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THE IRON AND MANGANESE REQUIREMENTS

OF PLANTS
BY E. S. T W Y M A N
Department of Botany, The University, Birmingham

{Received 9 November 1950)

A. INTRODUCTION
Since the paper by Somers & Shive (1942), on the iron-manganese relation in plant
metabolism, numerous workers have carried out investigations directed to test the validity
of their hypothesis which suggests that the physiologically active iron is in the ferrous
state and that manganese regulates the concentration of ferrous iron in the cell. Arnon
(1943), in his consideration of this hypothesis, points out that it is doubtful whether
plants can utilize only ferrous iron, since the iron porphyrin enzyme systems involve the
reversible reaction ferric to ferrous. Since manganous manganese is supplied to plants,
on the basis of Somers's and Shive's hypothesis, the manganous ion must be oxidized to
the manganic ion which can then oxidize the ferrous iron; a system whereby manganese
can be oxidized in higher plants has been demonstrated by Kenten & Mann (1949).
Nevertheless, as pointed out by Hewitt (19480), hypotheses based on oxidation-reduction
potentials of simple ions are probably inadequate to explain the effect of certain metals
(e.g. copper) in inducing iron deficiency, and much more information is required regarding
the nature of the complex metalo-compounds formed. With regard to the heavy-metal
induced iron chlorosis, it has been suggested by Sideris & Young (1949) that manganese
instead of iron may react with porphyrin compounds thereby inactivating them for
subsequent conversion to chlorophyll; Hewitt (19486) envisages manganese and other
heavy metals with similar ionic radii, valency and electronic configuration competing
with iron and blocking a reaction leading to chlorophyll formation. Such reactions with
heavy metals may intensify the chlorotic state, but reactions in iron metabolism result in
the formation of at least two iron fractions. Oserkowsky (1933), Jacobsen (1945) and
Bennett (1946) have all postulated the existence of 'active' and 'inactive' forms of iron,
the active form being a protein in close association with or in the chloroplasts. The
results of the experiments reported here support, in general, the existence ot active and
inactive forms of iron.
Somers & Shive (1942) suggested that manganese deficiency was iron toxicity and iron
deficiency was manganese toxicity. Several workers (Morris & Pierre 1947; Berger &
Gerloff, 1947; Hewitt, 1948 a) observed the symptoms of manganese toxicity to be quite
distinct from those of iron deficiency, but mild forms of the toxicity are in my experience
identical with iron deficiency. Morris & Pierre (1947) showed that manganese toxicity
could be prevented by increasing the iron supply; this was due to antagonism whereby
the amount of manganese absorbed was reduced by 50%. Wallace & Hewitt (1946)
state 'although manganese has been clearly shown in solution culture to be antagonistic
to iron it appears unlikely to be an important factor in lime-induced chlorosis'; in the
experiments described in this paper the effect of manganese on the absorption of iron is
 The iron and manganese requirements of plants 211
not predictable and is not always explained by antagonism. Nicholas (1949) found that
the ratio of total iron to total manganese in plant tissues was not important in diagnosing
deficiency or toxicity of manganese, and the results presented here are in agreement with this
finding. Hewitt (19486), working with oats and sugar beet in sand culture supplied with
various concentrations of iron and manganese, claims that excess of one and deficiency
of the other are not synonymous; this may well be, but the iron/manganese balance in the
medium and in the tissues may be pre-disposing factors in the development of iron
deficiency, manganese deficiency or manganese toxicity.
Some of the experiments described in this paper had been completed before some of
those reviewed above but were mentioned by Twyman (1946). Three aspects of the work
recorded below are worthy of particular attention and further consideration: {a) the rate
of iron absorption in relation to iron deficiency; {b) the antagonism between iron and
manganese; and (c) the Fe/Mn ratio in the medium and in the tissues on the growth and
development of normal plants.

B. EXPERIMENTAL METHODS
The water-culture technique employed was essentially that described by Stout & Arnon
(1939) utilizing good quality distilled water and purified stock solutions of the major
nutrients (i.e. calcium nitrate, potassium nitrate, magnesium sulphate, and di-potassium
phosphate). Arnon's (1938) minor element solutions A4 and B7 were used, omitting the
manganese from the A4. The manganese and iron were added separately according to
experimental requirements. The iron source was one of the following solutions: {a) ferrous
sulphate and tartaric acid, {b) ferric citrate, or {c) ferric ammonium citrate, while the
manganese source was manganese chloride.
The seeds or fruits were germinated on filter-paper moistened with the major nutrient
solution and when the seedlings were handleable (about 2 in. long) they were transferred
to the 11. culture beakers. The experimental treatment was then commenced, each culture
receiving suitable aeration and complete changes of solution being effected three times
weekly.
At harvest the plant material was dried, ground and stored in readiness for analysis.
Two or three grams of the dried and powdered plant material were ashed in an electric
muffle at a low temperature (approximately 500° C.) and the ash taken up in warm 5 %
sulphuric acid; if insoluble manganese dioxide appeared it was dissolved by the addition
of sulphurous acid solution and heating continued to expel sulphur dioxide. The solution
of the ash was filtered and made up to volume, usually ioo ml., from which suitable ali-
quots were taken for the estimation of iron and manganese. The colorimetric methods of
analysis employed were: for iron the thioglycollic acid method (B.D.H. 1939), and for
manganese, the periodate method (Snell & Snell, 1936).

