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II. Nutrient Ion Accumulation in Roots
CECIL H. WADLEIGH
types: simple and that involving Donnan equilibrium. The magnitude
of either type of diffusional process will be conditional upon the concentrations of ions a t the surface of the absorbing roots as well as the concentrations of the ions in the absorbing cells in addition to the permeability of the root cell membranes to the diffusate. Exchange adsorption
is involved in ionic accumulation by roots, but there is some question
regarding the extent to which this process is limiting in ion accumulation. Ion accumulation through metabolic processes is the most intriguing of the three categories mentioned. It is the mechanism by
which chemical energy released through catabolism effects the accumulation of ions in the root cells against concentration gradients. To the
extent to which one can precisely differentiate between the roles of the
three types of processes of ion entry into roots, metabolism probably has
the predominant influence under most conditions.
Let us consider the factors that are known to affect the metabolic
accumulation of ions by cells capable of absorption. These may be listed
1 . Supply of metabolite in roots.
Hoagland and Broyer (1936) provided evidence that absorption
of K+ and NO, by barley roots was dependent on the supply of
respiratory substrate such as sugar.
2. Oxygen supply.
Numerous investigations in addition to Steward (1937), Hoagland and Broyer (1936), and Lundegardh (1940) reported that
a supply of oxygen to plant roots is essential for absorption of inorganic ions. It is of interest that the level of soil oxygen may
modify selectivity of roots with respect to ions absorbed. Hopkins
et al. (1950) found that decreasing the partial pressure of oxygen in air supply to the root zone was associated with an increase in the concentration of sodium in the tops of tomato,
soybean, and tobacco plants; whereas the opposite effect was
noted for the concentration of potassium, calcium, and phosphorus.
3. Carbon dioxide accumulation.
The inhibitory action of carbon dioxide in ion absorption has
been studied by Chang and Loomis (1945). They concluded
that increasing carbon dioxide content of the air supplied to
nutrient cultures may be toxic per se to plants over and above
any effect of inadequacy in oxygen supply. Under alkaline soil
conditions, increasing partial pressure of carbon dioxide brings
about an increase in concentration of the bicarbonate ion in the
soil solution. The presence of the bicarbonate ion i n the sub-
MINERAL NUTRITION O F PLANTS
strate was associated with an abnormally high level of potassium
accumulation and a low level of calcium content in the leaves
of pear trees (Lindner and Harley, 1944), and bean plants
(Wadleigh and Brown, 1952).
4. Supply of nutrient ions.
Numerous studies in addition to those of Hoagland and Broyer
(1936) and Robertson and Wilkins (1948) have shown that
metabolically controlled salt accumulation is conditioned by the
concentration of ions in the external medium.
5 . Level of internal accumulation.
One would deduce that cells with a relatively low concentration
of salt are more effective in displaying metabolically induced
salt accumulation than those having a relatively high salt level.
Hoagland and Broyer (1936) presented clear-cut evidence that
this is so.
6. Competition between ions.
Antagonism among ions diffusing through a membrane has
long been a field of investigation by physiologists. Studies of
this type have now evolved into ascertaining degrees of competition among ions for protoplasmic binding sites that effect
ion transport across the cytoplasm. Since this is a relatively active field of inquiry at the present time, it will be discussed at
7. Stimulative effects.
Viets (1944) found that increasing levels of Cat+ in the culture
solution up to 200 meq. per liter were associated with increasing
accumulation of potassium uniformly supplied at 5 meq. per
liter. This effect was not observed, however, under conditions
that inhibited oxidative metabolism. Caplin and Steward ( 1948)
observed that there was an active principle in coconut milk that
stimulated the mature secondary phloem tissue of carrot to
active and very rapid growth-conditions favorable to enhanced
absorption of nutrient ions.
