analyses of the tissues of the various plant organs, and it is therefore rea-