C. RESULTS
The results of the experiments are classified and will be discussed under the following
four main headings:
(1) The relation between iron and manganese supplies and the yield.
(2) The relation between the visual symptoms of nutritional status and the Fe/Mn
ratio in the substrate and in the tissues.
212 E. S. TWYMAN
(3) The iron and manganese content of the plant tissues at various levels of supply
of these elements.
(4) The visual symptoms of nutritional status and the iron and manganese content of
the plant tissues.
(i) The relation between iron and manganese supplies and the yield
This relationship may be discussed by considering the general trend of the yield at
a constant level of one of the elements when the other is varied; and also by observing the
fluctuations in yield as the Fe/Mn ratio in the culture solution is increased or decreased.
Tables 1-4 record the relevant data.
Table i. The iron to manganese ratio in the mediitm, and the dry weight yield in grams
for each culture of eight oat plants (1944)
Fe concentration in the medium
Mn cone,
in the 0-005 p.p.m. 0-50 p.p.m. 3-00 p.p.m.
medium
(p.p.m.) Fe/Mn* Dry wt. Fe/Mn* Dry wt. Fe/Mn* • Dry wt.
in medium (g-) in medium (g-) in medium (g-)
0-000 459-0000 4-43tt 459-00 7-87t 459-00 i5-i6t
0-002 2-5000 6-17: 162-00 22-i8t 351-00 2i-S3t
0010 0-5000 5-22J 45-00 30-961- 182-00 36-i6t
0-250 0-0200 5-62§ 2-00 40-89 12-00 39-66
2-000 0-0025 4-47§ 0-25 42-38 1-50 46-36
5-000 o-ooio 4-59§ o-io 41-53 0-60 49-80
* The Fe/Mn ratios have been corrected for the manganese impurity in the iron salt.
t With manganese-deficiency symptoms.
X With iron-deficiency symptoms.
§ With manganese-toxicity symptoms.

Table 2. The iron to manganese ratio in the medium and the average
dry weight in grams of single oat plants (1947)
Fe concentration in the culture solution
Mn
concentration
0 005 p.p.m. o-oio p.p.m. 0-500 p.p.m. 3-000 p.p.m.
in medium
(p.p.m.) Fe/Mn Dry wt.
Fe/Mn Dry wt.
Fe/Mn
Dry wt.
Fe/Mn
Dry wt.
medium (g.) medium (g-) medium (g.) medium (8-)

0 000 cc 0-75 (±0-32) cc 0-69 (±0-18) OC 0-67 (±o-oq) OC 1-02 (±0-44)

0 002 250 0-63 (±o-is) 5-00 0-65 (±0-12) 250 2-64 (±0-38) 1500 1-72 (±0-37)
0 005 i-oo 0-84 ( ±O'I2) 2 00 0-71 (±012) IOO 5-69 (±c-88) 600 4-72 (±1-35)
0010 0-50 0-40 ( ±0-08) I 00 0-55 (±O-OQ) 50 9-54 (±0-68) 300 8-07 (±1-20)
0 050 O-IO 0-39 ( ± o i o ) 0 20 0-69 ( ± 0 2l) 10 14-12 (±1-68) 60 13-44 (±1-75)
0250 0-02 o-si ( ± 0 1 6 ) 0-04 0-70 (±0-09) 2 10-07 (±2-22) 12 16-18 (±0-61)

See Table 8 for symptoms of nutritional disorders. Standard deviations are in brackets.

{a) Yield trends at constant manganese and varying iron supplies

Oat and tomato plants grown without manganese showed an increase in yield as the
iron supply was increased (Tables i and 3). This was probably due to the manganese
impurity in the iron salt used and not to a direct effect of the iron concentration. Never-
theless, at higher manganese supphes (greater than 0-25 p.p.m.), where the manganese
impurity in the iron salt could be neglected, increasing the iron supply up to certain
levels resulted in increased yields. Thus, for oat plants (Tables i and 2) grown with
O-O-5-O p.p.m. Mn in the medium, increasing the iron to 0-5 p.p.m. was beneficial. With
tomato plants and lettuce plants at constant manganese supply the iron concentration
 The iron and manganese requirements of plants 213
could be raised to 3-0 and 10 p.p.m. respectively with advantage (Tables 3 and 4), but in
the case of oat plants this was not always the case. In 1944 (Table i) increasing the iron
supply to 3-0 p.p.m. from 0-5 p.p.m. had a slight favourable effect or had no effect at all
on the yield, irrespective of the manganese level; in 1947 (Table 2), however, there was
a tendency for the yields to be depressed at manganese concentrations between 0-002 and
°'°5 P-P-m. when the iron supply was increased from 0-5 to 3-0 p.p.m., but when the
manganese supply was 0-25 p.p.m. an increase in yield was obtained at the highest iron
level. Lettuce plants receiving 30 p.p.m. Fe and over showed a tendency to have their
yields depressed when the manganese supply was 0-25 p.p.m. (Table 4).

Table 3. The iron to manganese ratio in the medium and the mean dry weight {four replicates)
in grams of single tomato plants {data obtained in collaboration with M. Ahmed)
Fe concentrations in the medium
Mn
concen- 0-05 p.p.m. 0-5 p.p.m. 3-00 p.p.m.
tration
in medium Fe/Mn* Dry wt. Fe/Mn* Dr}' wt. Fe/Mn* Dry wt.
(p.p.m.) in in in
medium (g-) medium (g-) medium (g-)

0-000 400 2-24 400 3-19 400 7-41

0-005 10 S-95 80 6-85 240 8-71
0050 I 6-02 10 8-97 53 11-93

All values in the above table vertically and horizontally are significantly different at the 5 % level except
that the treatment receiving 0-05 p.p.m. Fe and 0-005 P-p.m. Mn equals the treatment 0-05 p.p.m. Fe and
0-05 p.p.m. Mn.
See Table 9 for symptoms of nutritional disorders.
* The Fe/Mn ratios have been corrected for the manganese impurity in the iron salt.
Table 4. Lettuce plants {December—April 1945). The percentage increase in dry weight due
to the addition of manganese (0-25 p.p.m.) at various concentrations of iron in the medium
Iron Mean dry wt. in g. of eight plants (tops only) Percentage
concentration increase
in medium due to
(p.p.m.) No Mn With 0-25 p.p.m. Mn added Mn
2-0 1-87 (±0-30) 5-58 (±0-98) 197-5
lO'O 1-46 (±0-23) 7-10 ( + 1-40) 386-8
300 i-6i (±0-15) 5-58 (±0-81) 247-3
50-0 1-90 ( + 0-34) 6-31 (±0-69) 232-4

Standard deviations in brackets.