8. Toxic effects.
Since salt accumulation by roots is usually dependent on concomitant aerobic respiration, Machlis ( 1944) proceeded to
study the effects of the oxidase inhibitors, cyanide and azide,
and of the dehydrogenase inhibitors, iodoacetate and malonate,
on the respiration and bromide accumulation by excised barley
roots. Cyanide and azide were found to inhibit approximately
two-thirds of the respiration and, at the same time, prevent any
accumulation of bromide. Machlis suggested that cytochrome
CECIL H. W A D L E I G H
oxidase was the enzyme affected. Iodoacetate and malonate were
found to inhibit both respiration and salt accumulation. Malate,
succinate, fumarate, and citrate reversed iodoacetate inhibition
of both respiration and accumulation of salt and neutralized
malonate inhibition of accumulation. This suggested that the
citric acid phase of Krebs' cycle was an integral part of the
respiratory system essential for salt accumulation.
9. Membrane specificity.
Specificity in ion absorption is especially evident in the wide
range in accumulation of sodium by different species when
grown at a given level of sodium supply. Collander (1941) determined sodium content of the herbage of 21 species supplied
with 2 meq. of sodium per liter in the culture solution. Very
little sodium was found in buckwheat, with the other species
ranging up to the very high accumulation of this ion found in
Ulrich (1941) showed the concurrence among trends of 0, consumption, CO, evolution, and salt absorption by excised barley
roots as affected by temperature. At 5O C., 100 g. of fresh roots
consumed 41 ml. of oxygen, produced 36 ml. of CO,, and accumulated K+ to a level of 13 meq. per liter of sap during 8
hours. These three effects increased concurrently with increasing temperature and at 35O C. 100 g. of fresh barley roots consumed 246 ml. of oxygen, evolved 247 ml. of CO,, and accumulated K+ to 41 meq. per liter of sap for the 8-hour period.
The role of soil moisture in the mineral nutrition of plants has
been reviewed by Wadleigh and Richards (1951) . Water in the
soil affects ionic accumulation in plants in three ways: (a) by
conditioning the availability of nutrient ions; (b) by regulating
plant growth processes dependent on soil moisture availability;
and (c) by the effects of anaerobiosis resulting from excess
water in the soil.
The foregoing factors affecting metabolic ion absorption are interrelated in their effects on the conflux or processes involved. Lundegardh
(1940) has developed an intriguing concept pertaining to the mechanism by which chemical energy released through the respiratory
process is used in ion accumulation by plant cells. Since this theory has
received considerable attention in recent reviews (Broyer, 1951 ; Robertson, 1951; Burstrom, 1951; Overstreet and Jacobson, 1952) on the min-
O F PLANTS
era1 nutrition of plants, it is well to consider it briefly, even though
recent investigations have emphasized its inadequacies.
The Lundegardh hypothesis assumes that the cytochrome systems
are an integral part of ionic transfer from the outer cell surface across
the cytoplasm into the vacuole. The chain of events from energy release by respiration to inward transfer of cations is set forth briefly as
follows: When the hydrogen atom liberated by the dehydrogenase
phase of respiration reaches the cytochrome system, the cytochrome
picks up the electron and the hydrogen ion is freed to exchange for
external cations. The resulting ferrocytochrome effects movement of
the electron to the outer cell surface, where the electron is lost via
cytochrome oxidase to externally supplied oxygen; and the oxidized
ferricytochrome acts as an inward carrier for anions. Robertson and
Wilkins (1948) pointed out that if the Lundegardh theory is valid, the
maximum rate of anion accumulation in the cell should take place
when each electron leaving via the cytochrome system is exchanged for
an anion from the external medium. On the assumption that respiration
is proceeding by the cytochrome system, all molecular oxygen involved
in the process becomes combined as water, and each molecule of oxygen
requires four electrons and four hydrogen atoms. Hence, the maximum
rate of salt accumulation should be 4 gram moles of monovalent salt
accumulated for each gram mole of oxygen used, or salt accumulation/salt respiration = 4. Robertson and Wilkins (1948) studied chloride intake by carrot tissue in relation to respiration and found that the
value of the aforementioned ratio tended to approach a value of four if
neither the rate of respiration nor the rate of chloride accumulation was
limited by the external concentration. These results were confirmatory of the Lundegardh theory.
In a later contribution, Robertson ef al. (1951) presented evidence
that the Lundegardh concept is not adequate to explain the complexities
of the mechanism of ion absorption. They found that 2,4-dinitrophenol
inhibited the accumulation of KC1 by carrot cells while enhancing the
rate of respiration. Furthermore, 2,4-dinitrophenol increases the leakage of ions from cells transferred from salt to water. The respiration
stimulated by dinitrophenol is sensitive to inhibition by cyanide. Although cyanide inhibits salt accumulation, it apparently does not induce leakage of ions from the cells. On the evidence (Bonner, 1949)
that dinitrophenol blocks the transfer of energy-rich phosphate to
growth processes, Robertson ef al. ( 1951) suggest that the Lundegardh
theory requires modification to allow for the participation of energyrich phosphate.
In their review of the mechanisms on ion absorption by roots, Over-
CECIL H . WADLEIGH
street and Jacobson (1952) point out that probably the greatest service
of the Lundegardh concept has been in showing the very close relationship between salt accumulation and the cytochrome system. Because of
the quantitative and qualitative linking of ionic accumulation with
cytochrome-mediated respiration, it seems probable that any theory of
salt absorption will involve the cytochrome system. They regard the
actual identification of the cytochrome-cytochrome oxidase system with
the actual ion carrier as premature.
These reviewers emphasize Rosenberg’s ( 1948) thermodynamic
treatment of ionic accumulation involving the postulation of ionic
donators and acceptors in diverse parts of the system: external substrate-membrane-internal
phase. The mechanisms of transport so
analyzed are involved in the Lundegardh theory. Nevertheless, Overstreet and Jacobson (1952) point out several objections to this theory,
viz.: (1) different rates of absorption of diverse ions with the same
charge, (2) unequal rates of absorption of cations and associated anions,
and (3) the mutual reciprocal effect of ion pairs. Furthermore, as
Epstein (1953) points out, there is now valid evidence for specific binding sites on protoplasm for cations in contrast to the concept that cations
move passively through the protoplasm as mere electrovalent companions to the anions that are transferred metabolically. Epstein, by supplying cations on exchange resins, has also accrued unpublished evidence that “salt respiration” is just as much associated with cation accumulation as with anion accumulation.
It is pertinent to consider the current evidence on the importance
of cytoplasmic binding sites in ion accumulation. Jacobson and Overstreet ( 1 947) have set forth the properties that must characterize hypothetical compounds or reactive groups that are capable of fixing in plant
cells inorganic ions taken from the culture medium in exchange for
equivalent ions released by the cells:
“1. The ion fixing compounds must be related to the oxidative
metabolism of the plant since there is a parallelism between ion absorption and oxygen tension. Under conditions of arrested metabolic activity
such as at low temperature or in solutions in equilibrium with low oxygen tensions, very little or no accumulation takes place in root systems
or in a variety of other cells.
“2. The ion fixing compounds in the protoplasm must form compounds with ions such as K+, Rb+, Ca++,Br-, NO,-, SO,=, HPO,=, and
others, in which the ions are held by relatively strong bonds since plants
are able to accumulate these mineral nutrients offered at extremely low
.levels of concentration. Furthermore, plant roots can compete with soil
colloids which bind nutrient cations very firmly.
MINERAL NUTRITION O F PLANTS
“3. The ion fixing compounds should account not only for the
absorption of anions and cations, but also for the very wide range in
the absorption of different ions of the same sign. This would presumably
require two or more classes of compounds or groups.
“4. Although the ions may be bound quite strongly to the ion fixing
complex, the combinations so formed must nevertheless possess a high
degree of instability since the evidence is that the ions pass into free
solution in the vacuoles. Moreover, it is a familiar fact that in response
to injury and death, ions absorbed in cells freely diffuse into the surrounding medium. For example, radioactive K could be completely
leached from ether killed barley roots.”