{b) Yield trends at constant iron and varying manganese supplies

(i) At low iron supplies (0-005-0-01 p.p.m.). At low iron supplies increasing the man-
ganese supply from 0-0 to 5-0 p.p.m. had little influence on the dry weight yields of oat
plants (Tables i and 2). There might be a tendency for the yields to be depressed at the
higher manganese levels but the general low level of response was due to iron deficiency.
(ii) At medium iron supplies (0-05-0-5 p.p.m.). There was a marked progressive in-
crease in the yield of oat plants as the manganese supply was increased, to 2-0 p.p.m. in
1944 (Table i), to 0-05 p.p.m. in 1947 (Table 2) and to 0-05 p.p.m. in 1949 with tomato
plants (Table 3). There was some indication that the yields of the oat plants were de-
pressed by increasing the manganese supply to 5-0 and 0-25 p.p.m. in 1944 and 1947
respectively.
New Phytol. 50, 2 14
214 E. S. TWYMAN
(iii) At high iron supplies (3-0 p.p.m.). In the three species investigated (Tables 1-3)
increasing the manganese supply from 0-0 to 5-0 p.p.m. at 3-0 p.p.m. Fe level resulted in
an increased yield.
{c) The iron to manganese ratio in the medium and the yield
The Fe/Mn ratio in the culture solution responsible for producing the greatest yields
varied with the particular occasion on which experiments were conducted; thus with
oat plants the best responses obtained were associated with an Fe/Mn ratio of 0-6 in 1944
and 12-0 in 1947 (Tables i and 2). Fe/Mn ratios of the same order as those most suitable
for high yields and normal growth and development were also found associated with
plants showing chlorosis and low yields; for example, oat plants in 1944 (Table i) supplied
with o-oi p.p.m. Mn and 0-005 P-P-m- Fe (Fe/Mn = 0-5) had a yield of only 5-2 g.
compared with 49-8 g. at higher levels of iron and manganese where the Fe/Mn ratio was
0-6. Similar results were obtained in the experiment with tomato plants (Table 3), where
those supplied with 0-05 p.p.m. Fe and 0-005 p.p.m. Mn (Fe/Mn = 10) had average yields

Table 5. Oat plants 1944 and 1947. Iron to manganese ratio, in the culture solutions
producing normal plants in relation to the dry weight yields in grams
1944
Fe/Mn
O-I 0-25 0-60 1-5 2-0 1 2 -0

Dry wt. (g.) 41-53 42-38 49-80 46-36 40-89 39 -66

1947
Fe/Mn 2-0 io-o 12-0 60-0

Dry wt. (g.) 10-07 14-12 16-18 I3'44

of 5-95 g. compared with 8-97 g. when the ratio was the same, but the supplies of both iron
and manganese were ten times greater. It is clear, therefore, that the absolute levels of
supply of iron and manganese were more important in determining the yield than the
Fe/Mn ratio when the supplies of iron and/or manganese were low. This is to be expected
according to the law of limiting factors.
When the supplies of these elements were adequate and normal healthy plants were
produced the Fe/Mn ratio in the medium appeared to have some effect on the yield. This
is seen in Table 5, where it is evident that the particular ratios at which maximum yields
were obtained (i.e. 0-6 in 1944 and 12-0 in 1947) varied considerably; it is therefore
suggested that the optimum ratio is probably determined by factors other than nutritional
such as temperature, day length and light intensity.

(2) The relation between the visual symptoms of nutritional status and the
iron to manganese ratio in the substrate and in the tissues
The following discussion is concerned with the incidence and the rate of development
of the symptoms of manganese deficiency, iron deficiency and manganese toxicity, with
respect to the Fe/Mn ratio in the medium and in the tissues The relevant data available
are presented in Tables 6-9, and they will be discussed under the two headings: {a) man-
ganese deficiency; {b) chlorosis.
 The iron and manganese requirements of plants 215
{a) Manganese deficiency
All cases of manganese deficiency in oat plants and tomato plants (Tables 6-9) were
associated with high iron to manganese ratios in the medium (i.e. greater than 45) and in
the tissues (i.e. greater than 8-o). As the Fe/Mn ratio in the medium and in the tissues
became smaller it took longer for the first signs of grey speck to appear. Thus, when the
iron supply (3-0 p.p.m.) greatly exceeds a low supply of manganese (o-o-o-oi p.p.m.),
manganese-deficiency symptoms will appear more rapidly than when iron is in moderate
supply (0-5 p.p.m.). Manganese deficiency, as in the medium, was correlated with high
Fe/Mn ratios in the tissues where iron accumulated to at least an extent eight times that
of manganese. Accumulation of iron, however, as will be mentioned later, is associated
with other nutritional disorders.

Table 6. Oat plants [February-April 1944). Showing the relation between

the iron to manganese ratio and the nutritional status of oat plants

Fe/Mn in Concentration in
the medium Symptoms: (a) at harvest,
Dry wt. (6) when first seen in
(a) (g.) days after sowing
(b) (*)
Medium Tissues Fe Mn 8 plants
(p.p.m.) (p.p.m.) (a) (b)
459-000 oc 0-005 0-000 4-43 Severe grey speck 38
459-000 30-00 0-500 0-000 7-87 Severe grey speck 44
459-000 24-00 3-000 0-000 15-16 Severe grey speck 52
351-000 21-60 3-000 0-002 21-53 Grey speck 54
182-000 10-25 3-000 0010 3616 Slight grey speck 66
162-000 15'4° 0-500 0-002 22-18 Grey speck 57
45-000 7-66 0-500 o-oio 30-96 Slight grey speck 66
12-000 200 3-000 0-250 39-66 Normal —
2-500 3-30 0-005 0-002 6-17 Chlorotic 50
a-000 1-13 0-500 0-250 40-89 Normal —
1-500 0-66 3-000 2-000 46-36 Normal —
0-600 0-53 3-000 5-000 49-80 Normal —
0-500 1-65 0-005 O-OIO 5-22 Chlorotic —
0-250 0-55 0-500 2-000 42-38 Normal —
o-ioo 0-55 0-500 5-000 41-53 Normal —
0-020 0-21 0-005 0-250 5-62 Chlorotic (Mn toxicity) 44
0-002 O-IO 0-005 2-000 4-47 Chlorotic (Mn toxicity) 44
O-OOI 0-08 0-005 5-000 4-60 Chlorotic (Mn toxicity) 44
The Fe/Mn ratios in the medium are corrected for manganese impurity in the iron salt.

In 1947 most of the oat plants (Table 8) grown at low concentrations of iron (0-005
and O-OI p.p.m.) and manganese levels of 0-0—o-oi p.p.m. did not develop manganese-
deficiency symptoms although plants at the higher iron supplies (0-5 and 3-0 p.p.m.) and
the same manganese concentrations did develop grey speck.
This evidence suggests some relation between the occurrence of manganese deficiency
and the iron supply. This is due to at least two causes: (i) the increase in dry matter due
to adequate iron supplies at low manganese levels; and (2) the retardation of manganese
uptake by high iron concentrations. Thus at a constant low manganese level a plant may
remain small and free from manganese-deficiency symptoms due to low availability of
iron, but if the iron supply is increased then the manganese becomes the limiting factor
to the development of the resultant larger plant and manganese-deficiency symptoms
appear. In addition to this process the absorption of manganese is antagonized by in-
creasing the iron concentration in the external solution. Thus at a low manganese supply,
2l6 E. S. TWYMAN
manganese deficiency would be expected to arise sooner at high iron concentrations than
lower ones.
Table 7. Oat plants. Showing the relation between the iron to
manganese ratio and the nutritional status of oat plants

Fe/Mn in Concentration in
the medium of Dry wt. Symptoms at harvest
yield and remarks
(0) (b) (g-)
Medium Tissues Fe (p.p.m.) Mn (p.p.m.)
CC 21-000 0-20 0000 6-S All Mn deficient. First
seen 17 days after
sowing
100-000 12-40 0-20 0002 u-3 All Mn deficient. Fitst
seen 20 days after
sowing
20-000 6-10 0-20 o-oio 24-7 Normal plants
4-000 2-40 0-20 0-050 26-2 Normal plants
0-800 0-67 0-20 0-250 27-5
Normal plants
o-ioo 0-27 0-20 2-000 22-4 Normal plants
0-040 016 0-20 5-000 249
Normal plants
0-020 0-13 0-20 10-000 24-1 Slight chlorosis of upper
leaves
0-013 0-065 0-20 15-000 15-3
Severe chlorosis of
upper leaves
0010 0-090 0-20 20-000 Slight chlorosis of upper
25-4
leaves
0-000 0-00 0-050 7th leaf and tillers
5-3 chlorotic
0-000 0-00 0-000
Chlorotic and very
2-5 slight Mn deficiency
41 days after sowing

Table 8. Sytnptoms of nutritional disorder and the mean fresh weight of single oat plants
at different levels of supply of manganese and iron {four replicates)

Mn in Fe in the culture medium

medium
(p.p.m.) 0-005 p.p.m. 001 p.p.m. 0-50 p.p.m. 3-0 p.p.m.
o-ooo Chlorotic, grey speck Chlorotic, grey speck Very severe grey Severe grey speck.
on lower green on lower green speck, plants dying plants dying
leaves of one plant leaves of one plant
only only
6-03 g. 5-2 g. 4-8 g. 5-O g-
O-OO2 Chlorotic, no grey Chlorotic, no grey All plants with grey Severe grey speck
speck speck speck
4'6 g. 4'9 g- 28-9 g. 17-9 g-
0-005 Chlorotic, no grey Chlorotic, no grey Fair green, all plants Fair plants, but all
speck speck with grey speck with grey speck
6-9 g. 5'9 g- 62-6 g. 54'° g-
O-OIO Chlorotic, no grey Chlorotic, no grey Good plants, slight Good plants, slight
speck speck grey speck grey speck
2-2 g. 3-8 g. 97-1 g- 85-2 g.
0-050 Chlorotic Chlorotic Good plants, normal Good plants, normal
2-0 g. 5-5 g- 127-2 g. 122-3 g-
0-250 Chlorotic Chlorotic Good plants, normal Good plants, normal
3-2 g. 4-5 g- 87-8 g. 132-0 g.