Jacobson and Overstreet (1947) studied intake and loss of radioactive strontium and iodide by barley roots to gain some insight into the
nature of ion fixation within the plant. Many of the determinations
were carried out at Oo C. in order to avoid complications due to longitudinal translocation. Observations on live roots at Oo C. and 2 5 O C. indicated the marked dependence of intake of radioiodide on metabolic
activity. There was a marked difference between behavior of iodide
and strontium in that the intake of iodide increased by a factor of 7.8
for the 25O C. rise in temperature, whereas intake of strontium increased by a factor of only 1.5. The difference between absorption of
iodide and strontium was further emphasized by observations on etherkilled roots. Dead roots showed relatively little intake of iodide at 2 5 O C.,
whereas they were found to take in more strontium at 25O C. during 15
minutes than live roots. Dead roots were found to release absorbed ions
to the external medium much more rapidly and completely than live
roots. The data suggest that strontium is held differently in live than
in dead roots; but even in living tissue, the exchange curves indicate that
no appreciable fraction of strontium or iodide is held in a nonexchangeable form. On the basis of these data, Jacobson and Overstreet (1947)
were able to make certain observations on the nature of ion carriers:
“(a) The ion carriers are intermediate metabolic products or closely
related substances; (b) the carriers are not stable in vitro; ( c ) they
undergo chemical alteration in the course of their carrier function; and
(d) they probably function as chelated complexes.”
Studies on the kinetics and characterization of binding sites for ions
in absorbing membranes now constitute a most promising field of inquiry in the mineral nutrition of plants. In order to evaluate the influence of microbial activity on the entry of nutrient ions into plant
roots, it would be helpful to consider briefly a few recent observations
on the nature of entities in protoplasm capable of binding ions. Roberts
and co-workers (Roberts et al., 1949; Roberts and Roberts, 1950; Cowie
CECIL H. WADLEIGH
et al., 1949) have produced evidence that the binding sites for potassium
in Escherichia coli are closely associated with carbohydrate metabolism.
Jacobson et al. (1950) investigated the competition between K+
and Ca++and found that although the presence of K+ reduced the absorption rate of Ca++,the effect of Cat+ on K+ was more complicated. I n
certain concentration ranges, Ca++depressed absorption of K+, but at
other levels it enhanced entry of K+. It was concluded that probably a
single binding substance serves both Ca++and K+, but a n additional special role must be assigned to Ca++in the absorption process. This is in
line with the earlier observations of Viets (1944).
Epstein and Hagen (1952) devised a novel approach to the kinetics
of ion binding during absorption by considering the reaction of an ion
with the binding entity as analogous to the equilibria between enzyme
and substrate. They hypothesize that the absorption involves the formation and breakdown of an intermediate labile complex, MR, of the
metal ion, M, with a metabolically produced binding compound or carrier, R. The analysis is essentiaIly identical with the kinetic treatment
of enzyme reactions presented by Lineweaver and Burk (1934) for
analysis of the equilibria between the substrate, S, the enzyme, E, and
the labile complex, ES. In such an analysis, the interfering ions assume
the role of inhibitors or alternate substrates.
Epstein and Hagen (1952) concluded from their work as follows:
“It was found that K and Cs interfere competitively with Rb absorption, and it is concluded that these three ions are bound by the same
binding sites or reactive centers.
“Except at high Rb or Na concentrations, Na does not interfere competitively with Rb absorptions; that is, is not bound by the same sites.
At low Rb concentrations, Na over a wide range of concentrations entirely fails to interfere with Rb absorption. Li is not competitive with
regard to the K-Rb-Cs sites; i.e., it is not bound by them. At relatively
low concentrations of Rb and Li, Li increases the rate of absorption
“The findings are considered to be consistent with the hypothesis and
indicate the existence of several distinct binding sites of which one
group binds K, Rb, and Cs in preference to Na and Li.”
Epstein and Leggett ( 1954) have studied competition among Sr,
Ba, Ca, and Mg for binding sites in metabolic absorption by barley
roots. They found that Ca, Sr, and Ba compete for identical binding
sites, but that the Mg ion does not compete for these same sites. Furthermore, Epstein (1953) has presented evidence on the absorption of
anions indicating that Br and C1 compete for the same binding sites,
but the NO, ion is noncompetitive on the halide binding.