{b) Chlorosis
There were two types of chloroses encountered in these water culture experiments, one
due to iron deficiency and the other caused by manganese toxicity, the symptoms of which
were practically indistinguishable in the early stages from iron deficiency. The latter was
 The iron and manganese requirements of plants 217
associated with Fe/Mn ratios in the medium and in the tissues which were also found with
normal or manganese-deficient plants (Table 6; treatments: (i) 0-005 p.p.m. Fe and
0-002 p.p.m. Mn, and (ii) 0-005 P-P-m. Fe and o-oi p.p.m. Mn). Manganese toxicity,
however, is usually associated with much lower Fe/Mn ratios (i.e. less than 0-02 in the
medium or less than 0-2 in the tissues, e.g. Tables 6 and 7). There was, however, no very
close relation between the Fe/Mn ratio and the incidence of these chloroses which were
mainly determined by the absolute quantities of iron and manganese available in the
medium.

Table 9. Tomato plants. Showing the relation between the iron

to manganese ratio and the nutritional status of tomato plants

Fe/Mn in Concentration in
the medium Mean
(a) dry wt. Observations at harvest
(a) (b) (g-)
Medium* Tissues Fe Mn
(p.p.m.) (p.p.m.)
400 OC 0-05 0-000 2-24 Severe manganese deficiency
400 645-0 0-50 0-000 3-19 Severe manganese deficiency
400 120-0 3-00 0-000 7-41 Manganese deficiency
240 83-0 3-00 0-005 8-71 Slight manganese deficiency
80 34-0 0-50 0-005 6-85 Manganese deficiency
53 7-6 3-00 0-050 11-93 Normal
10 3-8 0-50 0-050 8-97 Normal
10 4-4 0-05 0-005 5-95 Manganese and iron deficiency
I
2-4 0-05 0-050 6-02 Severe iron deficiency
(See Table 3 for significance of dry weights.)
* The Fe/Mn ratios in the medium have been corrected for the manganese impurity in the iron salt.

(3) The iron and manganese content of the plant tissues at various
levels of supply of these elements
This aspect of the work may be considered under the following four headings:
{a) The manganese concentrations in the tissues as affected by increasing the iron
supply at a constant level of manganese in the medium.
{b) The manganese concentration in the tissues as affected by changes in the manganese
supply at constant levels of iron in the medium.
(c) The iron concentrations in the tissues as affected by changes in the manganese
supply at constant levels of iron in the medium.
{d) The iron concentrations in the tissues as affected by changes in the iron supply at
constant manganese levels in the medium.
{a) The most general effect of increasing the external iron supply to plants (oats,
lettuce and tomato) receiving a constant level of manganese in the culture solution was the
progressive depression of the manganese concentrations in the tissues (Tables 10-14).
This result may be explained by considering the effect of two factors, namely: (i) a dilution
factor, whereby the available manganese is diluted by the increasing quantity of tissue
produced as the iron supply is increased; (ii) an antagonism factor, whereby the absorp-
tion of manganese is progressively retarded by increasing the iron supply.
In these experiments, at any particular constant manganese level from 0-0to ioo-o p.p.m.,
the growth and development was controlled by the supply of iron. For example, where
iron was limiting growth (i.e. 0-005-0-01 p.p.m. Fe, Tables 10 and 11) manganese was
2i8 E. S. TWYMAN
accumulated in the tissues of these small chlorotic plants in quantities ranging from low
(io and 12 p.p.m.. Table 11) to excessive quantities (780 p.p.m.. Table 10; and 529 p.p.m..
Table 11). When, however, the iron supply was increased, considerable increase m growth

Table 10. Manganese concentrations {p.p.m. dry weight) in the stem and leaf tissues of oat
plants (1944) grown with various iron and manganese concentrations in the culture
solution
Fe concentration in medium
Mn concentration
in medium
p.p.m. 0-005 0-5 3-0
p.p.m. p.p.m. p.p.m.

0-000 o*t 5* 4*
0-002 7* 6*
23t 9* 8*
o-oio 37t
0-250 318I 68 42
2-000 635t 166 114
5-000 78ot 233 135

* With manganese-deficiency symptoms.

f With iron-deficiency symptoms.
X With manganese-toxicity symptoms.

Table 11. Manganese concentrations {p.p.m. dry weight) in the stem and leaf tissues of oat
plants (1947) grown at various iron and manganese levels in the culture solution
Fc concentration in mediurr1
Mn concentration
in medium 0-005 o-oio 0-500 3-0
(p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.)
0-000 5*t 6*t 4* 6*
0-002 I2t lot 3* 2*
0005 34t 3ot 7* 8*
0010 53t 54t 9* 6*
0-050 22of 34 21
i83t
0-250 529t 454t 92 66
* With manganese-deficiency symptoms,
f With iron-deficiency symptoms.

Table 12. Mean manganese concentrations in the stem and leaf tissues of tomato plants
grown with various concentrations of iron and manganese in the culture solution {data
obtained in collaboration with M. Ahmed)
Fe concentration in medium
Mn concentration
in medium 0-050 0-500 3-00
(p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.)
o-ooo 0-0* 0-2* I-o*
0-005 2-ot 3-0* 2-0*
0-050 31-of 200 12-0

* Manganese-deficiency symptoms,
-f With iron-deficiency symptoms.

took place and so greater demands for manganese were made, but whether these demands
were met or not obviously depended on the total quantity presented to the plant during
its existence in the nutrient medium. If this quantity was sufficient to maintain a con-
centration of 14-0 p.p.m. Mn in the tissues then normal development ensued, but if the
 The iron and manganese requirements of plants 219
quantity of manganese absorbed was unable to maintain this minimum concentration
then manganese deficiency occurred. This was the process mainly in operation at low
manganese concentrations in the medium (o-o-o-oi p.p.m.). When iron and manganese
were adequate (o-5-3-o p.p.m. Fe and Mn greater than 0-05 p.p.m.) then the antagonism
factor as well as the dilution factor began to influence the concentration of manganese in
the tissues. This appeared to be the case in Tables 10-14, where lower concentrations of
manganese in the tissues were in association with the higher iron levels at a particular
manganese concentration in the medium, but from the data available it was difficult to
determine which of the factors was mainly responsible for the results. That antagonism
is a factor has been verified by analysis of the external solution from which the absorption
of manganese by intact tomato plants at the same state of development was measured at

Table 13. The mean manganese concentrations in the stem and leaf tissues of lettuce and oat
plants supplied with 0-250 p.p.m. manganese and various iron concentrations in the
culture solution
Lettuce Oats
Fe in the Mn in the Fe in the Mn in the
medium tissues medium tissues
(p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.)
2-0 150 ( + ii-o) 0-05 444 (±66-4)
IO-O 97 (±11-4) 0-50 77 ( + 28-4)
30-0 57 (±17-4) 2-00 66 ( + 12-8)
50-0 34 ( ± 6-6) 25-00 56 (+12-8)
Standard deviations in brackets.

Table 14. Manganese and iron concentrations in the stem and leaf
tissues of oat plants {in p.p.m. dry weight)

Fe concen- Mn concentration in the medium

tration in
medium 10 p.p.m. 15 p.p.m. 20 p.p.m. 50 p.p.m. IOO p.p.m.
(p.p.m.)
Mn Fe Mn Fe Mn Fe Mn Fe Mn Fe

0-2 780 99 1246 102 1438 94 1999 96 4064 92

0-4 655 87 1049 73 1451 86 1405 119 2237 108

All cultures showed symptoms of manganese toxicity.

different iron concentrations in the medium. The results of this work may be published
later.
{b) As the manganese concentration in the culture solution is raised at a given iron
level, the concentration of manganese in the tissues is increased (Tables 10-15). -^t
low iron levels manganese was absorbed and accumulated in the small chlorotic plants to
very high values (e.g. 318, 635 and 780 p.p.m. in Table 10), and when manganese was
presented with adequate iron to oat plants in very high concentration (Table 14)
manganese may accumulate to as much as 4000 p.p.m.
(c) When considering the iron concentration in the tissues as affected by changes in the
manganese supply at constant levels of iron, there seem to be two main trends depending
on whether the iron concentration in the culture solution is low or high. At low iron levels
(i.e. O-OO5-O-OI p.p.m. Fe, Table 17) increasing the manganese in the medium up to
220 E. S. TWYMAN
O-OI p.p.m. appears to increase the concentration of iron in the tissues, whereas at high
iron levels the concentration in the tissues falls as the manganese in the medium is raised
(Tables 15-19). There are obvious exceptions to these generalizations and the factors

Table 15. The iron and manganese concentrations in the dry stem and leaf tissues of oat
plants supplied with 0-2 p.p.m. iron and various concentrations of manganese

Mn concentration Concentration in the tissues of

in medium
(p.p.m.) Fe (p.p.m.) Mn (p.p.m.)
0000 105 5*
0-002 87 7*
0010 67 II
0-050 92 39
0-250 60 89
2-000 71 256
5-000 49 297
10-000 56 447t
15-000 45 6781
20000 49 5o6t
* With manganese-deficiency symptoms,
f With manganese-toxicity symptoms.

Table 16. Iron concentrations {p.p.m. dry weight) in the stem and leaf tissues of oat plants
(1944) supplied with various iron and manganese concentrations in the culture solution
Fe concentration in nnedium
Mn concentration
in medium 0-005 0-500 3-00
(p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.)
0-000 84*t 150* 95*
0-002 76t 108* 130*
O-OIO 6it 69* 82*
0-250 68t 77 83
2-000 65t 92 75
5-000 7ot 130 71

* With manganese-deficiency symptoms.

t With iron-deficiency symptoms.
X With manganese-toxicity symptoms.

Table 17. Iron concentrations {p.p.m. dry weight) in the stem and leaf tissues of oat plants
{ ) grown with various iron and manganese concentrations in the culture solution

Mn concentration Fe concentration in the medium

in medium 0-005 O-OI 0-500 3-00
(p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.)
0-000 I46*t I73*t 234* 258*
0-002 i65t II2t no* 165*
0-005 2O3t 158* 157*
I9it
O-OIO 127* 138*
368t 277t
0-050 82 141
344t
0-250 193 91 126
255t
* With manganese-deficiency symptoms,
t With iron-deficiency symptoms.

involved in the absorption and the establishment of tissue concentrations of iron are
imperfectly known and complex. There is some indication that concentrations of
 The iron and manganese requirements of plants 221
manganese above 5-0 p.p.m. may retard the absorption of iron by oat plants when the iron
supply in the medium was 0-2 p.p.m. (Table 15), but this is not so obvious in Table 14
where concentrations up to 100 p.p.m. had no effect on the iron concentrations in the
tissues. In an experiment with tomato plants approximate estimations were made of the
percentage iron supplied and subsequently absorbed; these figures showed that very

Table 18. Average iron concentrations in the stem and leaf tissues of lettuce plants grown
with (0-25 p.p.m.) and without manganese at four different iron levels in the culture
solution

Fe Mn concentration in medium
concentration
in medium o-ooo
(p.p.m.) 0-250
(p.p.m.) (p.p.m.)
2-O 331* 236
lO-O 386* 239
30-0 427* 256
SO-O 396* 242

* With manganese-deficiency symptoms.

Table 19. Tomato plants. Average iron concentrations in the stem and leaf tissues of tomato
plants supplied with various concentrations of iron manganese {data obtained in col-
laboration with M. Ahmed)

Mn Fe concentration in medium
concentration
in medium 0-05 0-50
(p.p.m.) 3-00
(p.p.m.) (p.p.m.) (p.p.m.)
0-000 132* 129* 120*
0-005 88*f 130* 106*
0-050 74t 75 91
* With manganese-deficiency symptoms,
t With iron-deficiency symptoms.