MINERAL NITTRITION O F PLANTS
These recent investigations emphasize the diversity and specificity
of labile “ion carriers” or binding sites that undoubtedly occur in the
absorbing membranes of plant cells. There is sound evidence that these
“carriers” are metabolically energized in the protoplasm and they appear to be the key determinants in mineral nutrient absorption by plant
A N D IONACCUMULATION
The soil microbiologists have accrued information that would aid
them in predicting or indicating the manner and extent to which microbial activity may affect or be involved in the eleven previously listed
factors conditioning metabolically induced ion absorption by roots. Norman ( 1951) and Clark ( 1949) have recently reviewed the influence of
soil microorganisms on the availability of mineral nutrients to crop
plants. Let us consider briefly, nevertheless, some possible and probable
effects that soil microbes may have on these eleven factors.
1. Supply of metabolite in roots.
Since the population of soil microorganisms is especially intense
at the surface of roots, presumably owing to the supply of energy
material sloughed off or emanating from the roots, it is conceivable that the supply and quality of metabolite in the root may
affect the density of population in the rhizosphere, which, in
turn, may affect availability of minerals in the soil. Furthermore, microbial activity under certain conditions, influences the
availability of minor elements in the soil (Clark, 1949), which,
in turn, may affect chlorophyll formation in the leaves (Brown,
1953) supplying metabolite to the roots.
2. Oxygen supply.
Microorganisms carry on respiration and may contribute to depleting the oxygen supply under conditions of poor aeration
(Norman, 1951). Low oxygen supply will not only affect the
metabolism of root cells but will also condition the availability
of nitrogen and such minor elements as iron and manganese
( Wadleigh and Richards, 1951) .
3. Carbon dioxide accumulation.
Norman (1951) points out that “under optimum conditions in
soils well supplied with organic matter, carbon dioxide evolution may attain rates as high as 100 pounds per acre per day,
though figures of 20-30 pounds per day are more general.” Under poor soil aeration, such activity could contribute to bringing
about a level of CO, at the root surface that is inhibitive to ion
absorption (Chang and Loomis, 1945).
CECIL H. WADLEIGH
4. Supply of nutrient ions.
Clark (1949) has reviewed the evidence on this point. The activities of microorganisms can be a major consideration in the
supply of nitrogen, phosphorus, sulfur, and minor elements to
5. Level of internal accumulation.
Soil microbes would have an indirect effect on this point in that
they may have affected the supply of nutrients to the plant at
an earlier stage of growth, thereby influencing the facility with
which the plant effects ion accumulation at the later stage.
6. Competition between ions.
The production of CO, by microbes may effect an increase in
hydrogen ion concentration in the soil and thereby alter the
relative availability of cations and phosphate. Any effect microbial activity may have on depleting oxygen supply may also
have the further effect of modifying any inherent capacity the
plant root possesses with respect to selectively excluding sodium
(Hopkins et al., 1950).
7. Stimulative effects.
Schmidt ( 1951) reviewed the evidence relating the effects of
growth-stimulating substances produced by bacteria. Numerous
investigators have shown that various growth regulators for
higher plants are produced by bacterial synthesis. Substances
so produced have been shown to stimulate seed germination and
nodule formation on legumes. It is quite possible that certain of
these growth regulators could promote metabolically controlled
8. Toxic effects.
Steinberg (1947) grew tobacco seedlings in aseptic culture in
the presence of diffusates of certain common soil bacteria and
found that various types of chlorosis developed on the leaves. In
a later work, Steinberg (1951) collected samples of soil adjacent
to the roots of tobacco plants which were either growing normally or were severely frenched. In six of seven paired samples
of rhizosphere soil, higher populations of 3acillus cereus were
found for the frenching soil than were found in normal soils.
Roots of frenched tobacco showed especially large populations
of B. cereus, with values several times those hitherto reported
for normal plant roots. The study indicated a good probability
that B. cereus has a causal relationship in frenching of tobacco.
As Norman (1951) points out, organic acids are a product of
bacterial activity under anaerobic conditions. Little is known as