Table 20. Tomato plants. Estimated percentage of the iron supplied and subsequently
absorbed from the medium {data obtained in collaboration with M. Ahmed)
Iron concentration in the medium
Mn 0-05
concentration 0-5 3-0
(p.p.m.) (p.p.m.) (p.p.m.)
in medium
(p.p.m.) Fe absorbed Dry wt. Fe absorbed Dry wt. Fe absorbed Dry wt.
(%) (g-) (%) (g-) (%) (g-)
0-000 24-6 2-2 13-7 3-2 6-8 7-4
0005 43-7 5-9 23-5 6-8 7-1 8-7
0-050 37-4 6-0 22-4 9-0 8-4 n-9

little, if any, antagonism took place (Table 20). At the 0-05 p.p.m. Fe level the percentage
iron absorbed at the two manganese levels was approximately the same; thus iron absorp-
tion was unaffected. When the iron supply was raised to 0-5 p.p.m. an increased yield
was obtained by raising the manganese supply from 0-005 ^^ °'°5 P-P-m., but very little
difference in the percentage iron absorbed was recorded. This may be interpreted as
antagonism since a greater percentage of iron would be expected to be absorbed by a
222 E. S. TWYMAN

larger plant than a smaller one but this was not so. At the 3-0 p.p.m. Fe level increasmg
the manganese in the medium resulted in an increase in dry weight yield. Thus in the
absence of more critical data it seems unlikely that manganese in concentrations less than
5-0 p.p.m. adversely affect the absorption of iron. It will be of interest to examine directly
the absorption by intact plants of iron as affected by manganese in culture solutions.
{d) Increasing the iron supply may cause an increase in the iron concentration in the
tissues (Table 17, e.g. O-5-3-O p.p.m. Fe), but this is not always the case (Table 16,
e.g. 0-5-3-0 p.p.m.). When iron is in low concentration in the medium it may accumulate
in the tissues to very high levels (e.g. 368 p.p.m.. Table 17), and bear no relation at all
to the concentration in the substrate.
(4) The visual symptoms of nutritional stattts and the iron and
manganese content of the plant tissues
Three disorders may be recognized: {a) manganese deficiency, {b) iron deficiency, and
(c) manganese toxicity.
{a) Manganese deficiency
With oat, lettuce and tomato plants manganese-deficiency symptoms were always
present when the concentration in the dry stem and leaf tissues was less than i2-o p.p.m.
It was also observed that manganese-deficient plants usually have a somewhat high iron
content of over 90-100 p.p.m.

{b) Iron deficiency

A wide range of iron concentrations were found in iron-deficient plants which frequently
accumulated iron in higher concentrations than normal plants (Tables 17 and 19). The
main factors responsible for the development of the symptoms are: (i) the total amount
of iron presented to the plant during its life in the culture solution, and (ii) the rate of
supply of iron, that is in practice the frequency of changing the culture solution; these
factors will be discussed further, later on. The concentration of iron in the tissues bears
no relation to the incidence of chlorosis, and as mentioned previously, where iron limits
growth and development and manganese is available in the substrate, this element may
accumulate to quite high levels (300-400 p.p.m.).

{c) Manganese toxicity

Oat plants exhibiting manganese toxicity had a content of manganese in the dry tissues
of 300 p.p.m. or more, when the iron supply was such that normal plants were produced
if the manganese supply was lower and accumulation in the tissues of the element did not
occur (Table 15). It may be assumed rather tentatively that smaller quantities of man-
ganese in the tissues may be toxic under iron-deficient conditions. The concentration of
iron in the tissues of plants with manganese-toxicity symptoms was very variable (Tables
15 and 16).
D. DISCUSSION

In the course of this investigation five states of supply and apparent utilization of iron and
manganese were recognized:
(1) Low supplies and inadequate utilization of both manganese and iron.
(2) Adequate manganese and low iron supplies with inadequate utilization of iron.
(3) High manganese and adequate iron resulting in inefficient utilization of iron.
 The iron and manganese requirements of plants 223
(4) Low manganese and adequate iron supplies resulting in increased iron utilization
and the appearance of manganese-defieieney symptoms.
(5) Adequate supplies of both manganese and iron resulting in the optimum utilization
of iron and manganese.

(i) Low iron and manganese supplies

If a comparison is made between the iron and manganese contents of the tissues of
plants receiving low iron and manganese and those of plants receiving adequate supplies
of both elements it is apparent that there is a tendency for iron to accumulate in the
tissues (Table 21). These plants are never normal, and under the conditions of these
experiments they are always small and chlorotic, yet the concentrations of iron and
manganese are such that by more than doubling the dry weight yield with no further
absorption of iron and manganese, the concentration of these elements could be main-
tained at levels usually associated with normal healthy growth and development. The

Table 21.
Concentration in Concentration in
Treat- medium Mean tissues
ment Description of supplies dry wt.
no. Mn Fe (g-) Mn Fe
(p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.)
I Low Mn and Fe 0-005 0-005 0-85 34 203 Chlorotic
2 Low Mn and Fe 0-010 0-005 0-40 368 Chlorotic
53
3 Low Mn and Fe 0-005 o-oio 0-71 30 191 Chlorotic
4 Low Mn and Fe o-oio O-OIO °'55 54 277 Chlorotic
S Adequate Mn and low Fe 0-250 0-005 0-51 529 255 Chlorotic
6 Adequate Mn and low Fe 0-350 O-OIO 0-70 454 193 Chlorotic
7 Low Mn, high Fe 0-005 3-000 4-72 8 157 Mn deficient
8 Low Mn, high Fe O-OIO 3-000 8-07 6 138 Mn deficient
9 Adequate Mn and low Fe 0-050 0-500 14-12 34 82 Normal

fact remains that under these conditions of low iron and manganese supplies, plants are
not even small and normal in appearance, they are always chlorotic.
It is clear, therefore, that although the supply of iron in the early stages of growth and
development is a limiting factor because of the low concentration in the tissues, the iron
accumulated during subsequent stages of growth is never fully and efBciently utilized.
This leads to the conclusion reached by previous investigators that iron is inactivated
within the plant. Since large ' inactive' accumulations of iron occur under conditions of
low intermittent iron supply (culture solution changed every 2 or 3 days) it appears that
the slow rate of entry of iron favours 'inactivations'. It is suggested that there may be
a primary iron acceptor responsible for transferring iron to active metabolic processes.
If this primary or active iron acceptor does not react with iron it becomes irreversibly
converted to a secondary product which forms a very stable compound with iron. If A is
the active iron acceptor and B is the inactive iron acceptor then A in the absence of iron
is slowly and irreversibly converted to B; thus with a slow rate of iron entry where the
production of A exceeds the number of iron ions necessary to react with all the active
acceptors, the B or inactive acceptors will accumulate and eventually compete with A
for the small amounts of iron entering the plant. It is apparent, therefore, on this
hypothesis, how iron becomes progressively inactivated within the plant under con-
ditions of low iron supplies.
224 ^ - ^- TWYMAN

(2) Adequate manganese and low iron supplies

Plants supplied with adequate manganese are usually more chlorotic than plants
supplied with low manganese. It is suggested that manganese competes with the available
iron for reaction with the active iron acceptors forming stable inactive metabolites and
this would intensify the chlorosis and both iron and manganese would accumulate
(e.g. Table 21, treatments 5 and 6).

(3) High manganese and adequate iron supplies

Under conditions of high manganese and adequate iron supplies plants usually show
chlorosis, the intensity of which is correlated with the manganese concentration in the
medium. This chlorosis of manganese toxicity may be due to one or more of the following
processes:
(i) Competition between the iron and manganese for the active iron acceptors.
(ii) Antagonism between iron and manganese whereby manganese in high concentra-
tion in the medium may slow up the entry of iron thus encouraging the formation of the
inactive iron acceptors. This antagonism will be further studied.
(iii) A direct toxic action of manganese on processes concerned in iron metabolism.

(4) Low manganese and adequate iron supplies

Under these conditions manganese deficiency develops due to the small quantities of
manganese accumulated in the tissues. The low level of manganese in the tissues is aggra-
vated by the frequent changes of culture solution, a procedure which may prevent the
complete absorption of the available manganese in the medium because of the antagonistic
influence of the iron. Thus the total quantity of manganese in the plants and the time of
the appearance of the symptoms is determined to some extent by the external iron
concentration.
(5) Adequate supplies of both iron and manganese
Normal plant growth and development, as far as can be seen, is characteristic of
adequate supplies of iron and manganese. Analysis of the tissues shows that considerable
variation in the concentration of iron and manganese is to be expected. In this connexion
it might be as well to remember that these concentrations merely measure the degree of
dilution of the absolute quantities of iron and manganese absorbed by the plant up to the
time of sampling, the absorption of these elements usually taking place under very ill-
defined conditions. Many factors will probably determine the actual values obtained, for
example, light intensity, temperature and day length; these factors are usually not
controlled in the field or in greenhouse experiments. Any one of these or other factors may
influence the processes of absorption, growth and development, with the result that the
concentrations of iron and manganese in the tissues will be affected. It is, therefore, to
be expected that normal plants, even under the conditions of these experiments, will have
very different iron and manganese values between themselves in one experiment and
between the same treatments in different experiments. It has been noted that within
a particular experiment the Fe/Mn ratio in the medium may be important in determining
the maximum yield amongst normal plants. This point needs further examination, but it
may be mentioned here that it is doubtful whether the ratio will, in the immediate future,
be of practical importance in the diagnosis of the nutritional status of plants in the field.
 The iron and manganese requirements of plants 225
It may well be that the Fe/Mn ratio will only be related to dry weight yields under strictly
controlled conditions.
E. SUMMARY
1. The results of investigations on the iron and manganese requirements of oat, lettuce
and tomato plants are presented and discussed under the following headings:
{a) The iron and manganese supplies and the yield.
{b) The visual symptoms of nutritional status and the Fe/Mn ratio in the substrate
and in the tissues.
(c) The iron and manganese content of the tissues at various levels of supply of these
elements.
{d) The visual symptoms of nutritional status and the iron and manganese content of
the tissues.
2. The rate of entry of iron in relation to the development of iron deficiency is
emphasized as important in the accumulation of inactive iron.
3. The part played by the iron-manganese and manganese-iron antagonisms in the
development of iron deficiency, manganese deficiency, and toxicity is discussed.
4. It is suggested that the Fe/Mn ratio in the substrate and in the tissues may be
important in the metabolism of apparently normal plants.

During the course of this work I was fortunate enough to receive help from {a) The
Agricultural Research Council, and {b) The Research Committee of the University of
Birmingham. The Agricultural Research Council made a grant which enabled a technical
assistant to be employed on this work, while the Research Committee provided funds for
the purchase of special equipment. I am indebted to these two bodies and wish to thank
them for their financial assistance.
To Prof. W. Stiles, F.R.S., I am most grateful for his interest and encouragement
during the course of this work. I also wish to thank Mr M. Ahmed for carrying out the
experiments on tomato plants referred to in the text.

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